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+{"text": "To evaluate the association between mood and anxiety disorders and thyroid autoimmunity in a community sample. Methods: A community based sample of 222 subjects was examined. Psychiatric diagnoses were formulated using the International Composite Diagnostic Interview Simplified (CIDIS), according to DSM-IV criteria. All subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO).16.6% of the overall sample had an anti-TPO value above the normal cut-off. Subjects with at least one diagnosis of anxiety disorders  or mood disorders  were positive for serum anti-TPO more frequently than subjects without mood or anxiety disorders. A statistically significant association with anti-TPO+ was found in Anxiety Disorder Not Otherwise Specified , in Major Depressive Episode  and Depressive Disorder Not Otherwise Specified .The study seems to suggest that individuals in the community with thyroid autoimmunity may be at high risk for mood and anxiety disorders. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. Should our results be confirmed, the findings may be of great interest for future preventive and case finding projects. Autoimmune thyroid disease may be linked to depression  and anxiThe purpose of this investigation was to evaluate the relationship between mood and anxiety disorders and thyroid autoimmunity in a community survey. This research was carried out on the data base of two epidemiological studies aimed at defining the prevalence of psychiatric  and thyrThis paper present the results of the cross psychiatric and endocrinological evaluation from the common areas of the two surveys.The sample was extracted by randomization (1/10) subsequent to stratification according to age and sex, from the records of 2 Sardinian villages. Probands were interviewed face to face in their homes by specifically trained physicians. Two standardized forms were used to acquire information concerning: demographic data, state of health and use of social and health services. Psychiatric diagnosis was made using the Italian Simplified version of the Composite International Diagnostic Interview (CIDIS) . The comAnti-thyroid peroxidase autoantibodies (anti-TPO), considered as the most sensitive and specific marker of thyroid autoimmunity  was deteAll subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO). FT4 and FT3 were measured by means of a chromatographic method based on separation of free T4 on Lisophase columns . TSH was measured by a chemiluminescent method  with normal values ranging from 0.3\u20133.0 \u03bcU/ml. Thyroid echography was performed using a \"real time\" echograph .2 test in 2 \u00d7 2 tables. Odds Ratio confidence intervals were calculated through application of the method of Miettinen [The association of anti- TPO+ with the main diagnoses deriving from CIDIS interview was calculated using Odds Ratio. Statistical significance was calculated using the Xiettinen .Multivariate Logistic Regression was performed in order to evaluate the possible influences of gender and age on the association between anti-TPO+ and mood or anxiety disorders. The analysis was carried out considering mood (or anxiety) disorders as dependent variable, and anti-TPO+ (presence vs absence), gender  and age (\u2264 44 vs > 44) and their second order interactions as independent variables, by means of backward stepwise procedure; interactions lacking evidence of association (p > 0.20) were eliminated from the models.From a total of 261 subjects identified (age >18 years), 222 (85.1%), 127 females (57.2%), and 95 males (42.7%); over 44 years 127 (57.2%), 79 females  48 males (37.7%), agreed to take part in the study whilst 20  refused to participate and 19 (7.3%) could not be traced. The final sample did not differ respect to the population of origin with reference to the variables applied in stratification.The lifetime prevalence of anxiety disorders in the sample was: Generalized Anxiety Disorder (GAD) 11.3%, Panic Disorder (PD) 2.7%, Anxiety Disorder Not Otherwise Specified (ADNOS) 5.4%, Social Phobia (SP) 5.4%; 18.5% had been diagnosed with at least one of these anxiety disorders. With regard to mood disorders, Major Depressive Episode (MDE) was present in 14.4%, Dysthymic Disorder (DD) in 2.7%, Depressive Disorder Not Otherwise Specified (DDNOS) in 4.0%; 18.9% had at least one of the above mentioned depressive disorders. 1.1% of the overall sample was affected by hypothyroidism, 16.6% had an anti-TPO value above the normal cut-off (anti-TPO+).Table The Multivariate Logistic Regression showed that gender and age do not interact with anti-TPO either in mood , or in anxiety disorders . Interactions were therefore eliminated from the models.The final Logistic Regression models clearly indicated that gender and age do not influence the risk of one mood or anxiety diagnosis either as independent variables or as confounders Table .The present study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+ in a general population sample which had not been selected from medical or psychiatric health facilities. This association is independent by gender and age. Regarding specific diagnosis, MDE, DDNOS and ADNOS were associated with anti-TPO+.This finding is consistent with several previous clinical studies providing evidence for a significant association of mood disorders or post-partum depression and symptomless autoimmune thyroiditis with or without sub-clinical hypothyroidism . AssociaA sub-clinical dysfunction of axis Thyrotropin Releasing Hormone (TRH) \u2013 Thyroid Stimulating Hormone (TSH) with consequent alteration of circadian rhythms of TSH has been hypothesized in some depressive disorders. Indeed, this hypothesis may explain why some forms of mood disorders were associated with anti-TPO+ or thyroid autoimmunity without hypothyroidism, as defined by routine blood tests. A slight reduction in thyroid hormone secretion such as that found in sub-clinical hypothyroidism may affect cognition and mood . At variMoreover, a study carried out in a large community sample found no association between thyroid dysfunction, including hypothyroidism defined by thyroid blood test, and the presence of depression or anxiety symptoms . This suWith regard to the public heath aspects of this research, should these findings be confirmed, they would constitute a most important public health issue, due to the high attributable risk found. The attributable risk is a useful measure to document the burden of risk to a community. Attributable risk depends both on the magnitude of relative risk and on the prevalence of the risk factor in the population . The higThe potential of the study is reduced by the small sample size, particularly regard to psychiatric diagnoses less frequently observed in the general population, such as Panic Disorder; the extension of the findings is therefore rather limited.This study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. If the findings are confirmed, they may prove to be of interest for future projects of case finding: a systematic screening for mood disorders in anti-TPO+ subjects and a systematic evaluation for thyroid diseases and thyroid autoimmunity in subjects with mood disorders may be advisable.None declared.MGC conceived the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. AL, SM and CS participated in the statistical analysis and drafted the manuscript. MCH, SM, MC, BC, LDO participated in its design and coordination. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Vitiligo is an acquired depigmenting disorder due to destruction of melanocytes. Although many theories have been suggested for its pathogenesis, the role of autoimmunity is the most popular one. The association of vitiligo with autoimmune thyroid diseases and the increased prevalence of autoantibodies including thyroid autoantibodies in vitiligo favor this role. Our objective was to compare the frequency of thyroid peroxidase antibody (anti-TPO) in vitiligo patients with healthy subjects in Iran.Ninety-four cases of vitiligo  and 96 control subjects  were enrolled in this controlled study. Patients with known thyroid disease, history of thyroid surgery and those receiving thyroid medications were not included. The two groups were matched regarding gender and age. The demographic data, symptoms related to thyroid diseases and results of skin and thyroid examinations were recorded in a questionnaire for each subject. Thyroid function tests including free T3, free T4 and TSH-IRMA were performed. Anti-TPO levels were assessed as well. The collected data were analyzed by SPSS version-11 in vitiligo patients and subgroups according to gender, age, extent, and duration of the disease compared with the control group.Anti-TPO was detected in 17 (18.1%) of patients affected by vitiligo, while this figure was 7 (7.3%) in the control group; the difference was significant with p-value < 0.025 (Phi & Cramer's V = 0.162). When analyzing subgroups, the difference in the frequency of anti-TPO remained significant only in females  (Phi & Cramer's V = 0.207) and in patients in the age ranges of 18\u201325  (Phi & Cramer's V = 0.28) and 26\u201335 year-old  (Phi & Cramer's V = 0.304).The difference of the frequency of anti-TPO was not significant regarding the duration and extent of vitiligo. In addition, there was no significant difference in the levels of free T3, free T4, and TSH in vitiligo patients compared with the control group.According to our study, anti-TPO was shown to be significantly more common in vitiligo patients especially in young women, compared with control group. As this antibody is a relatively sensitive and specific marker of autoimmune thyroid disorders including Hashimoto thyroiditis and Graves' disease, and considering the fact that vitiligo usually precedes the onset of thyroid dysfunction, periodic follow-up of vitiligo patients for detecting thyroid diseases is further emphasized especially in young women with increased level of anti-TPO. Vitiligo is an acquired depigmenting disorder due to destruction of melanocytes and the resultant absence of pigment production affecting skin and mucosal surfaces with a prevalence of about 1%. Different theories regarding its pathogenesis have been put forward, autoimmunity being the most popular one. The latter is based mainly on the association of vitiligo with known autoimmune diseases and the presence of organ specific antibodies in affected patients . VitiligWe conducted this controlled study for comparison of anti-TPO in vitiligo and healthy subjects in Razi Hospital, a university hospital in Tehran, Iran, from September 2004 till March 2005. Ninety-four vitiligo  patients without a history of thyroid surgery or taking medication for thyroid diseases were enrolled. The control group  comprised healthy medical students, medical staff and outpatients' relatives; cases with any history or sign of vitiligo and positive family history of vitiligo in their first and second degree relatives as well as those with a history of thyroid surgery or taking medication for thyroid diseases were excluded. All the subjects in the two groups underwent a complete skin and thyroid examination. The thyroid function tests  and anti-TPO were assessed for all. Radio-immunoassay was used for thyroid function tests . Anti-TPO was assessed using enzyme linked immunosorbant assay (ELISA) (Kit from Monobind) . The study was approved by the Committee of Ethics of the vice -chancellor of Tehran University of Medical Sciences. Consent was obtained from all participants. The data were analyzed using SPSS software version 11 with Mann-Whitney U, t, chi-square, Fisher's exact, Kolmogorov-Smirnov and Phi & Cramer's V tests.Some of the demographic and clinical findings of vitiligo patients are presented in table Anti-TPO was detected in seventeen cases (18.1%) in vitiligo patients compared with 7 cases (7.3%) in the control group: The difference was statistically significant with a p-value of 0.025. The intensity of the association of the presence of anti-TPO with vitiligo was 16.2% (Phi & Cramer's V = 0.162). Five cases (10.4%) of males in the vitiligo group were anti-TPO positive while this figure was 2 (4.3%) in the control group with no significant difference (Fisher's exact test: p = 0.226). Twelve female cases (26.1%) in the study group had anti-TPO compared to 5 cases (10.2%) in the control group. There was a significant difference in anti-TPO positivity in female cases between the study and control groups (chi-square = 0.044)  ( Phi & Cramer's V = 0.207). In order to assess the variation of anti-TPO with age, each group was divided into four equal subgroups: lower or equal to 17 year old, between 18 and 25, between 26 and 35 and equal or greater than 36 year-old. In the age range of 18 to 25, 20.8% of the study group and 3.2% of the control subjects had positive anti-TPO results showing a significant difference (Fisher's exact test: p< 0.05) ( Phi & Cramer's V = 0.28). Likewise, 18.5% of vitiligo patients in the age range of 26 to 35 versus 0% of controls showed positive anti-TPO with a significant difference (Fisher's exact test = 0.042) ( Phi & Cramer's V = 0.304). No significant difference was noted in the two other age ranges. Furthermore the frequency of positive anti-TPO showed no significant difference regarding the extent of the disease, the age of onset, the mean duration of disease and its clinical manifestations . Demographic and clinical findings of vitiligo patients with and without anti-TPO are presented in table Clinical autoimmune thyroid disease was diagnosed subsequent to positive anti-TPO results in 4 out of 24 (17%), 2 in the case and 2 in the control group. There were one case of autoimmune thyroiditis , one case of Graves' disease  and a case of subclinical hypothyroidism  in the vitiligo group and two patients with Hashimoto thyroiditis , one case of transient thyroiditis  and one patient with subclinical hypothyroidism  in the control group.Vitiligo is a generalized autoimmune disease manifesting acquired white patches due to loss of melanocytes. It is the most prevalent pigmentary disorder with an incidence rate between 0.1\u20132% showing multifactorial etiology and polygenic inheritance . The exaVarious thyroid autoantibodies including thyroid stimulating antibody, anti- thyroglobulin antibody and anti-thyroid peroxidase antibody, are detectable in autoimmune thyroid diseases the latter being the most sensitive test for the diagnosis and follow-up of this group of diseases. Thyroid peroxidase is responsible for the iodination of tyrosine residues in the thyroglobulin molecule. Anti-TPO antibody has been shown to mediate thyroid cell destruction in vitro. This antibody, historically referred to as the anti-microsomal antibody, is established as a sensitive tool for the detection of early subclinical autoimmune thyroid diseases, follow up of the response to immunotherapy and identification of at-risk cases for autoimmune thyroid diseases .Clinical as well as functional abnormalities of the thyroid gland have been reported by many authors to be significantly more frequent in vitiligo patients compared with control group ,4. We diAccording to our study the rate of positive anti-TPO was significantly higher in vitiligo patients compared with control subjects. Mandry et al assessed the presence and frequency of organ specific antibody in 20 patients with vitiligo and their relatives. They detected anti-microsomal and anti-thyroglobulin antibodies in 50% and 40% of their cases, respectively; they showed increased prevalence of organ specific antibodies in the relatives, as well . Morgan According to our study, the difference in the prevalence of anti-TPO was significant only in female cases and patients in the age ranges of 18 to 35 year-old, findings not previously reported in the literature. In our study we found no relationship between the presence of anti-TPO antibodies and the extent, duration, age of onset and anatomical location. Morgan found autoantibodies especially in generalized vitiligo , Mandry According to our study, anti-TPO was shown to be significantly more common in vitiligo patients especially in young women, compared with control group. As this antibody is a sensitive tool for the detection of autoimmune thyroid disorders including Graves' disease and Hashimoto thyroiditis, and considering the fact that vitiligo usually precedes the onset of thyroid dysfunction, periodic follow-up of vitiligo patients for detecting thyroid diseases is further emphasized especially in young women with increased level of anti-TPO.The author(s) declare that they have no competing interests.DM conceived the study. DM, MM, AM and BJ participated in its design and coordination. RRM and MM performed the data collection. MM and DM participated in the statistical analysis. DM and RRM drafted the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Aelurostrongylus falciformis (Schlegel 1933) in Fennoscandian badgers is described. Routine parasitological examination of nine Norwegian badgers, at the National Veterinary Institute during 2004 and 2005, identified A. falciformis in the terminal airways of five of the animals. The first stage larvae (L1) closely resembled, in size and morphology, those of Angiostrongylus vasorum (Baillet 1866). The diagnosis for both A. falciformis and A. vasorum is frequently based on the identification of L1 in faeces or sputum. The potential for misclassification of an A. falciformis infection as A. vasorum, where larval identification is the only diagnostic method used, is discussed.The first report of  Aelurostrongylus falciformis (Schlegel 1933) is a metastrongyle lung nematode of European badgers (Meles meles) and has been reported in continental Europe  i0 of 19 5% Italiane animal  reported"}
+{"text": "We have analysed 15 infiltrating duct carcinomas of the breast, 10 gastrointestinal adenocarcinomas, one each of the thyroid and larynx, and four mesenchymal tumours for the presence and the nature of procoagulant activity (PCA). The metastatic tumours had a significantly higher PCA (P = 0.01-0.001) as compared to the non-metastatic tumours in the respective groups, and almost 20-25 times the activity as compared to normal tissue (P = 0.001). Although the majority of the tumours had FVII-dependent tissue thromboplastin-like activity, some of the tumour homogenates revealed the presence of an FVII-independent PCA. Unlike the known alternate PCA, which acts via factor X activation, this PCA was factor X independent. It caused clot formation in FX-deficient plasma (six cases) and purified fibrinogen solution (four cases), indicating the presence of a Xa-like enzyme or a thrombin-like activity respectively."}
+{"text": "Although the value of routine antithyroglobin antibody (TG-Ab) testing is being questioned, in future epidemiologic studies looking at the role of PCBs in neurodevelopment perhaps TG-Ab should be included in the design as well. It might prove enlightening to also routinely test for TG-Ab at several research/medical institutions to continue to explore this immune connection with the thyroid economy. Also, perhaps it is time to explore the nutritional state  of the mother and her unborn child during gestation, which might contribute to the conflicting findings among the various cohort studies about the role of PCBs in neurodevelopment. In the meantime, until more is understood about neurodevelopmental impairment, I would like to take this opportunity to reinforce the need to routinely test all pregnant women and those planning to become pregnant for fT4, free triiodothyronine, thyroid-stimulating hormone, and TPO-Ab. Information such as this would allow for intervention, if needed, to prevent irreversible brain damage.I thank Koppe for raising the question of the significance of the presence of increased thyroid peroxidase antibodies (TPO-Ab) during neurodevelopment or even later in life. I have wondered for years why medical practitioners and laboratories do not routinely quantify TPO-Ab in blood screening for thyroid disorders. High priority should be given to learning more about the relationship between the combination of high TPO-Ab and low free thyroxine (fT"}
+{"text": "Recent studies in the field of autoimmune thyroid diseases have largely focused on the delineation of B-cell auto-epitopes recognized by the main autoantigens to improve our understanding of how these molecules are seen by the immune system. Among these autoantigens which are targeted by autoantibodies during the development of autoimmune thyroid diseases, thyroid peroxidase is a major player. Indeed, high amounts of anti-thyroid peroxidase autoantibodies are found in the sera of patients suffering from Graves' disease and Hashimoto's thyroiditis, respectively hyper and hypothyroidism. Since anti-thyroid peroxidase autoantibodies from patients'sera mainly recognize a discontinuous immunodominant region on thyroid peroxidase and due to the complexity of the three dimensional structure of human thyroid peroxidase, numerous investigations have been necessary to closely localize this immunodominant region. The aim of the present review is to summarize the current knowledge regarding the localization of the immunodominant region recognized by human thyroid peroxidase-specific autoantibodies generated during the development of autoimmune thyroid diseases. In vitro and in vivo cytotoxic effector functions have also been described such as C3 complement activation ..33].To improve our knowledge of IDR and to understand how the TPO is seen by the immune system during the pathogenesis of AITD, it was also important to determine the amino acid residues involved in the epitopes recognized by human anti-TPO aAbs. Among the major regions identified over time, contributing amino acid residues have been identified. By expressing recombinant hTPO mutants with single amino acid replacements, several groups participated to the characterization of critical amino acid residues. ,34,40,41The two main discontinuous IDR (known as IDR/A and IDR/B) have been studied for several years to identify the TPO amino acid sequences involved in the binding of human anti-TPO aAbs. Over time, a body of evidence demonstrated that IDR/A and IDR/B, although different, overlap in part ,27. ThusSuch observations emphasize, once again, the high complexity of the IDR on the TPO molecule. It has been now well demonstrated that the IDR is stretched along the MPO- and CCP-like domains of hTPO. Consequently, some regions such as the region 599\u2013617, 713\u2013720, and 772\u2013775 seem to be too far, on the three dimensional model of hTPO, to compose a single epitope, albeit discontinuous. Furthermore, a rapid inspection of the three dimensional TPO model shows that region 599\u2013617 (the main anchor point for the IDR/A-specific aAbs) is located on the opposite side of the MPO-like domain with regard to the immunodominant binding surface composed of three auto-reactive TPO peptide sequences  Figure . Until nin vivo, by using animals models for AITD, whether it will be possible to influence the diseases' course  by using these peptidic immunomodulators interacting with antigen presentation of B-cells to T-cells.Numerous efforts have been made to precisely characterize the regions, as well as the amino acid residues forming the IDR of hTPO. This could lead to the rational design of therapeutic peptides able to modulate or block immune responses such as antigen presentation. Thus, it would be of great interest to determine However, a major question remains: What is the clinical significance of the anti-TPO aAbs which are present in the two well characterized AITD (Graves' disease and Hashimoto's thyroiditis)? Although, there is a general consensus that one of the most important markers of thyroid autoimmunity is the production of such thyroid-specific aAbs. Their role during the pathogenesis of AITD is still controversial.vice versa) during the course of the diseases or after treatment.Several lines of evidence show that there is no difference in the epitope recognized by anti-TPO aAbs produced during Graves' disease, Hashimoto's thyroiditis and Euthyroiditis -46, and Thyroid Peroxidase; TPO, Autoantibody; aAb, Autoantigen; aAg, Immunodominant Region ; IDR, Autoimmune Thyroid Disease; AITD, Complement Control Protein; CCP, Myeloperoxidase; MPO, Epidermal Growth Factor; EGF.The author(s) declare that they have no competing interests."}
+{"text": "Within the intensive care unit (ICU), arterial blood pressure (ABP) is typically recorded at different (and sometimes uneven) sampling frequencies, and from different sensors, and is often corrupted by different artifacts and noise which are often non-Gaussian, nonlinear and nonstationary. Extracting robust parameters from such signals, and providing confidences in the estimates is therefore difficult and requires an adaptive filtering approach which accounts for artifact types.Using a large ICU database, and over 6000 hours of simultaneously acquired electrocardiogram (ECG) and ABP waveforms sampled at 125 Hz from a 437 patient subset, we documented six general types of ABP artifact. We describe a new ABP signal quality index (SQI), based upon the combination of two previously reported signal quality measures weighted together. One index measures morphological normality, and the other degradation due to noise. After extracting a 6084-hour subset of clean data using our SQI, we evaluated a new robust tracking algorithm for estimating blood pressure and heart rate (HR) based upon a Kalman Filter (KF) with an update sequence modified by the KF innovation sequence and the value of the SQI. In order to do this, we have created six novel models of different categories of artifacts that we have identified in our ABP waveform data. These artifact models were then injected into clean ABP waveforms in a controlled manner. Clinical blood pressure  estimates were then made from the ABP waveforms for both clean and corrupted data. The mean absolute error for systolic, mean and diastolic blood pressure was then calculated for different levels of artifact pollution to provide estimates of expected errors given a single value of the SQI.Our artifact models demonstrate that artifact types have differing effects on systolic, diastolic and mean ABP estimates. We show that, for most artifact types, diastolic ABP estimates are less noise-sensitive than mean ABP estimates, which in turn are more robust than systolic ABP estimates. We also show that our SQI can provide error bounds for both HR and ABP estimates.The KF/SQI-fusion method described in this article was shown to provide an accurate estimate of blood pressure and HR derived from the ABP waveform even in the presence of high levels of persistent noise and artifact, and during extreme bradycardia and tachycardia. Differences in error between artifact types, measurement sensors and the quality of the source signal can be factored into physiological estimation using an unbiased adaptive filter, signal innovation and signal quality measures. Arterial blood pressure (ABP) is a basic hemodynamic parameter in intensive care unit (ICU) monitoring. ABP waveforms are frequently corrupted by artifacts, such as transducer flushing, catheter clotting, movement artifacts, and non-invasive cuff inflations . These eMultiple average observations of blood pressure increase the accuracy of ABP estimates . TherefoIn this study we present extensions of our data fusion framework ,15 whichFor the evaluation of our algorithm, we chose more than 6000 hours of high quality data , which is set to be equal to 200 mmHg \u00b1 10 mmHg, with an exponential-like curve. We therefore use the hyperbolic tangent function (tanh) to simulate this behaviour as follows:This type of artifact  is the rate of saturation, Adias is the diastolic ABP and fs is the sampling frequency of the ABP. A large value of \u03b7 therefore leads to a rapid saturation to ABPmax. Examples of the real and artificial asmax artifact are shown in Figures where ABPmin) with an exponential-like decay, 3) an exponential increase from ABPmin to some ABP value, and 4) a gradual transition back to the unaffected blood pressure. An artifact boundary is created with these four parts and then applied to the ABP. The upper boundary of the first part of the artifact is the maximum of ABP in the window, and the lower part is a right upside of a square function with the independent variable altered from 0 to 2.5. The upper boundary of the second part (asmin_2) is defined as:This type of artifact Figure  appears asmin_2 to 90%. The upper boundary of the third and fourth part (asmin_3) is defined as:And the lower part is obtained by decreasing N is the length of the third and fourth parts. The lower third part is created by decreasing asmin_3 by 20% \u2013 40% of its original value and the lower fourth part is the left downside of a square function. An example of the real and artificial asmin artifact is shown in Figures where app artifact is shown in Figures This type of artifact is similar to the systolic and diastolic ABP saturation artifact, gradually decreasing the pulse pressure. We simulated this artifact by decrementing the systolic ABP in a linear manner over a variable window length. A lowpass FIR filter with a passband cutoff of 5 Hz and a stopband cutoff of 10 Hz and a Kaiser window was also applied. An example of the real and artificial asw are shown in figure This type of artifact consists of a series of square waves with varying random duty cycles. Examples of the real and artificial 2 bandpass filter. The brown noise was further filtered using a Kaiser window FIR bandpass filter with a passband between 1.5 Hz to 18 Hz. The resultant signal was then added to the real ABP signals. Examples of the real and artificial ahf are shown in figures This type of artifact is simulated by a brown noise generator (produced by Brownian motion), implemented through a 1/fsinc function as shown in Eq. (4). The central lobe of the sinc function was used as aimp artifact and was added to the real ABP signals. The impulse artifact is given byThis type of artifact is simulated by the \u03b7(0.05 <\u03b7 < 0.35) is the approximate percentage of pulse that central lobe occupies, n defines the frequency at which the sinc function oscillates, and \u230a\u230b denotes selecting the central lobe of the sinc function. An example of the real and artificial aimp is shown in figures where wSQI [jSQI [wSQI algorithm was designed to reduce the incidence of false ABP alarms by rejecting low-quality ABP segments. The algorithm consists of an open source ABP pulse onset detection routine, wabp [T), pulse pressure (the difference between the SBP and the DBP in a beat) and ECG-ABP delay time .Two previously developed ABP signal quality assessment methods, wSQI  and jSQIQI [jSQI  were comne, wabp , a wavefwSQI algorithm was previously trained on data from the MIMIC DB [true blood pressure alarms and a sensitivity of 98.2% and a positive predictivity of 99.4% for detecting false alarms. A wSQI value is associated with each beat and possesses a continuous value between 0 and 1 (bad to good). Values for wSQI greater than 0.5 generally correspond to good signal quality  [The MIMIC DB . It was MIMIC DB  with a snalysis) .jSQI algorithm is a binary abnormality index  and uses the same beat detection algorithm as wSQI, after which features in each ABP pulse are identified  ~ N and p(v) ~ N. The matrices A, B, H are the coefficient state transition matrices, Q being the state noise covariance, R the measurement noise covariance and u an optional control input to the state x.The random variables The KF algorithm is given by the following equations:a priori and a posteriori state estimates before and after a given measurement zk; Pk are the error covariances of a priori and a posteriori estimates, Kk is the gain required to minimise the a posteriori error covariance, Pk.where \u03c8, to adjust the measurement noise covariance, R, when Kk is updated. When the SQI is low, zk should be trusted less, so Kk should be small, and hence we force R to be large. This is achieved by modifying R as follows:We employed the KF to estimate the systolic, mean and diastolic blood pressure derived from the ABP at each pulse. However, in order to more heavily weight estimates derived from cleaner data, we propose the use of the SQI, R0 is chosen to be equal to unity and is the unaltered value of R. In other words, we do not assume the noise is stationary and instead the state noise covariance is adaptive based upon our signal quality measures.where R tends to unity as the SQI, \u03c8, tends to unity, and therefore doesn't affect the measurement noise covariance. When the SQI is high (near unity), the KF is forced to trust the current measurement, zk, and elevates the Kalman gain, Kk. At low values of \u03c8, R tends to infinity  and forces the KF to reduce Kk and hence trust the previous measurements more than the current measurement. Furthermore, an upper limit that defines the cusp between good and bad data, \u03c8t, is defined. When \u03c8 <\u03c8t, the KF is not updated. The determination of the value of \u03c8t is performed in the same manner as for the ECG signal quality metrics described in [\u03c8t = 0.5 provides an acceptable level of error (on average less than 20 mmHg). It should be noted that some types of artifact cause different levels of error and affect the SBP, MBP and DBP in different manners; see discussion.It can be seen from Eq. (13) that this nonlinear transformation of ribed in . This inA\u22481). After neglecting the control input, Eq. (8) then reduces to Q, the state noise covariance matrix, and R, the measurement noise covariance, to calculate Kk. R was similarly initialised to unity, noting that it is immediately modified by the SQI to reflect our trust in the data. Q was empirically adjusted to have an initial value of Q = 0.1. Values of Q < 0.1 lead to the KF trusting the state estimate too much and not adapting to the new initial observations. Values of Q > 0.1 lead to the KF trusting the new observations too much, and simply following the new values too closely. Setting H to unity then allows us to estimate the Kalman gain, Kk, from Eq. (10) and hence the a posteriori error covariance estimate, Pk, from Eq. (12). The filter can then be run online with only a few iterations (heart beats) for convergence. The Kalman residual is then given as Following Tarassenko and Townsend ,23, we pwabp [wSQI algorithm. The SQI for each beat was derived using a 10s window, centered on each beat. Second-by-second ABP features and SQI were acquired by calculating the median values of these beats within a moving 10s window, centered on each second. Then, the ABP values  together with the SQI, \u03c8, were inputted to the KF to obtain the optimal ABP estimation on a second-by-second basis. The final ABP values and SQI of each 10 second epoch were derived by calculating the median of the window's second-by-second output of the KF estimate of the ABP and SQI.The beat-onset detection method wabp  was applX, using the technique of Townsend and Tarassenko [In general it is possible to fuse any number of independent Kalman filtered observations, rassenko ,23 such Xk is the kth independent estimate and \u03c3k is the innovation . In a recent paper [k = 1) is corrupted by artifact and the corresponding parameter estimate (X1) is miscalculated, the SQI (SQI1) will be low and the sudden change of X1, will make the residual error (r1) large. The weighted innovation  sampling frequencies. Adjustments to the innovation update sequence can be made to adjust for the differing sampling frequencies, and the inherent confidences in the different recording equipment. In general, the innovation-based weighting function can be modified so thatIn theory, each of the \u03bbk \u2265 0 is a 'trust' factor for the kth channel of data. An example of the use of this parameter in blood pressure monitoring would be to fuse data from inflatable cuff measurements with direct arterial blood pressure. In this case, \u03bbk for the ABP could be set to 1.0, and the sphygmomanometer-based cuff pressure can be set to be 0.8 . In fact, the actual value of \u03bbk can be calibrated for different BP measurement devices, which have been shown to produce significantly different errors [\u03bbk.where 1 \u2265 t errors -27. For t errors . At low t errors . Such kn\u03bbk's, were set to be equal to unity, to reflect the fact that, in the absence of signal quality information, we trust the ECG as much as the ABP signal to provide an estimate of heart rate.) Figure \u03c8(ECG) is the signal quality of the ECG as described in [\u03c8(ABP) is the \u03c8 described in this paper.Of course, a continuous blood pressure waveform also carries more information than just blood pressure. Together with ABP, heart rate estimation is extremely important as a first-order estimate of cardiovascular system performance. In a recent paper , we demoribed in  and \u03c8 data segments, or 6084 hours total.The ABP estimate algorithm was evaluated on the MIMIC II database, found at . The fol is good The six artificial ABP artifact models described above were then separately added to the clean dataset at different percentages of noise duration to generate the noisy evaluation dataset. In order to provide a periodic training period, the artifacts were only added to every other 5 minute epoch in the clean data. Therefore, in each hour, 6 noisy 5 minute periods are created each followed by 5 minutes of clean data. The percentage of each 5 minute noise segment containing artificial noise was set to 20%, 40%, 60%, 80% and then 100% for each experiment. That is, for each 5 minute segment that is designated to contain noise, artifacts are simulated and added at random (in a non-overlapping manner) to these respective portions of that 5 minute segment. Each noisy (5 minute data) segment was chose to follow a clean 5 minute segment. The rationale for this distribution was to allow the KF a period on which to train on the clean data. However, it turns out that much shorter segments of clean data could be chosen (see results section). Figure The ABP estimation algorithm was also evaluated on an abnormal data subset of the MIMIC II database, which includes episodes of annotated arrhythmias . The preIn the MIMIC II database there are over 45,000 hours of simultaneous ECG and ABP data with associated alarms of the above types. These data include 707 episodes of extreme bradycardia alarms  and 1877 episodes of extreme tachycardia alarms . Such false alarm rates (28.4% for bradycardia and 23.1% for tachycardia) are typical of alarms in the ICU, which can be as high as 85% [gold standard by which to evaluate the different ABP estimation methods. These were:Following our previous work , we conswSQI.1. FE: Feature Extraction ABP estimate using wSQI and clipping the reported ABP value when \u03c8 <\u03c8t (SH). (This simulates the operation of monitoring equipment in the ICU.)2. SH: Sample-and-hold ABP estimate using ABP feature extraction routine of 3. KF: ABP estimate using the SQI-based Kalman filter.\u03c8 > 0.5 leads to a low ABP estimation error (less than 10 mmHg) using the SQI-based KF ABP estimation method described in this paper. However, the reduced pulse pressure artifact results in relatively large errors for the estimate of SBP when \u03c8 \u2264 0.9, indicating that we should trust the SBP less than the MBP or DBP, particularly in this circumstance. SBP estimates were corrupted mostly by saturations to ABPmin and reduced pulse pressure. The MBP was mostly distorted by saturations to ABPmax, square wave artifacts and saturations to ABPmin.In order to evaluate the accuracy of an estimation method in the presence of noise, we chose the root mean squared error (rMSE) of the difference between each ABP estimation method and the true ABP. As expected, the rMSE is larger the lower the signal quality. Table \u03c8 <\u03c8t) are almost as good as the SQI-modified KF algorithm. This is in part due to the accuracy of the signal quality metric, and in part due to the fact that the data do not vary much in terms of blood pressure (or heart rate) for long periods of time. Figures Note also that the results for the sample-and-hold algorithm  increase in true alarm suppression rate (for extreme bradycardia) and a drop in true alarm suppression rate (from 0.4% to 0.1%) for extreme tachycardia.Our artifact classification scheme, and resultant evaluations using artificial examples of each type of artifact, has highlighted the different accuracies in ABP  estimates in the presence of different types of artifacts. In general, the DBP is less noise-sensitive than the MBP, which is less noise-sensitive than the SBP. Slow saturations or decreases in pulse pressure  can lead to large errors. High frequency noise appears to be less problematical in assessing accurate ABP estimates. Therefore, an automated method for classifying the type of artifacts identified in this study would be useful in tuning the response of the system, and allowing adaptation of the KF to change based upon artifact types. Such schemes could involve running assessments of the frequency content of the data , step-chet al. [Note that although the KF-based approach presented in this article is, on average, only marginally superior to a sample-and-hold approach when the signal quality is low, which is reflective of the infrequent changes in ABP or HR in the type of data we are using (i.e. ICU data) . Of couret al. . One simIt is interesting to point out the generality of the results presented in this paper. The source data (the MIMIC II database) is a large ICU database consisting of a variety of patients one would expect to find in a top-level US teaching hospital. These include patient in the medical, surgical, coronary, trauma, and cardiac surgery care units. A full description of the data can be found in . Data weThe work in this paper may also be extended to analyze photoplethysmograms (waveforms derived from pulse oximetery), a more common and non-invasive method of measuring the pulsatile flow in the cardiovascular system. Although excellent proprietary systems exist for signal quality assessment in such signals, and in some cases an SQI is available from the oximeter, no information is publicly available concerning the response of monitors to different artifact types, and under different recording conditions. Furthermore, no data fusion framework of blood pressure with other cardiac-related signals (such as the ECG) has been published . Our approach may provide a general system for assessing the signal quality and using the SQI to automatically inform the validity of derived estimates and may be appropriate in many medical settings.\u03c8 \u2265 0.9) for selecting data that may be trusted to give accurate blood pressure estimates (with an average error less than 10 mmHg). We have also demonstrated that diastolic blood pressure estimates are more robust to artifacts than mean blood pressure estimates, which in turn are more robust to artifacts than systolic blood pressure estimates. SBP estimates are likely to result in large errors for even moderate to high signal quality levels for certain types of artifact without signal quality analysis. Saturation-type noise produced the largest errors for all three blood pressure estimates. Results demonstrate that stringent signal quality measures should be used to qualify all blood pressure estimates. We have also shown that fusing independent heart rate estimators from the ECG and ABP together with SQIs in a KF framework provides a large increase in performance when tracking real episodes of extreme bradycardia and tachycardia over conventional approaches used in the modern ICU.We have presented a phenomenological classification system for artifact types in the blood pressure, and artificial methods for generating these artifacts. We have also presented an updated online system for continuously estimating HR and ABP using an SQI-modified Kalman Filter, and robustly weighting the estimates based on our trust in a given data segment. Using an extensive database of simultaneous ECG and ABP signals, we have evaluated our HR and ABP tracking algorithm. The proposed algorithm is shown to be robust and provides a predefined threshold ; PP: Pulse-to-Pulse ; rMSE: Root Mean Square Error; SBP: Systolic Blood Pressure; SQI: Signal Quality Index; SH: Sample-and-Hold.The authors declare that they have no competing interests.GDC designed the experimental procedures, drafted the manuscript and provided overall editorial approval. GDC also provided coding assistance and a subset of the algorithms. RGM provided project guidance, interpretation of the ABP artifacts, and editorial assistance with the manuscript. QL provided descriptions of the techniques and generated the figures and editorial assistance. QL also implemented the majority of code, provided algorithm architecture input and designed some of the experiments. All authors read and approved the final manuscript.Matlab source code for generating artifacts described in article. Contains the following files: bp_art.m \u2013 Matlab routine for generating artifacts sigmoid_art.m \u2013 Matlab sub-function to bp_art.m brownnoise.m \u2013 Matlab sub-function to bp_art.m sinc_art.m \u2013 Matlab sub-function to bp_art.mClick here for file"}
+{"text": "Stimuli from different sensory modalities are thought to be processed initially in distinct unisensory brain areas prior to convergence in multisensory areas. However, signals in one modality can influence the processing of signals from other modalities and recent studies suggest this cross-modal influence may occur early on, even in \u2018unisensory\u2019 areas. Some recent psychophysical studies have shown specific cross-modal effects between touch and vision during binocular rivalry, but these cannot completely rule out a response bias. To test for genuine cross-modal integration of haptic and visual signals, we investigated whether congruent haptic input could influence visual contrast sensitivity compared to incongruent haptic input in three psychophysical experiments using a two-interval, two-alternative forced-choice method to eliminate response bias. The initial experiment demonstrated that contrast thresholds for a visual grating were lower when exploring a haptic grating that shared the same orientation compared to an orthogonal orientation. Two subsequent experiments mapped the orientation and spatial frequency tunings for the congruent haptic facilitation of vision, finding a clear orientation tuning effect but not a spatial frequency tuning. In addition to an increased contrast sensitivity for iso-oriented visual-haptic gratings, we found a significant loss of sensitivity for orthogonally oriented visual-haptic gratings. We conclude that the tactile influence on vision is a result of a tactile input to orientation-tuned visual areas. To gain a more unified and accurate perception of the world, information from different sensory modalities are frequently integrated et al.et al.(d\u2032) for detecting the visual stimulus increased, suggesting a genuine sensory interaction between touch and vision. Neuroimaging studies also show interactions between tactile and visual processing. For example, studies in blind people demonstrate activation in early visual cortices during Braille reading Interactions have not only been found between sound and vision, but also between touch and vision. For instance, Van der Burg, Olivers, Bronkhorst &Theeuwes et al.More recently, studies by Lunghi, Binda & Morrone et al.'s study et al.'s The orientation dependence in the visual-haptic interaction in Lunghi In the current study, we test for a genuine visual-haptic integration by investigating whether touch can influence visual contrast sensitivity using a two-interval, two-alternative forced-choice design to eliminate response bias. In addition, the orientation  and spatcongruent  or incongruent . We expected to find a lower visual detection threshold in the congruent condition compared to the incongruent condition.In this experiment we tested the hypothesis that task-irrelevant haptic stimulation can influence visual contrast sensitivity in a two-interval detection task. Participants were asked to indicate in which of two intervals a visual target stimulus was presented, regardless of its orientation. The target was an oriented grating  and the contrast of the target stimulus was adjusted after each trial using an adaptive staircase procedure (QUEST) This research was approved by the University of Sydney Human Research Ethics Committee. Written consent was obtained from each participant prior to the experiments. The experiments were approved by the local ethics committee of the University of Sydney.Thirteen subjects participated in this experiment . Nine subjects were not aware of the hypothesis being tested. One of the subjects was left handed, however all subjects conducted the haptic exploration with the index finger of their dominant hand. All subjects had normal or corrected-to-normal vision.Visual stimuli were generated using Matlab version 7.10.0  and the Psychophysics toolbox 2) in a soft circular aperture with an orientation of either 45\u00b0 clockwise or 45\u00b0 counter clockwise. The Gabor patch was embedded in static-random white noise (noise contrast 20%). The Gabor patch was presented with a temporally smooth on- and offset. This was produced by multiplying the stimulus by a Gaussian temporal profile with a standard deviation of 23.33 ms. Presented in a randomized order, one interval contained the Gabor patch plus noise, and the other interval contained only the noise. Both stimuli were presented on a black background (2.6 cd/m2) in the centre of the screen.The visual target stimulus see  was a GaThe haptic stimulus was a round disc (diameter 3 cm) with sinusoidal corrugations whose spatial frequency matched that of the visual stimulus (2.66 cycles per cm). The haptic stimulus was created with a 3D-printer. The haptic stimulus was mounted transversally on the Arduino motor shaft so that the haptic grating could change orientation and the motor was placed at the bottom of the box at the position overlapping with the apparent location of the visual stimulus. Subjects could not see the haptic stimulus nor their hand touching it.The experiment took place in a dark and quiet room. Subjects were first familiarised with the task by viewing the visual stimulus in full contrast in 5 practice trials. Each trial started with a green fixation cross . After a key-press, the fixation cross disappeared and the subject started to explore the haptic grating by making circular motions with their index finger. After 1.5 seconds, two intervals were presented on the screen for 1.2 s each, separated by a blank interval of 40 ms. Each interval started with 25 ms of random white noise frames and ended with 25 ms of random white noise frames. One of the intervals contained the visual grating embedded in noise and the other contained white random noise . The samThe contrast of the visual stimulus was varied logarithmically over trials using the adaptive QUEST Data from each observer's three sessions were pooled and contrast thresholds corresponding to 75% correct detection were determined for each condition (congruent and incongruent). To determine the 95% confidence intervals (CI) for each threshold, a bootstrap procedure was used: the data were resampled 1000 times and a cumulative Gaussian distribution was fit to each resampled data set. From the resulting distribution of 1000 resampled thresholds, the points bounding the central 95% of means defined the upper and lower CIs. The contrast thresholds were then compared using a paired t-test. The average slopes of the psychometric functions in each condition were also compared with a paired t-test.t(12)\u200a=\u200a\u22123.43, p\u200a=\u200a.005, \u200a=\u200a4.83, p<.001) . This analysis yielded no reliable difference in terms of slope and threshold and so the data sets were merged for further analysis. The group mean data  show tha p<.001) . FurtherIn Thirteen subjects participated in this experiment . Eleven subjects were not aware of the hypothesis being tested. All subjects conducted the haptic exploration task with their dominant hand, and all had normal or corrected-to-normal vision.The experimental procedure was the same as in F<1). The main effect of congruency was significant, F \u200a=\u200a7.43, p\u200a=\u200a.021, indicating that the overall performance was better in the congruent condition than in the incongruent condition. Importantly, the ANOVA yielded a significant interaction between congruency and orientation, F\u200a=\u200a5.92, p<.001, demonstrating that the congruency effect depends on the orientation of the haptic stimulus. The congruency effect was further examined for each haptic orientation by pairwise two-tailed t-tests. The t-tests (Bonferroni corrected for multiple comparisons), yielded a significant congruency effect when the haptic orientation was 45\u00b0, t(5)\u200a=\u200a\u22126.217, p<.001, but not for any other haptic orientations . The effect of orientation was further examined by separating the data into congruent and incongruent trials and conducting separate ANOVAs on each to examine whether 45\u00b0 differed significantly from the other orientations. The ANOVAs yielded significant main effects of orientation in both the congruent and the incongruent conditions, F\u200a=\u200a3.482, p\u200a=\u200a.009, and F\u200a=\u200a3.923, p\u200a=\u200a.004, respectively. To verify that this effect is indeed driven by the tactile and visual orientation of 45\u00b0, we tested the effect of tactile orientation without the tactile orientation of 45\u00b0. This analyses showed no significant effect of orientation for both the congruent and incongruent orientations , and a significant trough at 45\u00b0 for orthogonal haptic-visual gratings .Two participants were excluded from the analyses because their preliminary estimate of contrast produced performance that differed greatly from 75%. One of them performed near chance level (averaging 54% correct) and the second showed a ceiling effect (averaging 93% correct). This left a total number of 11 participants in the analyses. The results of The results of Thirteen subjects participated in this experiment . Eleven subjects were not aware of the hypothesis being tested.The experimental procedure was identical as in The haptic stimulus was presented at 45\u00b0 clockwise for the right-handed participants and at 45\u00b0 counter-clockwise for the left-handed participants. The visual orientation was either 45\u00b0 clockwise or 45\u00b0 counter-clockwise, 5 haptic spatial frequencies were tested, 14 times per spatial frequency for both visual grating orientations. Each participant conducted one session of 140 trials. The order of the haptic spatial frequency presented was randomized, as was the alternation of the visual orientation between congruent and incongruent.F\u200a=\u200a6.69, p\u200a=\u200a.027. This congruency effect was not dependent on the spatial frequency, as the two-way interaction was far from significant (F<1).Two participants were excluded from the analyses because their performance did not exceed chance . This left 11 participants in the analyses. The results of Recent research has demonstrated that stimulation in one modality can influence the activation of unisensory areas belonging to another modality. Intersensory integration between touch and vision has been demonstrated in several studies Could this haptic facilitatory congruency effect of haptic input on vision be due to attention? It is known from several studies that visual contrast sensitivity can be increased by attention et al.Top-down effects from imagery on haptic shape perception have been previously documented One of the most intriguing aspects of this study is the tight orientation tuning of the interaction between vision and touch. Even though high-level visual areas such as the parahippocampal place area In addition to orientation tuning, a further property of neurons in early visual cortex is spatial-frequency tuning An unexpected but interesting finding was the trough in the tuning curves in Experiments 2 and 3 when touch and vision were incongruently  oriented see  and 6. AFinally, functional activation of brain areas that are usually used for visual processing has been demonstrated in blind people during Braille reading Our results show that congruent haptic stimulation can improve performance on a simple visual grating detection task compared to incongruent haptic stimulation. The orientation dependence of this haptic enhancement of vision suggests that neurons in the visual cortex, where orientation-tuned responses are common, receive inputs from the somatosensory cortex, likely via multisensory areas. The results cannot be due to a response bias, and are unlikely to be due to attention. Several studies suggest tactile inputs to visual cortex exist but are usually weak and masked by strong visual signals. By conducting our experiments at visual contrast threshold, the relative strength of the tactile signal has been increased to the point where it can have a small but significant enhancing effect when congruent with vision. Analogous to a visual-tactile summation model suggested by Arabzadeh and colleagues"}
+{"text": "Information obtained from multiple sensory modalities, such as vision and touch, is integrated to yield a holistic percept. As a haptic approach usually involves cross-modal sensory experiences, it is necessary to develop an apparatus that can characterize how a biological system integrates visual-tactile sensory information as well as how a robotic device infers object information emanating from both vision and touch. In the present study, we develop a novel visual-tactile cross-modal integration stimulator that consists of an LED panel to present visual stimuli and a tactile stimulator with three degrees of freedom that can present tactile motion stimuli with arbitrary motion direction, speed, and indentation depth in the skin. The apparatus can present cross-modal stimuli in which the spatial locations of visual and tactile stimulations are perfectly aligned. We presented visual-tactile stimuli in which the visual and tactile directions were either congruent or incongruent, and human observers reported the perceived visual direction of motion. Results showed that perceived direction of visual motion can be biased by the direction of tactile motion when visual signals are weakened. The results also showed that the visual-tactile motion integration follows the rule of temporal congruency of multi-modal inputs, a fundamental property known for cross-modal integration. For eet al. reported a close interaction between saccade directions and the processing of tactile motion [et al. [et al. developed a visual sphere with visual ambiguity in its direction of rotation and results showed that touching the sphere disambiguates the visual percept [et al. investigated the temporal constraints of the visual-tactile crossmodal congruency effect in an experiment in which vibrotactile targets were presented to the index finger or thumb of either hand while visual distractors were presented from one of four possible locations. Participants made speeded discrimination responses regarding the spatial location of vibrotactile targets while ignoring the visual distractors. The results showed that the cross-modal effects were significant when visual and vibrotactile stimuli occurred within 100 ms. [Touch and vision are similar in that both sensory signals derive from a sheet of sensor arrays, cutaneous receptors in the skin and photoreceptors in the retina. Indeed, touch and vision are intuitively integrated to yield a holistic percept of the environment around us ,9. It hae motion  as well e motion , implyin [et al.  found th percept . Finally 100 ms. .Visual-tactile motion integration has been explored using several apparatuses, with tactile-array stimulators comprising a matrix of linear motors or actuators being the most sophisticated devices \u201315. The Using this apparatus, we presented visual-tactile motion stimuli in which the directions of motion were either congruent or incongruent between sensory modalities, and participants reported the perceived visual direction of motion. The magnitude of visual-tactile integration was then gauged by the degree to which the perceived visual direction was biased toward the tactile direction of motion. We hypothesize that the direction of perceived visual motion can be biased by the direction of tactile motion when visual signals are weakened. We also hypothesize that visual-tactile motion integration follows the rule of temporal congruency of multi-modal inputs: The effect of multi-sensory integration is most robust when sensory information from multiple modalities coincides ,24,25. A2.2.1.To present tactile motion stimuli in specified directions, speeds, and indentation depths we developed a tactile motion stimulator  with thr2.2.The surface of the stimulus drum was made from a grating whose orientation was orthogonal to the direction of surface motion. Specifically, the stimulus drum  had a diameter of 160 mm and was engraved with a square-wave grating of 3.9 mm wave length, 500 \u03bcm peak-to-peak amplitude, and a 0.4 duty cycle  or leftward (180\u00b0) motion. Use of the subject's own hand vivified the subsequent visual percepts. To eliminate possible cues elicited by the machine's shadow, the stimulus drum was visually presented through a specified aperture. The video was first transformed to gray scale, and different levels of Gaussian noise  were superimposed on each frame .The assumption was that superimposition of noise would degrade the signal-to-noise ratio in the visual signals, allowing us to explore how visual-tactile integration may depend on visual signal certainty. The Gaussian noise level is defined by the ratio between the standard deviation of superimposed Gaussian noise and the largest luminance difference observed in the original image :(1)Gaus2.6.Ten volunteer subjects , ranging from 24 to 40 years of age, were paid for their participation. Five participated in the visual certainty experiment and seven in the temporal congruency experiment. Informed consent was obtained from each subject and the protocol was approved by the Institutional Review Board of Human Research of the Chang Gung Medical Foundation.2.7.We performed two psychophysical experiments investigating the integration of visual and tactile signals in human observers. In the first experiment, subjects discriminated the direction of motion of visual stimuli when visual and tactile stimuli were simultaneously presented.In a factorial design, the visual direction of motion was rightward (0\u00b0) or leftward (180\u00b0), tactile direction of motion was rightward (0\u00b0) or leftward (180\u00b0), and visual Gaussian noise level was zero, 0.1, 0.2, 0.3, 0.4 or 0.5. Both visual and tactile stimuli were defined by retinotopic (eye-centered) coordinates. Each stimulus condition was presented 10 times. The experiment was split into five blocks to allow subjects to rest so that each block contained 48 trials . The surface-motion speed of the tactile stimulus was 40 mm/s and its indention depth was 1 mm. In each trial, the visual-tactile motion was presented for 1 s and then the subject reported the direction of the visual stimulus by pressing one of two buttons on a computer mouse in a left-right two-alternative forced-choice design. The stimulus duration was the total indentation duration of the rotating drum, defined as the time from initial contact to the offset of indentation. Ramp-down period, defined as the time from initial contact to full indentation, and ramp-up period, defined as the time from full indentation to leave-off, lasted 0.15 s. The inter-trial-interval was 1.6 s. It was hypothesized that strength of visual-tactile motion integration can be gauged by the degree to which the perceived visual direction of motion is affected by the direction of tactile stimulus motion.As a control experiment, we also performed a visual-only experiment in which the moving visual stimulus was presented as stated above, while the tactile stimulus was static (no surface motion), with a constant indentation of 1 mm. Each stimulus condition was presented 10 times. The experiment was split into five blocks and each block contained 24 trials . Thus, we can compare task performance with and without tactile motion stimulation. We first performed the visual-only experiment and then the visual-tactile experiment.2.8.tactileL and visualL are the onset latencies of tactile and visual stimuli, respectively. We first performed a pilot experiment to find the optimal visual noise level for individual subjects that could induce a specific level of visual uncertainty. The Gaussian noise level was chosen to induce accuracy range from 0.6 to 0.7 in the visual-only condition because the visual-tactile integration effect was observed robust within this Gaussian noise level in the visual uncertainty experiment. Each stimulus condition was repeated 10 times. The experiment was split into 10 blocks and each block contained 36 trials .We then examined whether the results obtained in the previous experiment were compatible with the rule of temporal congruency of multi-modal inputs . We perf3.3.1.3.1.1.p < 0.05). Results indicate that the perceived visual direction is biased toward the tactile direction especially when visual noise level is high, providing evidence of visual-tactile integration.We used the visual-tactile apparatus to perform direction-congruency experiment with a variety of visual noise levels. In the visual only condition, we found that the probability of choosing the veridical direction of visual motion (accuracy) peaked at zero noise, monotonically decreased as noise levels increased, and finally reached chance level (accuracy = 0.5) at the maximum level of visual noise (3.1.2.p < 0.001). Post-hoc analysis using paired t-test showed that accuracy differed significantly between the congruent and incongruent conditions when SOAs were -0.5, -0.25, 0, 0.25, 0.5 and 1 s (p < 0.05). That is, visual-tactile integration peaks when visual and tactile stimuli are presented simultaneously, a finding that is compatible with the rule of temporal congruency of multi-modal inputs. Also, the rule of temporal congruency for visual-tactile motion integration has a relatively wide tolerance of SOA up to 1 s.We then examined whether visual-tactile motion integration follows the rule of temporal congruency of multi-modal inputs. We performed the temporal congruency experiment with several SOAs and a fixed visual noise level. Across SOAs, the strength of visual-tactile integration, gauged by the degree to which perceived direction of visual motion is biased toward the direction of tactile motion, peaked when the SOA was close to zero (simultaneous presentation) and gradually decreased as the SOA deviated away from zero (asynchronous presentation)  and in Butz's study (960ms) while that in Shore's study was 10 ms.The results in the present study indicated that visual-tactile motion integration follows the rule of temporal congruency of multi-modal inputs. The temporal congruency is functionally relevant in that information from different senses occurring at approximately the same time most likely come from the same physical event . Butz etet al.  investign 100 ms . One posHuman brain imaging studies have showed robust increases of blood oxygen level-dependent (BOLD) signals in the extrastriate visual cortex when human observers are presented with tactile stimulus. These areas include the middle temporal (MT) ,35 and mA haptic approach is usually applied in multi-sensory scenarios. It is then of utmost importance to characterize how information is processed in biological systems to infer a holistic percept. Furthermore, this apparatus will help develop cross-modal inference algorithms to determine how robotic systems resolve conflicting visual and tactile sensory information in scenarios that could occur in the real world . However4.The present study introduces the design and demonstrates the validity of a novel visual-tactile motion integration apparatus that consists of a video display and tactile stimulator with three degrees of freedom. Using this apparatus, we showed that visual direction of motion is biased by the tactile direction of motion when visual signals are weakened. Additionally, the visual-tactile motion integration follows the rule of temporal congruency of multi-modal inputs. Further studies will be able to use this apparatus to investigate cross-modal motion integration mechanisms."}
+{"text": "Improving the energy efficiency in wireless sensor networks (WSN) has attracted considerable attention nowadays. The multiple-input multiple-output (MIMO) technique has been proved as a good candidate for improving the energy efficiency, but it may not be feasible in WSN which is due to the size limitation of the sensor node. As a solution, the cooperative multiple-input multiple-output (CMIMO) technique overcomes this constraint and shows a dramatically good performance. In this paper, a new CMIMO scheme based on the spatial modulation (SM) technique named CMIMO-SM is proposed for energy-efficiency improvement. We first establish the system model of CMIMO-SM. Based on this model, the transmission approach is introduced graphically. In order to evaluate the performance of the proposed scheme, a detailed analysis in terms of energy consumption per bit of the proposed scheme compared with the conventional CMIMO is presented. Later, under the guide of this new scheme we extend our proposed CMIMO-SM to a multihop clustered WSN for further achieving energy efficiency by finding an optimal hop-length. Equidistant hop as the traditional scheme will be compared in this paper. Results from the simulations and numerical experiments indicate that by the use of the proposed scheme, significant savings in terms of total energy consumption can be achieved. Combining the proposed scheme with monitoring sensor node will provide a good performance in arbitrary deployed WSN such as forest fire detection system. Wireless sensor network (WSN) has been considered as one of the key techniques in many applications such as environment monitoring, industrial control, and so forth . In a tyRecently, a technique motivated by improving the spectral efficiency named spatial modulation (SM)  is propoIn this paper, in order to take full advantage of these approaches, a new scheme named CMIMO-SM is proposed which involves the joint utilization of cooperative MIMO and SM techniques in WSN for energy-efficiency improvement. We first model the energy consumption of the CMIMO-SM communication and compare the results with the conventional CMIMO. The energy consumption is compared over different transmission distances under the requirement of the same throughput and bit error rate (BER). Three system configurations are considered in both CMIMO-SM and CMIMO system for validating the performance of the proposed scheme, and then the proposed CMIMO-SM scheme is extended to a multihop clustered scenario in which the optimal hop length is derived mathematically by considering the transmitted load. Results from numerical experiments indicate that by use of the proposed scheme, significant performance in terms of energy consumption can be achieved. Also it is demonstrated that the total energy balancing performance of the proposed scheme can be improved.The remainder of the paper is organized as follows. In In , for theThe basic idea of CMIMO started with a so called virtual antenna array (VAA) , a coopea gets into the SM system which consists of Mt transmitters and Mr receivers. The number of bits that can be transmitted using SM is log2(MMt), where M = 2b and b is constellation size. We can write this incoming bits a using a vector x = [x1x2 \u22ef xMt]T, where a power constraint of unity is assumed. According to the working principle of SM, only one antenna is active during the transmission which results in a single nonzero element in x. The signal is transmitted from the corresponding antenna over the Mr \u00d7 Mt MIMO channel H whose elements are assumed to be independent identically distributed (i.i.d.) complex Gaussian random variables with zero mean and unit variance, and Mr dimensional noise vector n are assumed to be i.i.d. complex Gaussian random variables with zero mean and variance of N0. Then the received signal can be written as y = \u0397x + n and the estimated symbol can be extracted by using optimal maximum likelihood (ML) detector. Mathematically, the estimated symbol can be expressed by F denotes the Frobenius norm of the vector.Spatial modulation is a practical way for transmitting information by using amplitude/phase modulation and antenna index techniques. This 3 dimensional signal expression approach is able to make SM achieve high spectral efficiency. Furthermore, due to the fact that only one antenna is active during the transmission, the ICI is effectively avoided. The basic working principle is explained using Let us consider a wireless sensor network in which each sensor node is equipped with a single antenna for data transmission. Typically, local sensor nodes inside the cluster collect the information and transmit it to the next relay cluster if the destination is far away. As we discussed in the last section, CMIMO can make single node with single antenna achieve MIMO performance and SM can provide high spectral efficiency and avoid ICI. Thus, if we allow CMIMO and SM techniques to be used among multiple nodes, the joint performance will be obtained. Such scenario is shown in Mt nodes on the transmitting side while Mr nodes on the receiving side. On the transmitting side, each sensor node broadcasts its information to all the other nodes inside the cluster using different time slots as the first stage. Once each node receives all the other information bits, the data sequence is ready to be transmitted through the MIMO channel. For each time instant, the data sequence is composed by the MQAM/MPSK modulated symbol part and antenna represented part. Only the MQAM/MPSK modulated symbols are transmitted and the symbol represented by corresponding antenna as the hidden information will be detected at receiver. On the receiver side, one destination node and Mr \u2212 1 nodes join the cooperative reception. The whole process of CMIMO-SM can be explained by using In our proposed model, each sensor node inside the cluster has a preassigned index using binary numbers to represent. In order to realize the MIMO function for saving energy, the local data exchange is necessary. The data flow inside the cluster is defined as local transmission while the data delivering between two clusters is defined as long-haul transmission. Suppose that Example: consider a CMIMO-SM based WSN with 4 sensor nodes on the transmitter and each sensor node has an index which is similar to the instance in PPA and the power consumption of all the circuit blocks Pc. PPA depends on the output transmission power Pout and has a linear relation:\u03b1 equals to \u03be/\u03b7 \u2212 1 with \u03be being the peak to average ratio (PAR) and \u03b7 being the drain efficiency of the RF power amplifiers. For MQAM, \u03be = 3(M1/2\u22121)/(M1/2+1). Pout can be calculated as below if the channel is a square-law path loss [d is the transmission distance, Gt and Gr are the transmitter and receiver antenna gains, respectively, \u03bb is the carrier wavelength, Ml is the link margin compensating the hardware process variations and other additive interference or background noise, and Nf is the receiver noise figure. It should be noted that Nf is given by Nf = Nr/N0, where Nr is the power spectral density (PSD) of the total effective noise at the receiver input and N0 is the single-sided thermal noise PSD at room temperature with a value N0= \u2212171\u2009dBm/Hz. In PoutPoutPPA Mt transmit and Mr receive antennas system is given byPDAC, Pmix, PLNA, PIFA, Pfilt, Pfilr, PADC, and Psyn are the power consumption values for the DAC, the mixer, the low noise amplifier (LNA), the intermediate frequency amplifier (IFA), the active filters at the transmitter side, the active filters at receiver side, the ADC, and the frequency synthesizer, respectively. The values of PDAC, PADC, and PIFA can be calculated using the mode introduced in [The total circuit power consumption for an duced in .The total energy consumption per bit can be expressed asrding to  and 2),,(4)Ebt=PEbt,csm consists of two components: energy consumption in the local phase El and energy consumption in the long-haul phase Elh, that is,Eit; (2) energy consumption of data collection for joint detection inside cluster in receiver side Ejr. Note that for the local phase communication, cooperative transmission is not used and Mt = Mr = 1, namely SISO communication. For the long-haul energy consumption, CMIMO-SM is utilized and note that the transmit antenna number Mt is always equal to one in the circuit power consumption, since only one antenna is active during each time instant. Assume that each sensor node has Ni bits to transmit; and then the energy consumption per bit for El is given byElh to El, the energy consumption per bit can be expressed asEit, Ejr, and Elh can be calculated according to (According to the transmission process described in rding to .EJ Joule per cycle [MtMr + (Mt + 1)M complex multiplications and Mt(Mr \u2212 1) complex additions are needed for one symbol detection [In this paper, for a realistic case we take into account the detection energy as well. The detection energy can be calculated by using operation complexity in terms of complex multiplication and addition. Processing ability of the multiplications and additions depends on the CPU speed and can be different for different devices. We refer to the TelosB mote  as an iner cycle . When SMetection . So the Mr complex multiplications and 2Mr \u2212 1 complex additions are needed for one symbol detection [Eitcand Ejrc represent the local transmission energy cost per bit for cooperation on the transmitter side and joint detection on the receiver side, respectively. The energy consumption per bit in long-haul transmission by using the Alamouti approach is denoted as Elhc. Eitc, Ejrc, and Elhc can be calculated according to  which is assumed to be much larger than dlocal. Let di represent the optimal transmission distance and then, for a transmission distance di, energy consumption per bit of long-haul is given byNow a multihop WSN composed of It will be assumed that two sensor nodes in one cluster are used in order to reduce the complexity of the calculation. However, it is important to note that the calculation can in principle be extended to any cluster size. For all nodes transmitting scenario, the total energy consumption can be expressed asdi from the source to the destination needs to be derived. First, through observation of the transmission distance in the proposed model, the constraint \u2211i=1ndi = d \u2212 ndlocal is obtained; and then the optimization problem can be formulated asIn order to minimize the total energy consumption, the optimal transmission distance d, the optimal transmission distance di is dependent on d and decreases as the cluster approaches to the destination.Under the all nodes transmission case, given the total distance between source and destination See the appendix.Therefore, setting hop distances using  for all d meters apart and n \u2212 1 clusters act as relays to forward the information. Assume that n is chosen to be 10, d and dlocal are set to be 1000 meters and 2 meters, respectively. The optimal transmission distances are obtained using (The numerical results of the optimization and multihop based CMIMO-SM are presented in ed using  and plotIn order to evaluate the performance of the proposed scheme, an equidistant scheme as a reference is compared with the proposed scheme in terms of energy consumption. In Figures A new cooperative transmission scheme CMIMO-SM has been proposed in this paper. The system model was designed and the energy consumption was analyzed in a cluster to cluster communication environment under three different system configuration cases. The superiority of using this scheme for minimization of total energy consumption is validated by simulations and numerical experiments. Results demonstrated that the proposed scheme always provides energy saving compared with when using the conventional one. Later, we integrated the CMIMO-SM into a multihop scenario to further optimize the total energy consumption. The results indicated that optimal hop scheme can reduce and balance energy consumption significantly, and thus the feasibility of using CMIMO-SM in optimal multihop scheme to boost the joint performance was proved. Therefore, it is concluded that the proposed scheme can be used as a guideline to design energy efficient communications in arbitrary deployed WSN for the reduction of energy consumption and extension of the lifetime."}
+{"text": "One of the possible approaches is to consider models of electronic separability, such as the bond charge model (BCM), which provides insight into the cohesion of covalent crystals in analogy with the classical ionic model. However, this model relies on empirical parametrization of bond and lone pair properties. In this contribution, we have coupled electron localization function (ELF) ab initio data with the bond charge model developed by Parr in order to analyze solid state compressibility from first principles and moreover, to derive general trends and shed light upon superhard behavior.Understanding the electronic nature of materials\u2019 compressibility has always been a major issue behind tabulation and rationalization of bulk moduli. This is especially because this understanding is one of the main approaches to the design and proposal of new materials with a desired  compressibility. It is well recognized that the softest part of the solid will be the one responsible for its compression at the first place. In chemical terms, this means that the valence will suffer the main consequences of pressurization. It is desirable to understand this response to pressure in terms of the valence properties (charge, volume,  Many phenomenological approaches have been developed to illustrate the advantage of this view . These scrystals . Li et aaterials . In conjaterials , Li\u2019s moaterials .etc. Efforts from the theoretical chemistry community have been devoted to reformulate these concepts from first principles. As an example, we can cite recent reformulations of electronegativity [etc.). Due to its direct link with Lewis theory, we have chosen the model by Borkman and Parr [Inherent to all these semi-empirical models, there are still elusive concepts to quantum-mechanical formalisms, as ionicity/covalent scales, electronegativities, covalent radii, gativity ,8,9. Thegativity  and the gativity . Anothergativity ,13 or thgativity ,15. Sincand Parr  as our sBriefly, our study is based on the success of the bond charge model ,17,18 inab-initio BCM  are derived from first principles. The ability of this model to predict solid state properties (compressibility) will be tested against experimental data in The remainder of the paper is organized as follows. Firstly, the original definition of the bond charge model for homo and heteronuclear binary molecules and covalent solids will be presented. Secondly, the computational details for the calculations performed over a broad group of diamond-type and zinc-blende-type solids from the IV, III\u2013V and II\u2013VI groups are given. In In this section, the two approaches to be merged are introduced: the bond charge model and the ELF topological formalism.A2) can be approximated by two cores holding a q/2 positive charge each, and a bond charge in between them, -q, that moves freely along a bond length RB = \u03bdR which is a fraction (\u03bd\u2264 1) of the interatomic distance R (see R \u2243 Re) is given in atomic units by:E0 represents the core energy , E1 refers to the coulombic attractive interactions and E2 is related to the kinetic energy of the bond electrons approximated as particles in a box of length RB . The constants, which are system independent, are then given by  and 2qB (1 \u2212\u03b4) at A and X atomic sites, and \u2212qB at each bond position, as dictated by the multiplicity and the charge neutrality condition. We should recall that EA and EX account for the energy of the corresponding core electrons not involved in the bonding, and, in practice, are independent of geometrical parameters. The greatest modification introduced in the application of BCM to solids is the presence of an infinite sum, accounting for all A, X, and B charged species, which is subsumed into the Madelung constant. It it also important to highlight that due to the inclusion of the bond charge in the summation, this Madelung constant is system dependant since the position of the bond charge will not correspond to a special Wyckoff position (invariable) except for homonuclear solids. The Madelung constants have been calculated thanks to the environ code [The model by Parr and Borkman was extended by Martin  to descrron code  and are From Equation (6), the bond energy of a zinc-blende-type lattice can be finally expressed within the framework of BCM as:It is interesting to highlight here that this expression is similar to the classical Madelung energy expression for ionic crystals. However, in our case the equation is done in terms of Lewis entities  instead of ions. The expression will then hold as long as the orthogonality of these entities holds too. In solids such as diamond, where for example, good localized orbitals can be found, Equation (7) will give an accurate description of the bonding energy in the crystal. However, as the localized picture fails , we should expect the fits to worsen. This equation will progressively fail as we go down the periodic table or move towards metallic solids, due to a less obvious separability.\u2202EB/\u2202R) \u2243 0 at equilibrium thenr1/r2 remains constant upon compression, not introducing changes in M  approach as implemented in the ELK (for electrons in k-space) code . The FP-ructures . We haveructures  local deructures  generaliIn a second step, the topology of the ELF functions coming from the previous calculations is investigated using CRITIC, a code developed by some of the authors ,33. CRITThe training set has been chosen to lay in the shared-electron range. This characteristic is necessary so that there is a bond basin linkable to each pair of nearest neighbors in the crystal. In other words, it is possible to assign bonding parameters within the ELF framework.r1/r2, (shifted from the center by the relative electronegativity of atoms) is determined by the local ELF maximum along the same direction. On the other hand, the distance between the two closest minima to the bond maximum along the internuclear direction provide the bond length, RB  depends neither on the lattice parameter of the crystal (which spreads a range of more than 2.5 \u00c5), nor on the two atoms involved in the bond.A similar value obtained for r1/r2 \u2243 1 ratios are obtained for the purely covalent families and those whose \u2206\u03c7 \u2243 0. There is one remarkable outlier: BN shows r1/r2 \u2243 1 with \u2206\u03c7 = 1. In order to understand this fact, an extra column has been added to RB, that is, taking away core contributions: Secondly, we observe that RB, as expected from their electronegativity difference .This means that we now decompose the bond length into Obtaining the charge associated with ELF entities through the integration over the corresponding basins might be considered in principle a straightforward and accurate method. However, for heavy elements, some ill-defined cases appear for integration, where the low symmetry of the basins and the appearance of spurious critical points at the muffin surfaces may lead to serious difficulties if we seek a quantitative analysis of its topology.qB values can be directly obtained by mere difference between the total and core number of electrons. Core values for the elements up to the 4th row involved in the solids under this study have been extracted from the ELF integrations gathered in [qB of 1.952 and 1.948 for C and BN, respectively, are in good agreement with the corresponding data in Based on pseudopotential principles, we assume that the core electronic distributions are practically unaltered by the environment, and thus hered in . Beyond hered in  and the hered in . Their chered in . This ashered in . For insBq correlating to the bond order does not agree with the definition of the Bq parameter in the traditional model. The step forward that ELF provides concerns also the evaluation of the charge transfer (\u03b4). In all cases .This is an important characteristic of this Non Empirical New Bond Charge Model (NEWBCM), parameters are not only obtained Since pressure can introduce significant changes in the stability of phases, it is most useful in the synthesis of novel phases and metastable materials. Pressure allows precise tuning of a fundamental parameter, the interatomic distance, which controls the electronic structure and virtually all the interatomic interactions that determine materials properties ,40,41. W\u03ba) or its inverse, the bulk modulus (B0), probably constitute the most widely used parameters. The inverse relationship between bulk modulus and volume has been widely explored [Among the description of the responses to pressure, compressibility .B\u03ba, whereas cores are assumed to remain untouched: \u03ba \u2243 B\u03ba [AXB4 unit.Following our BCM model, the valence charge is distributed along the chemical bonds, so that the compressibility of solids can be approximated to a good extent by the compressibility of the bonds, : \u03ba \u2243 \u03baB ,49. The R2 is 0.9774. A similar agreement was obtained by Gilman et al. [i.e., Bq/V ). The coherency between these two results can be perfectly understood from the analysis of Equation (15) if we take into account that V = a3 for these crystals:Equation 15) for our IV, III\u2013V and II\u2013VI compounds is analyzed in 5 for ourBq /V as would be expected from Lewis theory . Whereas the bond occupation has been found in our calculations to be very close to two, the dependence on the cell size can be improved by taking calculated bond lengths into account. Indeed, Equation (16) shows that the dependence on the lenght parameters should be B0 \u221d (BVR)\u22121 \u22121, exponent nearly \u20134), whereas Gilman took B0 \u221d V\u22121 alone (exponent on R = \u20133). Indeed, a logarithmic fit of the B0 experimental data vs.R gives a slope of 3.8 (\u00b10.1) (regression coefficient R2 = 0.9863) which explains the need to increase the length dependence from R\u22123 to R\u22123R4). It is interesting to note that, although calculations provide a better linear behavior (R2(NEWBCM) = 0.9927, R2 = 0.9830), the use of default or nominal (Bq = 2) values already provides all the physics.In his fit, Gilman made use of the cell volume and an average valence occupation Understanding the inherent relationship between (quantifiable) microscopic properties and macroscopic properties is the corner stone of material design.R \u221d V1/3, and bond lengths, RB, are found. While these results were already known from experience, the microscopic approach can also reveal new information. With this aim in mind, it is interesting to study the evolution of NEWBCM parameters  upon compression.We have shown that the concepts derived from ELF can be used to understand the microscopic origin of solids compressibility. If we analyze the dependencies of Equation (15), we see that the least compressible materials are found within the first period, where smaller interatomic distances, RB. Since RB = \u03bdR can be expressed as RB = R\u2212rA\u2212rX, and the core radii remain practicably untouched upon compression, a linear relationship necessarily holds for RB  with R for a given material.Let\u2019s start with r1 and r2 with R at constant \u03b4. According to Parr\u2019s electronegativity approach [AX, \u03c7AX, can be expressed in terms of the radii of the isolated atoms, rA and rX, as: [AX:K is a constant.Furthermore (and less intuitively), the linear relationship also holds for approach , the ele rX, as: (17)\u03c7AX=K, q, rA, rX, \u03c7A and \u03c7X are constants which only depend on the system, so that \u03b1 itsef is a constant as long as \u03b4 remains constant. In other words, r1 will scale linearly with the interatomic distance, as long as the charge transfer does not change upon pressurization. Of course, similar equations apply to r2, with r2 = \u03b2R.If we combine Equations (17) and (18) and reorganise, we see that:\u03c7 = 0.14) and polar  solids were chosen to compare their corresonding compression with respect to their bonding type. r1, r2 and RB. Negligible deviations from linearity are found in both cases, leading to three main conclusions:RB is the quantity showing the greatest compression of the model since it represents the valence size, i.e. the chemically sensitive part as pressure is applied [ applied ,49.\u03b1, \u03b2 and \u03bd are constant upon compression for the various types of bonding considered. Further analysis shows that \u03b1, \u03b2 and \u03bd can certainly be related to hardness, since they determine which ion compresses the most. Indeed a look at r1 and r2 compress at similar rates, whereas in BAs, the compression of As overcomes that of B.As a consequence of the previous observation and from Equation (19), we can consider that the charge transfer is also constant upon compression for the ranges considered.In order to check this hypothesis, we have repeated our DFT-ELF procedure upon compression of the zero pressure reference cell (no optimization needed due to the symmetry of the cell). Two prototypical examples of covalent  also plays an important role. Since these parameters are related to the relative hardness of atoms, the smaller \u03b1 and \u03b2 are, the less compressible the whole system becomes. What is most important, both parameters should be similarly small. Otherwise, the softest ion will subsume the pressure. Ideally, for a given r1, the compressibility is minimized when the relationship \u03b1 = \u03b2 is fulfilled. This condition is equivalent to locating the bond position in the middle of both atoms :A = X (or \u2206\u03c7 = 0), so that all lengths are the same.Perfectly covalent case (diamond): in this case A \u2260 X (\u2206\u03c7 \u2260 0). However, a relationship holds between the core and the bond location displacement. It is found that the cores differential size, \u03f5 = rX\u2212rA for \u03f5 > 0 is equivalent to the displacement of the bond location from the center toward the smaller atom A, r1 = Ar and r2 = Xr). In BN, a good combination of atomic sizes and polarity compensate to provide r1/r2 = 1.03.Polar case (BN): in this case These dependencies enable us to establish new relationships for materials with low compressibility, directly derived from microscopic conditions. From ab initio, but they are closer to chemical intuition and recover good agreement with experience.We have introduced the Electron Localization Function as the source of the necessary parameters for the analysis of the Bond Charge Model in solid state applications, named NEWBCM. The availability of charges and bond location has enabled us to develop a new BCM-based model in which, parameters are not only obtained The ability of NEWBCM to describe compressibility trends among these crystal families has been exploited in order to settle the directions for novel superhard materials design. Some of these hints are not related to macroscopic properties but to microscopic ones. Thus, quantitative relationships between electronic structure and macroscopic properties, such as compressibility, can now be proposed.\u03bd; (iv) Hard atoms; and (v) Atoms of similar hardness. Although most of these guidelines are related  and (iv)) and extremely intuitive, some of them, such as (iii) would not be deduced at first glance, and provide useful quantitative insight into the search and prediction of novel superhard materials from microscopic grounds.The micro-macroscopic properties that guide the quest for superhard materials can be summarized as follows: (i) Small atoms; (ii) Small bond; (iii) High r1/r2 ratio very close to one in spite of belonging to two very different bonding types . These conditions thus unite their different macroscopic properties into one relevant microscopic condition for superhardness.As an example, it has been shown that the hardest compounds within our study set, diamond and BN, both follow these principles, and more specifically, both show an Although this model is only applicable to zinc-blende types of structures, work is in progress to extend it to other structural types. This would, for example, enable us to understand the driving forces for chemical changes in phase transitions."}
+{"text": "In regard to our recently published paper entitled \u201cTreatment planning comparison of IMPT, VMAT and 4\u03c0 radiotherapy for prostate cases\u201d, a question was raised whether \u201c4\u03c0\u201d was used appropriately to describe the non-coplanar planning and delivery space. In this letter, the term use is explained from both theoretical and practical perspectives. It is concluded that the self-explanatory term provides a flexible description of non-coplanar radiotherapy with beam orientation optimization. Confusions with this term can be avoided by understanding the evolving and machine/patient specific nature of 4\u03c0 planning, Letter to the Editor:Dr. Sarkar\u2019s assessment of the solid angles is mathematically incorrect. Without considering collision, Dr. Sarkar estimated the theoretical accessible solid angles based on the observation for a typical C-arm linac that the gantry has 360\u00b0 access while the couch only has 180\u00b0 access. The observation is correct but Dr. Sarkar neglected the symmetry of the C-arm gantry geometry and the additional freedom of combining the gantry and couch rotation. In fact, the couch only needs 180\u00b0 rotation to provide the full 4\u03c0 access. Figure\u00a0Even with the collision consideration, the angles that can be safely accessed for head or foot treatment is significantly larger than a hemisphere. One can consider a typical brain treatment where not only the superior angles are accessible, some of the inferior oblique angles are also available by rotating the gantry towards the patient torso while the couch is rotated. It is true that the solid angle for body treatment is more limited such as the left lung treatment shown in Fig\u00a0selecting beams from the 4\u03c0 solid angles instead of using all beams in a single plan.Dr. Sarkar calculated the spherical surface covered by a 40\u00a0cm\u2009\u00d7\u200940\u00a0cm field in a 360\u00b0 arc. He then argued that the actual solid angle is much smaller due to the smaller actual field sizes. This is a misunderstanding of 4\u03c0 treatment planning, which indicates the freedom of There is a benefit of keeping 4\u03c0 as an open software and hardware platform to encourage and incorporate future development. The mechanical restrictions to approach 4\u03c0 solid angles are neither insurmountable nor static. Extended source-to-isocenter distances are readily achievable with existing C-arm platforms, although additional quality assurance and commissioning may be needed to safely use this mode. Robotic couches that are being introduced in the radiation oncology clinic  will cerFinally, there is a beauty in the nomenclature. 4\u03c0 is not a typical undecipherable acronym that one has to look up. It is simple yet self-explanatory. Different from the conventional term of \u201cnon-coplanar radiotherapy\u201d, it emphasizes the ability of automated beam orientation optimization in the non-coplanar solution space. It reflects continuously evolving effort in planning algorithms and delivery platforms advance radiotherapy dosimetry.Recently, Dr. Sarkar questioned our use of \u201c4\u03c0\u201d to describe non-coplanar external beam radiotherapy. This discussion brought the much needed attention to this topic. However, I disagree with his conclusion that 4\u03c0 should not be used to describe non-coplanar radiotherapy for the following reasons."}
+{"text": "Novel insights into the pathophysiology of schizophrenia are needed to move the field forward by providing the conceptual framework to facilitate development of new treatment strategies. It is well established that glutamatergic systems are disrupted in schizophrenia, which are intimately linked to metabolic function. While there are many promising new directions, accumulating evidence suggests that bioenergetic function is impaired in the brain in schizophrenia. There are multiple mechanisms in the brain to meet neuronal energy demands, including glycolysis, lactate uptake, and oxidative phosphorylation. In normal brain, neurons and astrocytes are coupled through the astrocyte-neuron lactate shuttle, where astrocytes metabolize glucose to lactate and pyruvate, primary energy substrates that are transported to neurons via monocarboxylate transporters (MCTs). Lactate generated by glycolysis in glial cells constitutively supports synaptic transmission even under conditions in which a sufficient supply of glucose and intracellular adenosine triphosphate (ATP) are present. Interestingly, working memory and other cognitive domains are dependent on the shuttling of lactate from astrocytes to neurons. This process highlights the bioenergetic coupling between astrocytes and neurons that develops as the brain matures, forming a critical biological process in the mature adult brain. We assessed elements of these systems in postmortem brain, testing the hypothesis that there are cell-subtype defects in bioenergetics function in the frontal cortex in schizophrenia.Well-validated assays were used to assess the activity of three glycolytic enzymes in postmortem dorsolateral prefrontal cortex (DLPFC) samples (n=16/group): lactate dehydrogenase (LDH), hexokinase (HXK), and phosphofructokinase (PFK). Each sample was assayed with and without a specific inhibitor (in duplicate) and normalized to protein loaded into the assay. We also probed for differences in protein expression using western blot analysis. Western blot analyses were run in duplicate using the following antibodies optimized for postmortem brain: MCT1, LDH, LDHA, LDHB, HXK1, glucose transporter 3 (GLUT3). We performed real time quantitative polymerase chain reaction (RT-qPCR) using TaqMan PCR assays  in duplicate on cDNA samples in 96-well optical plates on a Stratagene MX3000P . We also coupled laser capture microdissection (LCM) with RT-qPCR from superficial and deep layers of DLPFC using the Veritas Microdissection instrument and CapSure Macro LCM caps . Similar studies were performed in haloperidol-decanoate or vehicle (sesame oil) treated rats (intramuscular injection every 3 weeks for 9 months).We found a 24% decrease in PFK1 mRNA expression in the dorsolateral prefrontal cortex in schizophrenia (p=0.039). We also found decreases in HXK  and PFK  activity in the dorsolateral prefrontal cortex. These changes were not present in haloperidol treated rats. At the cell-level, in pyramidal neurons we found an increase in MCT1 mRNA expression , and decreases in HXK1 , PFK1 , GLUT1 , and GLUT3  mRNA expression. We found increases in MCT1  and GLUT3 , but not HXK1, PFK1, or GLUT1, mRNA expression in enriched pyramidal neuron samples of antipsychotic treated rats.As the brain develops, bioenergetic organization and the formation of synapses occur simultaneously, creating a fundamentally interdependent system. There is accumulating evidence of implicating a number of abnormalities associated with glucose metabolism, the lactate shuttle, and bioenergetic coupling in schizophrenia, suggesting energy storage and usage deficits in the brain. Bioenergetic deficits and genetic risk for synaptic dysfunction in schizophrenia could contribute to the pathophysiology of this illness. In normal brain, glucose enters cells through GLUTs and is processed by glycolytic enzymes resulting in bioenergetic substrates such as pyruvate. Pyruvate can then be converted to lactate and transported between cells or intracellularly by MCTs to be oxidized in the TCA cycle when neuronal energy demand is high. Our findings of decreased glycolytic enzyme and lactate transporter mRNA expression suggests a decrease in the capacity of pyramidal neurons to generate bioenergetic substrates from glucose via glycolytic pathways. Additionally, if neurons were unable to take up adequate amounts of glucose for glycolysis, the intracellular pool of available pyruvate/lactate for transport into mitochondria may be diminished, ultimately impacting energy supply. It is also possible that there is attenuated glycolysis in pyramidal neurons, with a shift towards pathways that boost protection from oxidative stress (pentose phosphate pathway). Other studies also report region and cell-subtype specific changes in the expression of genes encoding proteins involved in metabolism in this illness. Importantly, the above changes were not attributable to antipsychotic treatment. Both synaptic function and meeting of energetic demands are essential for cognition, and failure of either could contribute to the cognitive symptoms seen in schizophrenia. Augmenting affected systems such as glucose utilization pathways could offer a novel approach to restoring cognitive function in schizophrenia. This could include targeting pro-metabolic substrates pharmacologically."}
+{"text": "We report 1,327 probable cases of dengue in Burkina Faso in 2016. Of 35 serum samples tested by a trioplex test, 19 were confirmed dengue virus (DENV)\u2012positive: 11 DENV-2, 6 DENV-3, 2 nontypeable, and 1 DENV-2/DENV-3 co-infection. Molecular testing should be conducted to correctly identify causative agents in this complex infectious disease landscape. Dengue is an emerging viral disease mainly found in the tropical and subtropical zones, and a major public health concern worldwide , which detects DENV nonstructural protein 1 (NS1) and dengue-specific antibodies (IgM and IgG), in response to an outbreak of acute febrile illness in Burkina Faso. All patients with acute febrile illness during this period were suspected to have dengue; notably, some patients had biphasic fever with severe headache, myalgia, arthralgia, and rash. Patients who tested positive for NS1 or DENV antibodies were considered to have a probable DENV infection. All participants provided informed consent as specified by the Declaration of Helsinki, and approval of this study was obtained from the national ethics committee.Of the 1,947 blood samples tested, 1,327 were positive for NS1, DENV antibodies, or both. Of the 13 country regions investigated, the central region, which includes the city of Ouagadougou, was the most affected, having 1,679 of the 1,947 suspected cases  and 1,307 of the 1,327 probable cases. Of the 20 deceased patients, 18 were positive for NS1 and 2 were positive for NS1 and DENV IgM. The outbreak peaked November 11\u201214. Blood samples from 35 randomly selected patients were sent to the National Reference Laboratory for Influenza  for confirmation using the Centers for Disease Control and Prevention trioplex real-time RT-PCR protocol . Eleven In Burkina Faso, dengue represents an added burden to an infectious disease landscape dominated by malaria; therefore, implementation of molecular diagnostic testing is urgently needed to identify the correct etiologic agent associated with the disease. The trioplex real-time RT-PCR detected 19 cases of DENV. A total of 3 serum samples positive for NS1 were negative by this assay. These negative results can be explained in part by declining viremia levels that became undetectable around the time of molecular testing, although testing with a larger representative sample size could have provided more information.We found DENV-2 to be the dominant serotype in this outbreak, followed by DENV-3. No cases of DENV-1 or DENV-4 were found, although testing a larger number of specimens might have revealed the co-circulation of these DENV serotypes. Human cases of DENV-2 in Burkina Faso is supported by previous reports of DENV-2 circulating in mosquitoes (We speculate that increased international travel between neighboring countries and mosquito circulation has led to DENV-2 and DENV-3 successfully crossing the border into Burkina Faso. This pilot study shows DENV-2 and DENV-3 are both circulating in Burkina Faso and causing human disease. Molecular diagnostics, vector control strategies, and risk communication should be implemented in Burkina Faso in preparation for future outbreaks."}
+{"text": "Antibody therapies with high efficiency and low toxicity are becoming one of the major approaches in antibody therapeutics. Based on high-throughput sequencing and increasing experimental structures of antibodies/antibody-antigen complexes, computational approaches can predict antibody/antigen structures, engineering the function of antibodies and design antibody-antigen complexes with improved properties. This review summarizes recent progress in the field of in silico design of antibodies, including antibody structure modeling, antibody-antigen complex prediction, antibody stability evaluation, and allosteric effects in antibodies and functions. We listed the cases in which these methods have helped experimental studies to improve the affinities and physicochemical properties of antibodies. We emphasized how the molecular dynamics unveiled the allosteric effects during antibody-antigen recognition and antibody-effector recognition. Nowadays, monoclonal immunoglobulin gamma (IgG) antibodies have become a major framework in cancer therapy and therapy for many other critical diseases. IgG molecules bind to their cognate antigens and the immune complexes subsequently interact either with type I or type II Fc receptors on effector cells and on B cells, modulating both humoral immune processes and innate immune processes . StructuInterestingly, numerous evidences have shown that the antibody constant domain also plays a role in antibody antigen recognition . AntibodComplementarity-determining regions (CDRs) are part of the variable chains in IgGs, and a set of CDRs constitutes a paratope. Chothia and Lesk define the \u201chypervariable loops\u201d based on the relationship between amino acid sequences and three-dimensional structures around the antigen binding region ,19. TheySeveral widely used antibody structure prediction methods are developed from the industry, , Schr\u00f6dinger Inc.   and AcceUsing the three-dimensional structures of the antibody-antigen complexes, it is possible to enhance the antibody-antigen binding affinities by in silico mutations on antibody residues. In the best situation, when the antibody-antigen complex structures are available, it is relatively straight-forward to perform affinity maturation in silico. Firstly, as an initial step, the protein backbone was treated as rigid, and the conformation of the side chain was determined by discrete side-chain rotamer search. Secondly, the lowest-energy of the structures was further re-evaluated by using more accurate, but computationally more expense models,  or Generalized Born (GB) continuum electrostatics, unbound-state side-chain conformation search, and minimization). Based on the crystal complex structure between 11K2 and MCP-1 , all res5) of models to capture a global energy minimum. Encouragingly, the antibody-antigen complexes showed a strong energy funnel, with low energy structures corresponding to a low RMSD to the native structure, indicating that this method can recover the native conformation. Zhao et al. used RosettaDock to search the possible complexes between A\u03b2 fibrils/oligomers and a therapeutic antibody [One of the significant challenges in modeling antibody antigen complexes is docking the antibody onto its epitope on the surface of the antigen. As epitopes and paratopes are typically flat, the shape complementarity between antibody and antigen is not a good determinant of correct antibody placement, which limits the application of general protein-protein docking procedures. SnugDock , which iantibody , crenuzuAn alternative computational approach to physical modeling is the knowledge-based residue pair preference on epitope\u2013paratope interfaces. With the increasing crystal structures of antibody-antigen complexes in the Protein Data Bank (PDB), a statistical amino acid interaction preference matrix can be used to predict the antibody-antigen recognition. Wang et al. studied the physicochemical properties of regions on and far from the antibody-antigen interfaces, such as net charge, overall antibody charge distributions, and their role in antigen interactions. They found that amino acid preference in antibody-protein antigen recognition is entropy driven. The interface residues with low side-chain entropy are selected to compensate for the high backbone entropy when interacting with protein antigens Positively charged and polar antigen residues and bridging water molecules have a higher possibility of being selected on the antibody-protein antigen interface. Tyr, Ser, and Asp but few Lys are selected on the antibody-antigen interfaces . K. TharAlthough there are many successful cases for protein-protein affinity design, there are still challenges as the antibody-antigen recognition is not simply interactions between proteins; the solvent effect is also crucial. For instance, interfacial trapped water molecules, polar and charged side chains, and the balance between protein-solvent and protein-protein interactions from the unbound to bound state need to be considered during the modeling.In high concentrations of formulations for therapy or storage , antibodMD simulations have been used to study the mechanisms of antibody aggregation and amyloidosis ,65. In tThe process of antibody-antigen recognition is complicated and associated with conformational transitions of the antibody by the inherent flexibility ,71,72. RBesides antigen recognition, the effector functions, such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis, can also be engineered based on the structures between fragment crystallizable region (Fc) and receptors and the computational techniques in a high-throughput way. The engineerable parts are the hinge region, CH2 domain, N-glycans and N-glycan-attached residues. All\u2019acqua et al. introduced various modifications into the hinge region of mAb 12G3H11 to modulate the hinge\u2019s length, flexibility, and/or biochemical properties. They found that the upper and middle hinge are important, and mutations introduced to these regions can modulate the Fc\u03b3RIIIa or C1q binding . Lazar eIn silico methods are also widely used in vaccine design. Generally, structure-based in silico vaccine design includes epitope identification, immunogen design by epitope grafting, and antibody/antigen structure prediction. The epitopes include linear (both T-cell and B-cell) and conformational epitopes (mainly B-cell). The bioinformatics tools are well-established to predict the linear epitope ,103,104.High-throughput technology combined with computational technologies have dramatically advanced the development of biological therapeutics ,120,121.Improvements in conformational sampling methods and development of scoring functions used to estimate free energies are also much-needed. Sampling the conformation of flexible CDR loops, especially CDR-H3, is particularly important in antibody design, because large conformational changes can occur when antigen binding. Moreover, the evaluation of antibody allosteric effect requires extensive sampling about the motion and dynamic of antibody constant domains. Free energy perturbation (FEP) methodology can be used to estimate relative antigen binding affinity differences to the antibody variants , but theAllosteric effects and conformational change in antibody-antigen recognition and antibody effector functions are emerging and challenging to evaluate using experimental techniques ,132,133."}
+{"text": "In some biological networks defining importance is difficult, which then creates challenges in finding an appropriate centrality algorithm.Computing k centrality algorithms through our iterative algorithm MATRIA, producing a single ranked and unified set of central nodes. Through tests on three biological networks, we demonstrate evident and balanced correlations with the results of these k algorithms. We also improve its speed through GPU parallelism.We instead generalize the results of any Our results show iteration to be a powerful technique that can eliminate spatial bias among central nodes, increasing the level of agreement between algorithms with various importance definitions. GPU parallelism improves speed and makes iteration a tractable problem for larger networks. There are three core types of path-based centrality, each with different definitions of importance. Betweenness centrality  by approaching centrality from the perspective of a node\u2019s benefit to the network. We used a simplified economic Payment Model , definin,1 by appG is the maximum positive edge weight product over all paths between node i and node j, and L is the maximum negative edge weight product. We computed these paths using a modified Dijkstra\u2019s algorithm MOD_DIJKSTRA that used edge products and chose maximum path magnitudes. This is just closeness centrality using maximum paths, with \u201cpath length\u201d defined as G+L. Plugging CLO into X in Algorithm 1 represents our iterative closeness centrality algorithm ATRIA. We now define signed versions of other path-based backbones.where i, producing: Degree is easiest to define, with all local computations. For gains and losses we count incident positive and negative edges for a node W is the signed weight of edge .where MOD_DIJKSTRA algorithm to count the number of positive paths )) and negative paths ) that include i. The equation then becomes the sum of these terms: Betweenness is more challenging, but we can use the same BET or DEG for X in Algorithm 1 to respectively produce iterative betweenness or degree centrality. Since non-iterative path-based approaches produced extremely different results on our networks, we will use these iterative versions ITERCENT BET, ITERCENT CLO, and ITERCENT DEG to demonstrate MATRIA. Other centrality algorithms can be substituted for X, and we will in fact show that MATRIA can support any k centrality algorithms.We can then plug Table\u00a0g and produces the sets of central nodes SBET, SCLO and SDEG, then resolves disagreements between these sets through a procedure UNIFY to produce a final set S.Algorithm 2 shows our top-level MATRIA procedure that accepts a network x:x\u2208SBET\u2229SCLO\u2229SDEG. On network A the iterative backbones agreed on twelve central nodes, colored black in Fig.\u00a01b1-3 and labeled A1- A12. Recall this is already an improvement upon the non-iterative versions, which agreed on only five central nodes in the same vicinity. UNIFY first adds these twelve universal agreements to S.We define universal agreements as nodes discovered by all iterative backbones, or any c we label nodes found by one or two of the path-based backbones, but not all three . We use node color to indicate the backbone(s) that discovered them, with primary colors for nodes discovered by one backbone: Betweenness (4), colored red: B1- B4Closeness (5), colored yellow: C1- C5Degree (2), colored blue: D1, D2In Fig.\u00a01Betweenness & Closeness (1), colored orange: BC1Closeness & Degree (5), colored green: CD1- CD5Betweenness & Degree (1), colored violet: BD1We use secondary colors obtained by combining appropriate primary colors for nodes discovered by two backbones: x,y,z] in Fig.\u00a04a. In this case x, y and z were found as central by iterative betweenness, closeness and degree respectively. However, suppose centrality is actually a \u201ctoss-up\u201d between them, which would mean for example in iterative betweenness when x was found as most central, y and z had only slightly lower centrality values. In the next iteration x would be removed along with edge y\u2212z, causing y and z to lose all contributions from paths involving this triad . The same thing would happen when y was found by iterative closeness, and z by iterative degree. Adjacencies like the one in Fig.\u00a04b have the same issue for the same reason, with x (or y) losing contributions from its central neighbor upon its removal.We note patterns among these disagreements. Many times all three backbones are covered exactly once between two adjacent or three triad nodes. We argue that because of the fundamental properties of iteration, centrality is likely a \u201ctoss-up\u201d in these situations. Take for example the triad  and . UNIFY adds these to S (now 14 elements) as \u201ctoss-ups\u201d, and we also darken them in our updated Fig.\u00a01d to indicate they have been resolved. For supernode adjacencies there are three types: red-green , yellow-violet , and blue-orange . We have a total of six supernode adjacencies in Fig.\u00a01c and begin by adding them to S: , , , , , and .We define a B1 and B3). Having supernodes that share members is not helpful, because each supernode should provide multiple options for a central node. We now describe how UNIFY merges supernodes with common members, and specifically address the triad and adjacency in detail to handle this network. Supernode triads can also overlap with each other, as can supernode adjacencies, and we later briefly describe how to merge those.We now have an issue, because two of these adjacencies also include supernode triad members . Thus, the \u201ctoss-up\u201d becomes between (1) only x, (2) y and w, and (3) y and z. We merge these into one supernode triad , now allowing a single node to represent a set of nodes as shown in the Figure. Although the edges from x to {y, w} and {y, z} now become ambiguous, their weights are no longer relevant because we already ran the backbones.We first note that for a supernode adjacency Central Triad  with adjacency . We replace both elements in S by the supernode: .Upper Triad  with adjacencies  and . We replace all three elements in S by the supernodeB3,{C5,CD3,CD4},{D2,CD3,CD4}]. now has an overlap with adjacency . We similarly replace both elements in S by the supernode .We have several supernode adjacencies in our network where one of the two nodes is also in a supernode triad: d shows all resolved disagreements darkened. In addition, Table\u00a0S in UNIFY, which we now fully write as Algorithm 3.Figure\u00a01Ranking Supernodes: The final step of UNIFY is to rank the elements of S. We do this as follows: Universal Agreements: Mean ranking over backbones.Supernode Triads: Mean ranking of each node using the backbone that found it. For example in Fig.\u00a04a we would average the ranking of x in betweenness, y in closeness, and z in degree.Supernode Adjacencies: Same as supernode triads, except one node will have rankings for two backbones.Merged Supernodes: These have elements like {w,y} where w and y were said to both be important by a backbone. In this case use the ranking of whichever of w and y was discovered first as the ranking of {w,y}, then apply the above logic for the supernode ranking. Our results, shown in Fig.\u00a01e (red=high and violet=low rank), indicate that the top five entries  could correspond to leaders of the five most tightly connected components.Unresolvable Disagreements: Although most disagreements in Fig.\u00a0C3 and C4 that were found by closeness and not involved in a resolvable disagreement. These are still colored yellow in Fig.\u00a01d. Upon further investigation the disagreement resulted because iterative degree and betweenness found node A7 early (#2 and #7), but closeness found it later . With A7 directly connected to C3, removing it plummeted C3 in degree and betweenness centrality. But since A7 was also eventually discovered by closeness it became a universal agreement and could not be a supernode with C3. This seems to suggest forming supernodes on-the-fly, as opposed to waiting until the end. However the drop of C4 resulted from an indirect effect (removing A7 reduced many edges in that tight component), so that will not resolve all disagreements either. The other disagreement, BC1 and CD5, creates an interesting situation where two backbones each say one is important, but one (closeness) says both are important (i.e. not a \u201ctoss-up\u201d). We leave this as unresolvable for now, though could potentially add another type of element in S which encapsulates this. We will see however that even with our current approach, these unresolvable disagreements are quite rare in our networks.k centrality algorithms. In our example (k=3), we can view supernode adjacencies and triads as components of size 2 and 3. In general supernodes can be of sizes 2 to k.We also remark that UNIFY can be generalized to work with any We begin by evaluating the percentage of nodes for which UNIFY could reach an agreement on centrality. Table\u00a0We also checked some of the agreed important genes discovered by MATRIA in network B. Although gene essentiality statistics are limited for the Pacific Oyster, the results show promise. The gene for the most abundant and fundamental eukaryotic protein, Actin , was fouS correlates with the individual rank vectors SBET, SCLO, and SDEG, plus those found when including PN-Centrality and PageTrust (thus k=5). Table\u00a0We next verify that the rank vector for t thus k=. Table\u00a05i and j, where i<j. Each thread  first computes any maximum positive and negative paths between node i and node j in parallel. We then take N threads (for a network with N nodes), one per row, to compute the centrality of each element i. Next, we compute the most central node m on the CPU, followed by each thread  marking edge  if it (1) exists and (2) is in a stable triad with m. Finally each thread  removes edge  if it is marked. Table\u00a0As we realize that iteration is computationally expensive, we parallelize MATRIA for the GPU using a four-step process demonstrated by Fig.\u00a0x\u2192y would now be different from x\u2190y), an interesting question that we plan to immediately pursue.Our results illustrate that applying iteration to centrality algorithms with different definitions of \u201cimportance\u201d and unifying their results gives more meaning to their computed central node sets. By resolving disagreements MATRIA produces a ranked list of central nodes and supernodes, with a cardinality much smaller than the size of the network and several mutually agreed unimportant nodes removed. Rank vectors correlate well between this set and the individual iterative backbones and are much more consistent compared to just the iterative or non-iterative backbones. While cases of unresolvable disagreements can still occur in this unified set, they are rare. Through GPU optimizations MATRIA is currently practical for medium-sized networks, and we are exploring ways to push this boundary. We also plan to experiment with weighted averages when computing overall rankings. Finally, applying MATRIA to directed (i.e. metabolic) biological networks will require an extension of iteration and supernodes to incorporate direction (i.e. adjacency"}
+{"text": "Three dimensional chitosan scaffolds have been widely used in bone tissue engineering due to biodegradable and biocompatible properties. The present study aimed to evaluate osteogenic potential of NBP treated chitosan scaffold. The surface characteristics of scaffolds were analyzed by scanning electron microscopy (SEM) and X-ray diffraction (XRD), cell proliferation and differentiation was tested with osteoprogenitor cell line MC3T3-E1. The results show that NBP modified scaffold increase cell metabolic by MTT assay and live/dead assay. More importantly, we evidenced enhancement of osteogenic differentiation on NBP treated scaffolds by an increase of alkaline phosphatase (ALP) activity, high degree of extracellular mineralization and up-regulated osteogenic marker genes expression level. The findings in our study highlighted NBP as the innovative method to modified chitosan scaffold and to fine-tuning the scaffold a more suitable and beneficial biomaterial for  ROS and RNS has been investigated as the main role in many applications of NBP such as cancer therapy, wound healing, skin diseases, dental care, and sterilization10. In tissue engineering field, despite of the arising study of NBP acting directly with stem cells or progenitor cells to enhancing differentiation15, on the other hand, NBP has been extensively studied for surface modification of biomaterial, which is another key element in bone tissue engineering18.Within recent years, non-thermal biocompatible plasma (NBP) has focused on the development of biological and medical application. NBP was generated by ionizing working gas, composes of a variety of charged particles, reactive oxygen species (ROS), reactive nitrogen species (RNS), ultraviolet light, as well as electric and magnetic field19. The surface modification process also has shown as gas dependent manner. Inert gas (argon) incorporated oxygen-containing polar functional groups the poly PVA/chitosan surface whereas reactive gas (oxygen) increased surface roughness leading to improvement of surface wettability20. Similar results was reported by Choi et al.18, NBP treatment increased surface hydrophilicity which using nitrogen and air gas and resulting better osteoblast attachment and proliferation. Lauriault et al.21 demonstrated NBP induced miropatterned nitrogen-rich depositions to polymer film surface with enhancement of macrophages and chondrocytes adhesion and proliferation and can be used for tissue engineering. These evidence highlighted NBP as a useful tool for surface modification of biomaterial in tissue engineering.NBP based strategies as a novel arising method for surface modification of biodegradable polymers, and biomaterials demonstrate great potential. Typically, the consequences of NBP treatment of a material surface include increasing of surface hydropholicity, introducing functional groups to the surface and change the surface roughness22. Among those chitosan is an abundant natural polyheterosaccharide obtained from deacetylation of chitin23. The biological properties of chitosan, including biodegradability, biocompatibility and no toxicity has been well-established presently under extensive investigations for bone regeneration. The improved osteogenic differentiation of stem cells was observed on chitosan scaffold, and in vivo study was also conducted25. The pervasive use of chitosan scaffold in bone tissue engineering has directed much effort towards the advancement of chitosan-base biomaterials through surface modifications or combination with other materials in order to reach higher biocompatibility and osteocondictivity. However, the biological effects, particularly osteogenic differentiation potential of NBP treated 3D chitosan scaffold have not been well studied.Three-dimensional (3D) scaffolds desired special properties like biodegradable, porosity, mechanical strength and surface chemistry to provide stem cells attachment, proliferation and differentiation in bone tissue engineering. Scaffolds consisting of natural polymers have attracted significant interest due to their flexibility in terms of chemical modification and the ability of degradation into low molecular weight fragments that can be resorbed or eliminated inside human bodyIn the current study, we applied NBP treatment to 3D porous chitosan scaffold and evaluated the osteogenic potential of NBP modified chitosan scaffold using MC3T3-E1 cells. For that, we assess cell viability, metabolic ability and particularly osteogenic differentiation by detection of alkaline phosphatase activity, extracellular mineralization and osteogenic marker gene expression of osteoprogenitor MC3T3-E1 cells on NBP treated scaffolds aimed to be used in bone tissue engineering field.2 gas achieved higher NO\u03b3 signals (240\u2013280\u2009nm) and N2 emission from the second positive (300\u2013400\u2009nm), and weak hydroxyl radical (OH) around 309\u2009nm26, as compared with air gas ) accumulation was gradually increasing in a time dependent manner with NBP discharging using nitrogen (N2) gas  accumulation was almost 10 times more when air was working gas than N2 was used . Discharging with Ngas Fig.\u00a0. Furthergas Fig.\u00a0. Howeversed Fig.\u00a0.Figure 1Scanning electron microscopy (SEM) micrographs of chitosan scaffolds presented as a 3D highly porous sponge-like microstructure with high degree of interconnection throughout the scaffold in all directions. 5\u2009min treated scaffold showed compactness microstructure with homogeneous pore distribution, and thickened barriers between pores Fig.\u00a0. Particu23, suggesting that NBP modification did not change the crystallinity of scaffolds. Whereas, the crystalline size was further calculated by Scherrer equation. The results showed that NBP treated groups the crystallite size reduced 22%, 51%, and 25% for 3, 5, and 10\u2009min treatment, respectively . Figure\u00a02 gas and air gas treatment (P\u2009<\u20090.05)  Fig.\u00a0. We did P\u2009<\u20090.05).Alkaline phosphatase (ALP) activity is a well-defined osteogenic differentiation marker and is assumed to reflect the degree of differentiation. In this study, ALP activity of MC3T3-E1 cells on untreated and 5\u2009min NBP treated scaffold was measured at 1, 4, and 7 days. The results showed that ALP activity of cells on both scaffolds gradually increased from day 1 to day 7 Fig.\u00a0. No signP\u2009<\u20090.05) and 5\u2009min (P\u2009<\u20090.001) modified scaffold significantly enhanced the formation of calcified nodules in MC3T3-E1 cells after 4 days under osteogenic inductive condition  staining where the binding of calcium ions in mineralized extracellular matrix (ECM) forms an ARS-calcium complex in a chelation process. When compared to original scaffold, NBP 3 , and maintaining the high level until day 7. Osterix expression exhibited significant increase at day 7 (P\u2009<\u20090.05). As a late stage maker, osteocalcin (OCN) was significantly upregulated at 7 days culture.In order to explore the differentiation effect of NBP treated scaffold on MC3T3-E1 cells, expression of osteogenesis related genes was analyzed by real-time PCR. As shown in Fig.\u00a0EDS analysis was used to analyze the mineralization in the osteogenic differentiation of the calcium and phosphate deposits in the extracellular matrix. Quantitative elemental EDS analyses showed that the amount of calcium deposition increased after 7 days incubation, and significantly increased in the 5\u2009min NBP treated chitosan scaffolds group Table\u00a0. However2 secondary positive system and NO\u03b3 spectrum. These exited species bombarded onto scaffold surface, offering an effective strategy to incorporate reactive functional groups on chitosan scaffold surface.NBP exists as a quasi-neutral particle system in the form of gaseous or fluid-like mixtures of free electrons, ions, and radicals, generally also containing neutral atoms and molecules. Some of the neutral particles are in an excited state, which can return to the ground state by photon emission. The detection of spectrum of these emissions by optical fiber can speculate the radical contained in NBP. In the present study, when nitrogen gas is used, OES shows excited N22. So chitosan turns out to be a highly reactive polysaccharide. Electrons and reactive oxygen and nitrogen species generated from NBP interacted with the surface molecules, resulting in increasing number of oxygen- and nitrogen- containing groups and polar groups on the surface, showing with increased the surface roughness with emergence of sharp protuberances of chitosan film27. Silva SS et al.28 has demonstrated that when nitrogen was used as feeding gas, not only the etching process but also functionalization with nitrogen- containing group was observed on NBP modified chitosan membranes, which is consistent with the SEM results in present study. NBP treatment increased the pore size and thickened the pore barriers and resulted in the structural compactness of entire scaffold. The structural compactness change and thickened interconnectivity may due to post NBP treatment effect of surface adaptation and oxidation reactions, leading to an increase of cell affinity to the surface and improving cell viability and proliferation.For chitosan scaffold surface, amino group and hydroxyl group in chitosan provide functionality. The free amino group confers a positive charge to chitosan, allows it for many electrostatic interactions with electrons and negatively charged molecules29. Cell viability on scaffolds is essential for cellular compatibility and suitability for tissue engineering applications19. In our study, the cell viability significantly increased on the 5\u2009min NBP treated scaffold (P\u2009<\u20090.05), indicating that NBP treatment improves cell adhesion and growth on the scaffolds.Recent studies have indicated that the surface characteristics and microenvironment of scaffolds, including chemical deposition, roughness, hydrophobicity and matrix elasticity, can profoundly affect cell behavior and functioning in directing the osteogenic differentiation of stem cellsP\u2009<\u20090.001), suggesting that NBP modification of scaffold promote osteogenic differentiation. Mineralization is the final phase of osteoblast differentiation, whereby the mineral matrix, which predominately contains calcium phosphate in the form of hydroxyapatite, is secreted and deposited by mature osteoblasts. Evidence of osteogenic differentiation of MC3T3-E1 cells can be found in their capability to mineralize inorganic calcium at the middle of the differentiation period, monitored as inorganic mineral deposition. In our study, the ARS staining and EDS results demonstrate the beneficial action of the NBP modified chitosan scaffolds on osteoblast ECM mineralization. Besides, BMP-2 plays pivotal role in osteogenic differentiation and bone regeneration30. A combined BMP-2 and NBP stimulation augmented mineralization to a greater degree than treatment with either single stimulus, suggesting the combined method in vitro could be considered as groundwork for in vivo bone development.Following cell attachment and proliferation on scaffold surface, cells start to differentiate into osteoblast phenotype under osteoinductive media. Three major transformation stages of cell proliferation, maturation, and mineralization of ECM exist during the growth and differentiation of MC3T3-E1 cells to osteoblast lineage, accompanied with up-regulation of certain related proteins and genes of each stage. ALP enzyme indicates early stage of osteoblast activity and maturation. ALP activity in MC3T3-E1 cells on both scaffolds were increased as an incubation time dependent manner, and at day 7 ALP activity was significantly higher on NBP treated scaffold than control scaffold (P\u2009<\u20090.05), whereas osterix showed significant increased upregulation at day 7 (P\u2009<\u20090.05), as compared with untreated scaffold. ALP is a well-known marker of osteogenic differentiation for cells at an early stage31 and osterix is an osteoblast-specific transcription factor essential for osteogenic differentiation and bone formation. Runx2 can act as an indispensable factor transcriptionally regulating osteocalcin (OCN) and ALP gene expressions, participating in controlling osteoblast function and ECM mineralization in osteogenesis32. The higher expression level of ALP, Runx2 and osterix by MC3T3-E1 cultured on NBP modified scaffold indicates that the modification has an enhanced ability to supported cell differentiating into osteoblast phenotype.Real-time PCR analysis of osteogenic gene expression further indicates that NBP modification to scaffold is favorable for osteogenic differentiation. In this study, gene expression of ALP, runt-related transcription factor 2 (Runx2) was significantly upregulated on NBP modified scaffold at day 4 .The chitosan scaffolds used in this study were prepared and provided by Dankook University. The preparation of chitosan scaffold was described in previous studiesCHT solution with mineral oil/Span80  was improved dispersion force and physical property. Blend solution (CHT/MS\u2009=\u200960:10% v/v) strong stirring 350\u2009rpm for 1\u2009h after the solutions were injected into the mold , frozen at \u221280\u2009\u00b0C for 3\u2009h. 0.5\u2009N NaOH were injected into the mold at 1\u2009\u00b0C for 8\u2009h after scaffold was washed in ethanol stage by for 5\u2009h each. Finally, after washing for 48\u2009h in distilled water, the scaffold was frozen at \u221280\u2009\u00b0C for 24\u2009h and then lyophilized for 72\u2009h.38. Figure\u00a02O3) with thickness of 1 \u03bcm. NBP was generated with a discharge voltage 500\u2009V and electric current of 13\u2009mA with alternating current (AC) electric power supply. Nitrogen (N2) or air gas with a flow rate of 1.5 liter per minute (lpm) was used as working gas. The technical data of DBD device discharging with N2 and air was shown in Table\u00a02 and air gas were measured by the spectrometer . The intensity of optical emission signals was recorded in terms of wavelength (range from 280 to 450\u2009nm). In Fig.\u00a03 and nitrogen oxides (NO and NO2) during various NBP discharging time measured using a Gastec AP-20 gas sampling pump  and Gastec detector tubes .Figure\u00a0The characteristic of original chitosan scaffolds and scaffolds treated with NBP for 3, 5 and 10\u2009min, respectively, were analyzed using SEM. 3 scaffolds of each group were coated with platinuim at 20 milliampere (mA) for 60\u2009sec, and then examined by SEM .c and Gt are crystallite size of control and treated samples, respectively.XRD pattern of 3, 5, and 10\u2009min NBP treated and non-treated chitosan scaffolds was analyzed using Bruker D8 Advance X-ray diffractometer system  at room temperature. The wavelength of the radiation was 0.154\u2009nm, from a broad focus Cu tube operated at a voltage of 40\u2009kV and a current of 25\u2009mA was applied to the samples for measurement. The data was obtained in the form of 2-theta versus intensity (a.u.) chart. Then the obtained data was used for calculation of crystallite size using the following Scherrer Equation:5 cells. After 3\u2009h incubation, another 900\u2009\u03bcl of culture media was added to each well, and the culture was maintained at 37\u2009\u00b0C in 5% CO2 and 95% humidity. For osteogenic differentiation assays , the culture media was changed into osteogenic induction media supplemented with 10% FBS, 10\u2009mM \u03b2-glycerophosphate, and 50 \u00b5M L-ascorbic acid 2-phosphate (Sigma-Aldrich).MC3T3-E1 cells from normal mouse osteoblasts were obtained from the American Type Culture Collection . Cells cultured in \u03b1-minimal essential medium  containing 10% (v/v) heat inactivated fetal bovine serum (FBS) (Gibco) and 1% (v/v) antibiotics (Gibco) until adequate cell number was obtained. The cells used in the all experiments were less than passage 8. Cells were seeded on the scaffolds (both untreated and NBP treated) placed in a 24-well plate with 100\u2009\u03bcl of cell suspension containing of 1\u2009\u00d7\u2009105 cells were seeded on each scaffold placed in a 24-well plate. The analysis was conducted for an incubation period of 24\u2009h culture after seeding using a live/dead viability/cytotoxicity kit  and following the manufacturer\u2019s instructions. Before the live/dead staining, the cell-scaffolds were washed with PBS. 2\u2009\u03bcl of 50\u2009\u03bcM calcein and 4\u2009\u03bcl of 2\u2009mM ethidium homodimer-1 in 1\u2009ml PBS were added to each well and incubated for 20\u2009min at room temperature (RT) in the dark. Images of the cells were then captured under a fluorescence microscope .Cell viability was assessed using live/dead assay for original and modified scaffolds. Initially, chitosan scaffolds were treated by NBP for 3, 5 and 10\u2009min. Immediately post treatment, 1\u2009\u00d7\u200910[3--5-(3-carboxymethoxyphemyl)-2-(4-sulfophynyl)-2H-tetrazolium] (MTS) assay was performed on cells seeded on original and NBP 3 and 5\u2009min treated scaffolds. MTS was used as a reagent to detect cell metabolic activity. The metabolically active dehydrogenase enzymes in the cells reduce MTS to soluble formazan compounds. Absorbance was detected at 490\u2009nm using a microplate reader . The results were present as relative viability (% to control).The effect of NBP on the ALP activity of the MC3T3-E1 cells was analyzed using an ALP assay kit . The MC3T3-E1 cells were seeded on non-treated and 5\u2009min NBP treated chitosan scaffolds with culture media. After 24\u2009h incubation, the medium was changed to osteogenic induction medium and cultured for 1, 4 and 7 days. At each time point, ALP activity was determined in the cell supernatant following assay kit protocol. The absorbance was read at 405\u2009nm in a microplate reader (Gen5), and calculated according to the p-nitrophenol (pNP) standard curve. ALP activity (U/ml)\u2009=\u2009A/V/T (A: amount of pNP generated by samples (\u00b5mol), V: volume of sample added to the assay well (ml), T: reaction time (min)).5 cells on original and 5\u2009min NBP treated chitosan scaffold with culture media. 24\u2009h post incubation, the medium was changed to osteogenic induction media, and incubated for 4 days. The cells were rinsed twice with PBS and fixed with 4% paraformaldehyde for 10\u2009min, followed by staining with 2% ARS solution (pH 4.2) for 1\u2009h at room temperature. After aspiration of the unincorporated dye, the wells were washed 4 times with PBS. For quantification of calcified matrix, 1\u2009ml of 10% (v/v) acetic acid was added to each well, and the plate was incubated at room temperature for 30\u2009min with shaking. Aliquots (100\u2009\u00b5l) of the supernatant were read at 405\u2009nm in 96-well using a microplate reader (Gen5).Mineralization in MC3T3-E1 cells on NBP modified scaffolds was determined by staining with ARS, which selectively binds with calcium and yields a bright red staining. Cells were seeded at a density of 1\u2009\u00d7\u200910Further ARS staining and quantitation was performed to MC3T3-E1 cells seeded on untreated scaffold (with or without BMP-2 supplement) and 5\u2009min NBP treated scaffold (with or without BMP-2 supplement), respectively. BMP-2 was supplemented in culture media at the concentration of 200\u2009ng/ml and cells were cultured for 4 days. Similar process was followed for ARS staining and absorbance reading. The images of calcium deposition on scaffolds were photographed using a camera.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control gene to normalize RNA expression. All reactions were run in triplicate for three independent experiments.Expressions of osteogenic marker genes were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) after ribonucleic acid (RNA) isolation and complementary deoxyribonucleic acid (cDNA) synthesis. Total RNA was isolated from the MC3T3-E1 cells using the RNeasy Mini Kit . cDNA was synthesized using the superscript VILO cDNA synthese kit  following the manufacturer\u2019s instructions. 4\u2009\u00b5l of each cDNA was used in the PCR reactions . The amplifications were performed using the following protocol: 40 cycles of 95\u2009\u00b0C for 15\u2009s and 60\u2009\u00b0C for 60\u2009s. The primers included ALP, Osterix, Runx2, OCN . The primer sequences were shown in Table\u00a0MC3T3-E1 cells were seeded on the non-treated and 5\u2009minutes NBP treated chitosan scaffolds in general media. After 24\u2009hours incubation, the media was changed to osteogenic induction media to evaluate cell mineralization at 3 time points . At each time point, the chitosan scaffolds with cells were gently rinsed with PBS and primary fixed with Karnovsky\u2019s fixative  at room temperature for 2\u2009hours. The samples were washed with PBS three times as the secondary fixation with 1% osmium tetroxide (OsO4) at room temperature for 30\u2009min, followed by washing with PBS twice. Alcohol gradient dehydrating was performed to remove water from the scaffolds at increasing ethanol concentrations , and the samples were then dried with hexamethyldisilazane (HMDS) twice (10\u2009minutes each). The samples (n\u2009=\u20093) were coated with platinuim at 20 milliampere (mA) for 60\u2009seconds and analyzed the presence of calcium and phosphorus elements at the surface by EDS .t-test was used for two groups comparison. One-way analysis of variance was used to compare the results between more than two different groups followed by Turkey\u2019s multiple comparisons test. The statistically significant differences were based on *P\u2009<\u20090.05, **P\u2009<\u20090.01, and ***P\u2009<\u20090.001. All graphing, calculation and statistical analysis were performed using GraphPad Prism version 7.00 . All assays involved at least three independent sets of tests for each group scaffolds.Graphs are represented by the mean\u2009\u00b1\u2009standard deviation of replicates. Student\u2019s"}
+{"text": "A DNA machine circuit and AND-NOT-AND-OR logic gates were successfully constructed, and the strategy was used to detect ATP in human urine. With the advantage of target recycling amplification and GO suppressing background signal without fluorescent label and enzyme, this developed strategy has great potential for sensitive detection of different proteins and small molecules.In this study, a target recycled amplification, background signal suppression, label-free fluorescent, enzyme-free deoxyribonucleic acid (DNA) machine was developed for the detection of adenosine triphosphate (ATP) in human urine. ATP and DNA fuel strands (FS) were found to trigger the operation of the DNA machine and lead to the cyclic multiplexing of ATP and the release of single stranded (SS) DNA. Double-stranded DNA (dsDNA) was formed on graphene oxide (GO) from the combination of SS DNA and complementary strands (CS\u2032). These double strands then detached from the surface of the GO and in the process interacted with PicoGreen dye resulting in amplifying fluorescence intensity. The results revealed that the detection range of the DNA machine is from 100 to 600 nM (R Adenosine triphosphate (ATP) plays an important role in various cellular metabolism processes and biochemical reactions in cells and biological organisms. Many diseases are related to ATP concentration, such as ischemia, hypoxia, Parkinson\u2019s disease, hypoglycemia, and some malignant tumors ,2,3. TheIn recent years, signal amplification methods, such as single primer isothermal amplification , rollingIt is also worth noting that much of the research on molecular logic gates has focused on DNA, due to it having a well-regulated structure, straightforward hybridization rules, and molecular recognition abilities, and DNA machines have calculated some math problems ,32. OverUridine triphosphate (UTP), ATP, cytidine triphosphate (CTP), guanosine triphosphate (GTP), and PicoGreen dye were purchased from Shanghai Yi Sheng Biotechnology Co., Ltd. . Nucleotide sequences  were pur2, pH 7.5). One micrometer of aptamer DNA strands (AS), 1 \u00b5M of SS, and 1 \u00b5M of helper DANS strands (HS) were mixed. The resulting mixture was subsequently heated to 95 \u00b0C for 5 min and then cooled down slowly to room temperature inside a furnace. Fuel stranded (FS) and complementary stranded (CS\u2032) DNA were heated to 95 \u00b0C for 5 min and were then quickly placed into a refrigerator set to a temperature of around \u221220 \u00b0C for 5 min, respectively. PicoGreen was diluted 200 times and GO (1 wt.%) was diluted 100 times. In all of the experiments, the volume of the buffer was 1800 \u00b5L; the AS, SS, and HS mixture totaled 70 \u00b5L; the PicoGreen volume was 25 \u00b5L; and the GO volume used was 10 \u00b5L. The amount of FS and CS\u2032 used was 40 \u00b5L in each case.All reagent dilutions were carried out with a buffer . The parameters used in the reaction time experiments were a 480 nm wavelength emission and excitation at 520 nm. The spectral parameters used were a 480 nm wavelength emission.Urine samples were obtained from a healthy adult at Hainan University hospital . The urine samples were centrifuged at 13,000 r/min for 3 min. The supernatants of the resulting samples were then diluted 10 times before ATP was incorporated into the diluted urine samples at concentrations of 100, 300, and 500 nM, respectively. The measurements were repeated three times to compute the urine recovery.To demonstrate whether it is feasible to use the DNA machine for ATP detection, the fluorescence spectra of different mixed solutions were tested after being incubated for 3 h. In order to further prove the feasibility of the principle, the time detection of the reaction process was undertaken. According to the a-segment shown in In terms of the logic gates, the input signal is 1 if ATP, CS\u2032 DNA, or FS DNA are present, if not, it is 0. Meanwhile, at a wavelength of 520 nm, if the change in the fluorescence intensity, \u0394F, is greater than 45, the output signal is 1, if not, it is 0. Using the results of According to According to Equations (1) and (2), the logic gates of the DNA machine can be represented in schematic form, as shown in After the sensing solutions were incubated for 3 h, the fluorescence intensities of different ATP concentrations were tested using the described method. 0, where F0 is the peak of the blank curve in 2 = 0.99108) with a LOD of 127.9 pM (3\u03c3/slope). Compared to most other methods  were tested against ATP. As shown in In order to evaluate the application of the DNA machine for ATP detection in real urine, recovery tests were carried out. The urine of healthy people was centrifuged, and the supernatant was diluted 10 times using a buffer solution. Different ATP concentrations were added to the dilute liquid and tested using the described method. 2 = 0.99108) with a LOD of 127.9 pM. This method has strong selectivity and is very suitable for ATP detection in real human urine. On the basis of this strategy, AND-NOT-AND-OR logic gates and a DNA machine circuit were successfully constructed. This method has the characteristics of being low cost due to not requiring any fluorescent labeling and biological enzymes. This recommended method could also be adopted for highly sensitive detection of various ions and proteins. In the future, the sensitivity of the DNA machine for ATP detection can be improved by catalyzing hairpin assembly.In summary, a target recycled amplification, background signal suppression, label-free fluorescent, enzyme-free DNA machine was developed for highly sensitive ATP detection. The DNA machine is triggered in the presence of both the target ATP and FS DNA. Target ATP cycle amplification and GO background signal suppression strategies are used simultaneously to improve the sensitivity of the DNA machine. The detection range was found to be from 100 to 600 nM (R"}
+{"text": "The AND DNA calculator was successfully used for the ATP detection in human urine. Compared with other methods, this DNA calculator has the characteristics of being label-free, non-enzymic, simple, and highly sensitive.Herein we described a deoxyribonucleic acid (DNA) calculator for sensitive detection of the determination of adenosine triphosphate (ATP) using gold nanoparticles (GNP) and PicoGreen fluorescence dye as signal transducer, and ATP and single-stranded DNA (DNA-M\u2032) as activators. The calculator-related performances including linearity, reaction time, logic gate, and selectivity were investigated, respectively. The results revealed that this oligonucleotide sensor was highly sensitive and selective. The detection range was 50\u2013500 nmol/L (R Many diseases, such as Parkinson\u2019s disease, ischemia, hypoxia, hypoglycemia, and some malignant tumors, are closely related to the concentration of ATP ,2,3. ConSignal amplification techniques, such as rolling circle replication  and straMolecular computers are very promising for applications in nanotechnology, medicine, and biotechnology. To date, certain math problems have been solved using DNA computers ,20. MoleFirstly, we used fluorescence spectroscopy to investigate the feasibility of the proposed assay. The differences between the peaks of the four curves and the peak of the a-curve in To further explore the feasibility of the assay principle, we monitored the reaction progress over time. y = 8.77379 \u00d7 10\u22124x \u2212 0.01613  with an R2 value of 0.993, and the limit of detection (LOD) was 46.5 nmol/L (3\u03c3/slope). Therefore, within this linear range, this equation can be used to quantitatively estimate the ATP concentration. Compared with most other methods  were evaluated under the same experimental conditions. To validate the viability of the proposed assay, we conducted standard addition recovery tests by adding ATP to samples of human urine and serum. As shown in Adenosine, adenosine diphosphate (ADP), adenosine monophosphate (AMP), Cytidine triphosphate (CTP), ATP, guanosine triphosphate (GTP), uridine triphosphate (UTP), and PicoGreen dye were supplied by Shanghai Yi Sheng Technology Co., Ltd. . All oligonucleotides were purchased from Beijing Genomics Institute . The DNA sequences are listed in 2, 50 mmol/L NaCl, pH 7.5). The volumes of buffer solution, PicoGreen (200-fold dilution), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and GNPs (10-fold dilution) were 1800 \u00b5L, 30 \u00b5L, 90 \u00b5L, 100 \u00b5L, and 10 \u00b5L, respectively, in all experiments. Components in each experiments illustrated in All reagents were diluted in buffer solution . The fluorescence spectra were acquired with an excitation wavelength of 480 nm and a detection range from 495 to 700 nm. The reaction progress was monitored using an excitation wavelength of 480 nm and an emission wavelength of 520 nm.Fresh human serum samples were supplied by Nanjing China Senbeijia Biological Technology Co., Ltd., which did not do any other pretreatment. We obtained urine from a healthy adult and did not add any detergent. The urine sample was centrifuged at 12,000 rpm for 2 min and the supernatant was diluted tenfold with buffer solution. ATP was then added to the diluted urine and fresh human serum samples at concentrations of 100, 300, and 500 nmol/L, and each measurement was performed in triplicate. Finally, the ATP recovery was calculated.We developed a label-free-fluorescence-based and enzyme-free AND DNA calculator for the sensitive and convenient determination of ATP concentration. This AND DNA calculator has many important features. First, the GNP-based DNA calculator partially eliminates the background signal and improves the sensitivity of the sensor. Second, this DNA calculator does not require fluorescent labeling or enzymes, leading to a lower assay cost. Third, this DNA calculator is highly selective for ATP in the presence of similar small molecules  and affords a lower detection limit compared with most of the previously reported methods. Most importantly, this DNA calculator can be successfully applied to the detection of ATP in urine samples, demonstrating its immense potential for practical clinical diagnosis. Owing to these advantages, the proposed method could also be extended to facilitate the detection of other small molecules and proteins in a label-free and enzyme-free manner."}
+{"text": "This method for the detection of ATP has the characteristics of being simple, low cost, and highly sensitive.In this study, a label-free fluorescent, enzyme-free, simple, highly sensitive AND logic gate aptasensor was developed for the detection of adenosine triphosphate (ATP). Double-stranded deoxyribonucleic acid (DNA) with cohesive ends was attached to graphene oxide (GO) to form an aptasensor probe. ATP and single-stranded DNA were used as input signals. Fluorescence intensity of PicoGreen dye was used as an output signal. The biosensor-related performances, including the logic gate construction, reaction time, linearity, sensitivity, and specificity, were investigated and the results showed that an AND logic gate was successfully constructed. The ATP detection range was found to be 20 to 400 nM (R Adenosine triphosphate (ATP) is a direct source of energy in the body and has a great influence on biological processes ,2. Many Fluorescent biosensors have been widely used to detect substances such as ions, nucleic acids, organic molecules, and proteins ,7,8. TheCompared with signal amplification, the background signal suppression method can lead to the design of a sensor with a simpler structure. The inhibition of the background signal can be achieved using graphene oxide (GO) ,19,20, gThe application of molecular computers in medicine, nanotechnology, and biotechnology is very significant. Molecular logic gates form the hardware basis of molecular computers and can perform various molecular Boolean operations. Up to now, many logic gates have been constructed, such as AND, INH+NINH, and INHIBIT ,31,32. AATP, cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and PicoGreen dye were purchased from Shanghai Yi Sheng Technology Co., Ltd. . All oligonucleotides were supplied by Beijing Genomics Institute . The sequences of DNA-T, DNA-M, and DNA-M\u2019 are listed in detail in 2, pH 7.5 and ultradeionization). 1 \u00b5M DNA-T and 1 \u00b5M DNA-M were mixed and the resulting mixture was then heated to 95 \u00b0C for 5 min. Subsequently the mixture was cooled to room temperature in a furnace before finally being stored in a refrigerator at 4 \u00b0C. DNA-M\u2032 was also heated to 95 \u00b0C for 5 min, and was then placed in a refrigerator at about \u221220 \u00b0C for 5 min, and was then finally stored at 4 \u00b0C in the refrigerator. the PicoGreen dye was diluted to 200-fold by buffer. The GO was diluted to 100-fold by buffer. In all of the experiments, the total amount of buffer used was 1825 \u00b5L, the 1 \u00b5M DNA-T and DNA-M mixture totaled 88 \u00b5L, the amount of 1 \u00b5M DNA-M\u2032 used was 90 \u00b5L, the spectral detection of PicoGreen dye was achieved using 40 \u00b5L of dye, the reaction time of the detection of the PicoGreen dye was achieved using 30 \u00b5L of dye, and a total of 10 \u00b5L of GO was used. Before each experiment, the buffer and reagent were placed in a 2 mL sample tube, and the reaction mixture was kept for at least 3 h at room temperature.All reagent dilutions were performed using a buffer . The urine samples were centrifuged at 13,000 r/min for 3 min. The resulting supernatants were subsequently diluted to 10-fold with buffer solution and the pH of the resulting solutions was 7.5. ATP was added in diluted urine at concentrations of 100, 300 and 500 nM, respectively, repeating the measurement three times. The recovery was then computed.The change in the fluorescence intensity of the sensing liquid in a 10 mm square quartz cuvette was recorded using a fluorescence spectrometer  with magnetic stirring. The spectral detection parameters used were an emission wavelength of 480 nm and a scanning range of 495 nm to 700 nm. The reaction time program detection parameters used were an emission wavelength of 480 nm and an excitation wavelength of 520 nm.ATP and DNA-M\u2032 were used as input signals, and the fluorescence intensity change of PicoGreen was used as an output signal to construct an AND logic gate, a schematic overview of which is shown in In order to verify the feasibility of the AND logic gate, spectral analysis was performed, as shown in To further construct the logic gate, ATP and DNA-M\u2032 were used as input signals. When they were present, the input signal was 1, otherwise it was 0. The differences between the peaks of the four curves in To further verify the feasibility of the principle, timed detection was carried out, as shown in 0, and the other curve peaks were used as the final F values. The linearity is shown in 2 was found to be 0.9943, which proves that the sensor has very high linearity. The linear equation was Y = 0.0278 + 0.00181X and the measurement range was found to be 20\u2013400 nM. In this linear range, the concentration of target ATP can be estimated quantitatively using an equation. The limit of detection (LOD) was 142.6 pM (3\u03c3/slope). Compared with the most of the other literatures , it can be seen that the 20 nM ATP sensitivity was lower than that of the 50 nM ATP sensitivity. From 0\u00b7C), it can be deduced that their sensitivity is less than that of the 50 nM of ATP. The sensitivity range of the sensor was found to be 1.846 \u00d7 106 to 2.988 \u00d7 106 M\u22121, as shown in In order to verify the practicability of the method, ATP was added into human urine in order to carry out standard recovery tests. As shown in In summary, a label-free, fluorescent, enzyme-free, highly sensitive AND logic gate biosensor was developed for ATP detection. By suppressing the background signal with GO, the sensitivity of the sensor was improved. The sensor was found to have high sensitivity and linearity and was able to detect lower concentrations of ATP with strong selectivity. Therefore, a simple, low-cost, highly sensitive method for the detection of ATP was developed that can be used as a universal detection method for the detection of ions, proteins, and other substances."}
+{"text": "Effective exchange of information about genetic variants is currently hampered by the lack of readily available globally unique variant identifiers that would enable aggregation of information from different sources. The ClinGen Allele Registry addresses this problem by providing (1) globally unique \u201ccanonical\u201d variant identifiers (CAids) on demand, either individually or in large batches; (2) access to variant\u2010identifying information in a searchable Registry; (3) links to allele\u2010related records in many commonly used databases; and (4) services for adding links to information about registered variants in external sources. A core element of the Registry is a canonicalization service, implemented using in\u2010memory sequence alignment\u2010based index, which groups variant identifiers denoting the same nucleotide variant and assigns unique and dereferenceable CAids. More than 650 million distinct variants are currently registered, including those from gnomAD, ExAC, dbSNP, and ClinVar, including a small number of variants registered by Registry users. The Registry is accessible both via a web interface and programmatically via well\u2010documented Hypertext Transfer Protocol (HTTP) Representational State Transfer Application Programming Interface (REST\u2010APIs). For programmatic interoperability, the Registry content is accessible in the JavaScript Object Notation for Linked Data (JSON\u2010LD) format. We present several use cases and demonstrate how the linked information may provide raw material for reasoning about variant's pathogenicity. Our immediate motivation came from the need for unique variant identifiers, particularly for variants not previously registered in ClinVar and for the variants undergoing pathogenicity assessment by the ClinGen\u2010supported expert panels. Assessment of a variant's pathogenicity frequently requires information derived from literature, population sequencing databases, high\u2010throughput experiments, curated databases such as ClinVar  Allele Registry. The Registry provides globally unique \u201ccanonical\u201d variant identifiers (the \u201cCAids\u201d) on demand via web (UI or API) services. A user\u2010friendly web interface provides various ways to query existing and register new variants. The canonical identifiers may be obtained either individually or in high volume via web APIs to meet the registration needs of external databases of any size. The canonicalization service is provided using a custom in\u2010memory index that is based on the alignment of hundreds of thousands of transcripts and genomic sequences. For each canonical identifier, there is typically a multiplicity of HGVS notations for the same variant in the context of common genomic and transcript reference sequences. The Registry also provides web UIs to support identification and registration of individual variants from partial descriptions present in the literature, genetic test reports, or in other sources.Another important Registry feature is the support for linking to information about registered variants in external sources frequently used by clinicians, diagnostic laboratories, and researchers. The Registry currently links to major resources such as gnomAD, ExAC  facilitate the linking of variant information by providing globally unique \u201ccanonical\u201d variant identifiers on demand. To identify groups of equivalent variant representations and assign them a unique canonical identifier, the Registry normalizes variant representations within individual reference sequences and maps them across known reference sequences, including different versions of genome assemblies, assembled loci, and transcripts. The amino acid sequences are not stored or aligned explicitly\u2014they are computed on\u2010the\u2010fly from protein\u2010coding transcript sequences. The registry provides search functionality to help identify a variant using common types of variant\u2010identifying information. A service is available for registering new variants, either individually or in batches. Registration requires a login that is instantly created on demand.Allele Registry services . Briefly, a contextual allele is defined in the context of a specific reference sequence. All contextual alleles corresponding to a same variant are associated with the same canonical allele. Each canonical allele has its own \u201cCAid\u201d number  and persistent and dereferenceable universal resource identifier (URI)  that serves as a Globally Unique Identifier (GUID) for the variant. The maximal nucleotide (transcript or genomic) allele size is 10,000\u00a0bp, a cutoff selected based on tradeoff between the need to accommodate large alleles and the need to efficiently store and process them. The Registry also allows for \u201ccomplex\u201d alleles including haplotypes ; however, it treats each haplotype as a single variant, not modeling individual variants that constitute a haplotype explicitly. The nucleotide (genomic and transcript) and protein variants are treated as different types of entities that may be joined by a one\u2010to\u2010many relationship .The Registry is based on the Allele Model developed by the ClinGen Data Model Working Group  files. The UIs are implemented in Ruby as Genboree plugins. Registration of new variants and of new sources of information about them requires authentication and authorization, whereas a query does not. Any user is currently authorized to register variants upon creating an account, a process that can be completed in less than 1\u00a0min. Although currently not implemented, Allele Registry will support widely used open authentication systems in future.The overall data flow of the Registry is summarized in Figure\u00a02.2The Registry parser takes as input either VCF representations or HGVS expressions representing the following four types of nucleotide variants: single nucleotide variants (SNVs), insertions, deletions, and indels  of contextual alleles in the context of a specific reference sequence, the canonicalization step provides a representation for nucleotide variants across multiple references Figure\u00a0. Because2.5The reference database consists of alignments of reference nucleotide sequences from key genomic databases  and supports the validation, normalization, and canonicalization steps. The nucleotide sequences are used as aligned against the latest human genome assembly, currently set to GRCh38. The alignments of reference sequences were imported from authoritative sources including EBI, NCBI, and the LRG consortium. To ensure high\u2010bandwidth operation and low\u2010latency response, the majority of sequences and alignments are represented as transformations\u2014consisting of edit operations according to the imported alignments\u2014of the latest human genome assembly, as described in detail below. This approach speeds up the mapping of alleles across different reference sequences and compresses the sequences and alignments so that the whole database can be kept in memory for fast access. To support a variety of queries, the database also includes all major reference sequence identifiers and sequence annotations such as exons, intron/exon boundaries, and coding sequences. Relations between nucleotide variants and their corresponding amino acid variants are computed on\u2010the\u2010fly.The largest part of the reference database is the GRCh38 assembly stored as FASTA format (roughly 3 GB). Other genomic sequences, such as older assemblies , transcripts, and gene regions, are encoded as key attributes that define their alignment to the GRCh38 reference. These attributes include (1) the GRCh38 locus to which the given reference sequence is aligned, (2) A CIGAR (Concise Idiosyncratic Gapped Alignment Report) string describing the alignment, (3) list of sequences corresponding to insertions or mismatches in the CIGAR string, and (4) sequence corresponding to the unaligned part of the sequence. Rather than being directly stored, protein sequences are calculated on the fly from source transcript sequences. The whole reference database currently occupies \u223c4 GB of memory.2.6The allele database stores variant definitions and identifiers from major resources . It is composed of a custom low\u2010level key\u2010value database engine with several indices that support fast querying. The database engine fulfills ACID  requirements, allowing the Registry to function as an OLTP (On\u2010Line Transaction Processing) system that supports real\u2010time registration of new variants.2.7The allele database Section  includes2.8https://reg.clinicalgenome.org)\u2014are freely available for public use. The source code is distributed under a GNU Affero GPL v3.0 license and is available at https://github.com/BRL-BCM.The Allele Registry services\u2014web app and APIs  for more than 650 million distinct variants, most originally identified in major variant databases, including gnomAD, ExAC, dbSNP, MyVariant.info, COSMIC, and ClinVar  currently offers 11 variant query options, one of which (\u201cHGVS\u201d) is illustrated in Figure\u00a0a. Queries using a canonical allele identifier (\u201cCAid\u201d) or HGVS expressions return unique results, while other queries  may result in more than one matching variant . The interface also generates variants that are not yet registered. Such variants may be immediately registered by a single click and their CAids or HGVS expressions may subsequently be used for their unambiguous identification, for example when performing variant classification.Some of the current and large part of older literature include incomplete references to variants that include a gene name and partial \u201cmutation\u201d description without a transcript or genomic reference sequence identifier. This poses a problem for generating an HGVS expression or variant descriptions in VCF format. This incomplete allele identification is a critical issue as variant classification for rare diseases often relies on data contained in the medical literature . Although a simple query does not require a login, registration does. Registry accounts are readily available through the web interface. An interface dedicated to bulk registration of thousands of variants using as an input a list of HGVS expressions is also available. Query and registration of millions or more variants per batch are best accomplished via the Registry APIs, as we describe next.A query for an individual variant or for multiple variants in the batch mode may identify variants that are well defined but absent from the Registry. In this case, a user has an option to register the variants Figure\u00a0b. Altho3.4The use of web APIs is the preferred interoperability method for regular and automated interactions with Registry services. The APIs are indispensable for query and registration of large number (millions) of variants: all variants, even from large resources such as dbSNP and myVariant.info can be queried or registered within 1 or 2 hr Table\u00a0.The ClinGen Pathogenicity Calculator  or an annotated VCF file format.3.5Several variant\u2010centric tools and databases currently interoperate with the Registry. ClinGen's Variant Curation Interface, CiVIC , and 450 thousand variants per minute for ClinVar. The largest percent (0.52%) of duplicate entries was found in dbSNP, a significantly lower (0.03%) in MyVariant.Info, while in ClinVar no duplicate records were found; it is consistent with the extensive curation performed by the NCBI staff of variants submitted to ClinVar before they provide a ClinVar identifier  were submitted using the stepwise process described in Supporting Information Sections 1.1\u20131.3. The Registry processed 1.3 million variants per minute for dbSNP indels, 4.6 million variants per minute for MyVariant.Info . Although there are 781 variants with consistent assertions of the that result in the same alternate sequence, we identified 65 with potentially clinically relevant discordant assertions in ClinVar Table\u00a0. For exa4The ClinGen Allele Registry enables effective exchange of information about human genetic variants by providing globally unique \u201ccanonical\u201d variant identifiers on demand. The web interfaces (web UIs) provide support for a number of key use cases, including variant query and registration, either individually or in large batches. The Registry cross\u2010references information about variants across major databases by collating alternate variant identifiers and supports publication and sharing of links to information about variants in any external source, large or small. The Registry is designed to scale to billions of variants and to meet the needs of all global sequencing projects and even computational prediction algorithms that output information about variants that are yet to be observed in humans or in vitro.The Registry is accessible programmatically via well\u2010documented web APIs in accordance with recently articulated FAIR  principles . This is a special case of a variant that can be described as a set of variants within a haplotype. In its present form, the registry currently does not explicitly model haplotypes and treats each distinct haplotype as a distinct variant. Finally, the registry also assumes that each variant is described at the base pair level of resolution and does not support variants such as CNVs that may not be described at that level of precision. Continuous development in coordination with key stakeholders are in process toward overcoming these limitations.In summary, the Registry web services create an innovative nexus for effective exchange and aggregation of information about human genetic variants, thus catalyzing the emergence of a commons of variant data and knowledge required for the advancement of genome research and the genomic medicine.SEP is a member of the Baylor Genetics Scientific Advisory Panel. AM is an employee of BCM and performs integration consulting services for BCM\u2010developed software including Genboree through IP Genesis, Inc. LB is employed by Sunquest Information Systems company. Sunquest is a commercial laboratory software vendor. Other authors do not have any conflicts of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Supplemental InformationClick here for additional data file."}
+{"text": "Age-related height loss is a normal physical change that occurs in all individuals over 50\u2009years of age. Although many epidemiological studies on height loss have been conducted worldwide, none have been long-term longitudinal epidemiological studies spanning over 30\u2009years. This study was designed to investigate changes in adult spinal deformity and examine the relationship between adult spinal deformity and height loss.Fifty-three local healthy subjects  from Furano, Hokkaido, Japan, volunteered for this longitudinal cohort study. Their heights were measured in 1983 and again in 2017. Spino-pelvic parameters were compared between measurements obtained in 1983 and 2017. Individuals with height loss were then divided into two groups, those with degenerative spondylosis and those with degenerative lumbar scoliosis, and different characteristics were compared between the two groups.p\u2009<\u20090.05). Height loss in both sexes was related to changes in pelvic parameters including pelvic incidence-lumbar lordosis , pelvic tilt , and sagittal vertical axis . Greater height loss was more commonly seen in women (p\u2009=\u00a00.001) and in patients with degenerative lumbar scoliosis (p\u2009=\u00a00.02).The mean age of the subjects was 44.4 (31\u201355) years at baseline and 78.6 (65\u201389) years at the final follow-up. The mean height was 157.4\u2009cm at baseline and 153.6\u2009cm at the final follow-up, with a mean height loss of 3.8\u2009cm over 34.2\u2009years. All parameters except for thoracic kyphosis were significantly different between measurements taken in 1983 and 2017 (This longitudinal study revealed that height loss is more commonly observed in women and is associated with adult spinal deformity and degenerative lumbar scoliosis. Height loss is a normal physical change with aging, but excessive height loss is due to spinal kyphosis and scoliosis leading to spinal malalignment. Our findings suggest that height loss might be an early physical symptom for spinal malalignment. Population aging is seen worldwide. Thus, it is important that an appropriate quality of life (QOL) is maintained for the entire lifespan of any individual. Adult spinal deformity (ASD) can affect the sagittal alignment of the spine, which may impact the QOL \u20133. The uIndividuals with ASD will receive lower scores in all domains of the 36-Item Short Form Survey as they age, while in other chronic conditions such a decline with age is not observed \u20139.Height loss is an age-related physical change that occurs in all individuals over 50\u2009years and that elderly people are aware of , and is Although both ASD and height loss are associated with aging, no study has examined the relationship between height loss and ASD, based on the abovementioned parameters. Besides, many epidemiological studies were conducted on height loss worldwide; however, over the 30\u2009years longitudinal no epidemiological studies have been conducted in this field. The purpose of this 34-year longitudinal cohort study was to investigate the relationship between ASD and height loss in community-based volunteers and to determine the effects of sex.This study is a follow-up of an epidemiological study by Takemitsu et al. in 1983, in Furano, Hokkaido, Japan [The inclusion criteria were as follows: no spinal or major joint surgery between 1983 and 2017, ability to walk independently to our hospital for the present study, and aged less than 55\u2009years in 1983 in consideration of current age.The exclusion criteria were being unable to stand in the correct posture during radiography, unable to tolerate the pain, osteoporosis fracture, paralysis of trunk or lower extremity, and not consenting to the study. Figure\u00a0The subjects underwent radiography of the entire spine in an upright position in 1983 and 2017. The participants were instructed to stand in a relaxed position and look straight ahead, with their fists at the level of the clavicle.The heights of all subjects were measured in meters without their shoes, with the back of their head, buttocks, back, and heels against an upright board. The back muscle was stretched as much as possible, and feet were supinated by about 30\u00b0. Participants with height loss were then selected and divided into two groups: those with \u22644\u2009cm  and those with >\u20094\u2009cm  loss of height.Standardized radiographic measurements of sagittal spino-pelvic parameters included thoracic kyphosis (TK), lumbar lordosis (LL), PT, pelvic incidence (PI), sacral slope (SS), and SVA , 21 . All data are expressed as mean and range. Variables that were normally distributed were analyzed using the Shapiro-Wilk test. When measured variables were found to have a non-normal distribution, Spearman\u2019s correlation was used to evaluate the association between height loss and spino-pelvic parameters. Sub-analyses were performed to evaluate the association between height loss and sex, DS, and DLS.P-value of <\u20090.05 was considered statistically significant.In comparing the height loss between the 2 groups, if lack of normality was rejected, a t-test was performed. If the lack of normality was significant, the Wilcoxon signed rank test or Mann-Whitney U-test was performed, as appropriate. A The mean age of the subjects was 44.4 (31\u201355) years at baseline and 78.6(65\u201389) years at the final follow-up. The mean follow-up period was 34.2 (34\u201334.4) years. The mean height of the subjects was 157.4 (140\u2013170) cm at baseline and 153.6(132\u2013169) cm at the final follow-up, with a mean height loss of 3.8\u2009cm over 34\u2009years.A summary of longitudinal changes in radiographic variables from baseline to the final follow-up is presented in Table\u00a0Spino-pelvic parameters including PI-LL, PT, and SVA were significantly associated with height loss in both male and female subjects Table\u00a0. Based oTo the best of our knowledge, this 34-year longitudinal cohort study is the first to examine the relationship between ASD and height loss. We observed that height loss was significantly correlated with changes in the sagittal modifiers of the SRS-Schwab classification [There was no significant change in the values for TK over 34\u2009years, which is similar to the findings of Kobayashi et al. who also reported small changes in TK over their study period of 12.3\u2009years , which mAging-related spinal changes begin with degenerative inter-vertebral disk changes . Later, The main cause for DLS is disc degeneration, leading to decreased disc height and the progression of DLS. Faraj et al. provided strong evidence that increased disc degeneration in DLS, the first sign of height loss, leads to progression of the lumbar scoliosis curve . In the Furthermore, it is reported that LL decreases and SVA increases as degenerative scoliosis progresses , with prIn our study, height loss was more pronounced in women, which might be because of the differences in the skeletal structure between the sexes. It should be noted that these differences may be observed over time; however, to date, no prospective studies have confirmed this change, although Takemitsu et al. have reported a higher prevalence of lumbar degenerative kyphosis in women in a previous study , which mThe complication rate of ASD surgery is high , 36, andOne of the strengths of our study is that height was measured accurately in both times, while in previous reports, participants recalled their previous heights from memory, which might have led to inaccurate height recording . Our stuWe observed that height loss was related to ASD and DLS. Height loss was more prominent in women than in men. Height loss is a normal physical change with aging, but excessive height loss is mainly due to the occurrence of spinal kyphosis and scoliosis leading to spinal malalignment. Hence, height loss may be considered as a physical symptom for spinal malalignment."}
+{"text": "Cryptosporidium genome (\u223c9 Mb) has over 20 copies of genes encoding insulinase-like proteases (INS), suggesting that these enzymes may have important biological functions in the pathogen and could be developmentally regulated. In this study, INS-5, a unique member of the INS family in Cryptosporidium parvum, was cloned and expressed in Escherichia coli BL21 (DE3). In addition to the predicted INS-5 of \u223c78 kDa, smaller fragments of \u223c70, \u223c55, and \u223c30 kDa were simultaneously generated. After purification through a nickel-nitrilotriacetic acid affinity column, the full recombinant protein obtained was used to prepare polyclonal antibodies. Antibodies raised against INS-5 recognized the recombinant protein and native protein in sporozoite extracts. Further characterization of INS-5 included qRT-PCR assessment of gene expression; immunofluorescence localization of the protein expression in sporozoites, merozoites, and other developmental stages; and neutralization of invasion of C. parvum in vitro. The results obtained indicated that although INS-5 was expressed in sporozoites and merozoites, the high gene expression was from 36 to 48 h of the in vitro culture after invasion. Anti-INS-5 antibodies partially neutralized the invasion (inhibition rate = 38.5%). Results of this study suggest that INS-5 plays some role in the invasion and growth of C. parvum.The small  Cryptosporidium spp. are apicomplexan parasites of the gastrointestinal epithelium, causing diarrhea in humans and various animals   . In partes (INS) . Most ofium spp. .Vibrio vulnificus secretes a novel insulinase, SidC, which contributes to the proliferation of this human bacterial pathogen  were purchased from Waterborne, Inc.  and stored in antibiotics at 4\u00b0C for less than 2 months before use. They were treated with 0.5% sodium hypochlorite for 10 min on ice and exposed to excystation solution containing 0.75% taurodeoxycholic acid and 0.25% trypsin at 37\u00b0C for 1 h to obtain free sporozoites. Human ileocecal adenocarcinoma HCT-8 cells (ATCC CCL-244) were obtained from the Shanghai Branch of the Chinese Academy of Sciences and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin\u2013streptomycin solution (PS) at 37\u00b0C under 5% CO2. The pET28a vector was obtained from Novagen, Inc. , while Escherichia coli strains DH5\u03b1 and BL21 (DE3) were obtained from Tiangen Biotech .cgd2_2760 gene from the genomic DNA of the C. parvum IOWA isolate. The primers used included 5\u2032-CGGGATCCATGACAGCTAATGGGAA-3\u2032 (the added restriction site BamHI is underlined) and 5\u2032-ACGCGTCGACAAGACCTAAGTCGC-3\u2032  and were synthesized by Sangon Biotech, Co., Ltd. . The template DNA was extracted from C. parvum oocysts by using the QIAGEN DNeasy Blood & Tissue Kit . The PCR cycle included one cycle of 95\u00b0C for 5 min; 35 cycles of 95\u00b0C for 45 s, 55\u00b0C for 45 s, and 68\u00b0C for 2 min; and one cycle of 68\u00b0C for 7 min. The PCR was performed in a GeneAmp 9700 . The amplified PCR product was purified using the SanPre PCR Product Purification Kit (Sangon Biotech) and digested with restriction enzymes BamHI and SalI. The resulting fragment was inserted between the BamHI and SalI restriction sites of pET28a vector to generate recombinant plasmid cgd2_2760-pET28a, which was transformed into the competent E. coli DH5\u03b1. Positive colonies selected on LB agar with kanamycin were identified by PCR and sequenced to confirm their identity and sequence accuracy.Polymerase chain reaction (PCR) was used to amplify the cgd2_2760-pET28a plasmid was isolated from E. coli DH5\u03b1 using the EZ-10 Spin Column Plasmid DNA Minipreps Kit (Sangon Biotech). E. coli BL21 (DE3) cells were transformed with the plasmid and grown in LB medium supplemented with 100 \u03bcg/ml of kanamycin at 37\u00b0C. When the A600 of the culture reached 0.6\u20130.8, the expression of the recombinant INS-5 protein was induced by adding 0.5 mM isopropyl-\u03b2-D-thiogalactoside (IPTG). The E. coli cells were cultured at 16, 25, and 37\u00b0C for another 5 h to identify the most favorable temperature for the expression of the recombinant protein. This led to the section of 25\u00b0C as the optimal temperature.The E. coli BL21 cells were inoculated into 1-L medium and cultured as described above. The cells were harvested by centrifugation at 4\u00b0C and 8,000 \u00d7 g for 20 min, resuspended in phosphate-buffered saline (PBS) at pH 7.4, and lysed by sonication on ice. The insoluble components were collected by centrifugation at 4\u00b0C and 10,000 \u00d7 g for 20 min. The pellet containing inclusion bodies was dissolved in 8M urea. After centrifugation at 4\u00b0C and 10,000 \u00d7 g for 20 min, the supernatant containing the denatured recombinant protein was filtered through a 0.45-\u03bcm cellulose acetate membrane filter  and applied to a 1-ml nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Novagen). The column was washed with five volumes of equilibration buffer and eluted with six volumes of 500 mM imidazole in 8M urea. These steps were repeated six times to obtain enough target protein of high purity. The renaturation of the purified protein was achieved by gradient dialysis from urea solution to PBS buffer at 4\u00b0C. The renatured protein was concentrated by ultrafiltration with Amicon\u00ae Ultra-15 10K Centrifugal Filter Devices (Millipore) and assessed for purity using SDS-PAGE stained with Coomassie Brilliant Blue and western blot analyses. The bands of the expected size and lower-molecular-weight species were excised from the gel and analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).For the production of recombinant protein, Polyclonal antisera were raised by GenScript, Co., Ltd.  using immunization of rabbits with the purified INS-5 protein of \u223c78 kDa dissolved in PBS buffer at the concentration of 0.35 mg/ml. The sera were collected from the immunized rabbits and used in the purification of polyclonal IgG antibodies through an affinity chromatographic column conjugated with recombinant INS-5. The final concentration of polyclonal antibodies was 0.4 mg/ml. The specificity of the antibodies was evaluated using western blot analysis.Western blot analysis was used to assess the purity of the recombinant INS-5 protein. The INS-5 protein was electrophoresed on 10% SDS polyacrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% non-fat milk in PBS and 0.5% Tween 20 (PBST) for 2 h, the membrane was probed with anti-His tag antibodies  at 1:1,000 dilution for 2 h. After incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies  at 1:5,000 dilution for 1 h, the antigen\u2013antibody complex was visualized using the DAB kit (Tiangen Biotech). The membrane was washed three times with PBST after each incubation.C. parvum oocysts was also assessed by western blot analysis (\u223c5 \u00d7 106 oocysts/lane), using the procedure described above. Anti-INS-5 polyclonal antibodies (0.16 \u03bcg/ml), immune sera  or pre-immune sera , were the primary antibodies, and HRP-conjugated goat anti-rabbit antibodies (Yeasen)  were the secondary antibodies in western blot analysis. Crude native proteins were released from free sporozoites after boiling them in 5 \u00d7 protein loading buffer for 5 min.The native INS-5 extracted from C. parvum oocysts that had been treated with 0.5% sodium hypochlorite as described above. The cultures were maintained at 37\u00b0C for 24 or 48 h to examine intracellular stages and for 36 h to collect free merozoites from the culture. The infected cultures, merozoites, oocysts, and free sporozoites were fixed at room temperature with methanol for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 15 min. After blocking of non-specific binding with 5% BSA in PBS at room temperature for 1 h, the slides were incubated with anti-INS-5 antibodies (0.4 \u03bcg/ml) in 5% BSA\u2013PBS for 1 h and Alexa Fluor\u00ae 594-conjugated goat anti-rabbit IgG  in BSA\u2013PBS at 1:400 dilution for another hour. After counterstaining with the nuclear stain 4\u2032,6-diamidino-2-phenylindole  at 1:2,000 dilution for 10 min, the slides were mounted with No-Fade Mounting Medium  and examined using differential interference contrast (DIC) and fluorescence microscopy on a BX53 microscope .HCT-8 cells were seeded into a plastic 12-well plate with coverslips, grown to 40\u201350% confluence, and exposed to cgd2_2760 gene in developmental stages of C. parvum. Data on the expression of C. parvum 18S rRNA (Cp18S rRNA) gene were used in data normalization. In this analysis, HCT-8 cells were seeded into a 12-well plate, grown to 80\u201390% confluence, and exposed to sodium hypochlorite-treated C. parvum oocysts. After the invasion, infected HCT-8 cells were collected at 2, 6, 12, 24, 36, 48, and 72 h of the in vitro culture. Total RNA was extracted from wells using an RNeasy Mini Kit (Qiagen) and measured for concentration using a NanoDrop 2000 . cDNA was synthesized from 1 \u03bcg of the extracted RNA using a GoScriptTM Reverse Transcription System  and analyzed by qPCR in a LightCycler\u00ae 480 (Roche), with the following cycling conditions: 95\u00b0C for 3 min and 45 cycles of 95\u00b0C for 30 s, 58\u00b0C for 30 s, and 72\u00b0C for 30 s. The 20-\u03bcl qPCR reaction contained 1 \u03bcl of cDNA, 0.1 mM primers, and 10 \u03bcl of SYBR Green PCR Mix . The primers used included 5\u2032-GAATTTGGAATTTGGGATGC-3\u2032 and 5\u2032-GCTCGCATGAATCATTTTGA-3\u2032 (amplicon size = 220 bp) for the analysis of the cgd2_2760 gene, and the published qPCR primers 5\u2032-CTC CAC CAA CTA AGA ACG GCC-3\u2032 and 5\u2032-TAG AGA TTG GAG GTT GTT CCT-3\u2032 (amplicon size = 256 bp) from the Cp18S rRNA gene (\u2013\u25b3\u25b3CT method (Quantitative PCR (qPCR) was used to evaluate the expression of the RNA gene . The datT method .Cryptosporidium parvum oocysts treated with 0.5% sodium hypochlorite were incubated at 37\u00b0C for 15 min in medium containing pre-immune serum or immune serum diluted at 1:100, 1:200, 1:500, and 1:1,000, with medium alone as controls. Oocysts and excysted sporozoites in 100 \u03bcl were seeded onto HCT-8 cells cultured to 80\u201390% confluence in a 12-well plate (with 900 \u03bcl/well of culture medium). After 2 h of incubation, the HCT-8 cell cultures were washed with PBS buffer and maintained in fresh culture medium for an additional 24 h. The cells were then fixed and permeabilized and blocked for non-specific binding as described above. Afterward, they were stained with Cy3-labeled Sporo-GloTM antibodies (Waterborne). The intensity of C. parvum infection in HCT-8 cells was measured by immunofluorescence microscopy, after the slides were mounted with a No-Fade Mounting Medium (Booster). Fifty visual fields were randomly selected and photographed under 200\u00d7, and the total number of parasites in each field was quantified using ImageJ 1.4.3.671. The neutralization efficiency of immune sera or antibodies at different dilutions was calculated by comparing the number of parasites between treated groups and controls in three replicate analyses. The data generated were compared using Student\u2019s t-test. Differences were considered significant at p \u2264 0.05.cgd2_2760 gene was successfully amplified by PCR from the genomic DNA of C. parvum using the forward and reverse primers with the added poly-histidine sequences (cgd2_2760-pET28a was transformed into E. coli BL21 (DE3) cells for the expression of INS-5 with a His-tag at both termini.The equences . The PCRE. coli BL21(DE3) cells transformed with the recombinant cgd2_2760-pET28a was in agreement with the predicted size of \u223c78 kDa  = 9.655, p = 0.011] at the dilution of 1:1,000, 29.0%  at the dilution of 1:500, 28.4%  at the dilution of 1:200, and 38.5%  at the dilution of 1:100 . Some of the small INS-5 products could be due to early termination of the protein translation because of the existence of several rare E. coli codons in the recombinant cgd2_2760-pET28a plasmid. The use of the Rosetta (DE3) strain, however, failed to increase INS-5 expression in E. coli. Another reason for the existence of small INS-5 fragments could be due to post-translational proteolysis of INS-5. Toxolysin 4, an INS in T. gondii, was shown to undergo proteolytic maturation (Cryptosporidium spp. further process INS in vivo requires validation. Multiple bands were also seen in the expression of human INS in E. coli (The INS-5 protein appears to have post-translational processing. Lower-molecular-weight species were observed in SDS-PAGE and western blot analyses of the protein when INS-5 was expressed exogenously in turation . In addi E. coli .Cryptosporidium INS members lack the active Zn-chelating \u201cHXXEH\u201d motif and thus are likely to be catalytically inactive copies (Cryptosporidium life cycle needs further research. INS genes in C. parvum are expressed at various levels and with different patterns (cgd2_930 gene, has been identified as a putative rhoptry protein of sporozoites (C. parvum, the biological functions of these INS proteins could be different from INS-5.It appears INS-5 may not have any classic INS activities. Although it is a member of the INS family, INS-5 contains only one inactive M16 domain, compared with four domains in classic INS, including the first domain that contains the enzyme activity site . Previoue copies . These Ipatterns , implyinpatterns . Anotherrozoites . In the C. parvum. Although INS-5 was observed to be expressed in the invasive stages of C. parvum by immunofluorescent microscopy and its gene showed the highest expression in later time points of the C. parvum infection by qRT-PCR analysis, the specific location of the protein remains unclear. Ultrastructural analysis is needed to determine the subcellular location of INS-5. Gene ablation or silencing is also needed in further assessing the role of INS-5 in the invasion and growth of C. parvum. This would allow better understanding of the developmental regulation and biological functions of INS proteins in Cryptosporidium life cycle stages.In summary, this preliminary study represents our initial attempts in characterizing the functions of INS-5 in All datasets generated for this study are included in the article/supplementary material.YF and LX conceived and designed the experiments. NN performed the experiments. RJ, YG, NL, and HW provided technical assistance. NN, YF, and LX analyzed the data. NN, RJ, YF, and LX wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "We propose a model that shows Miga interacts with Vap33 to mediate ERMCSs and excessive ERMCSs lead to neurodegeneration.Endoplasmic reticulum (ER)\u2013mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, and mitochondrial dynamics. However, the molecular organization, functions, regulation of ERMCS, and the physiological roles of altered ERMCSs are not fully understood in higher eukaryotes. We found that Miga, a mitochondrion located protein, markedly increases ERMCSs and causes severe neurodegeneration upon overexpression in fly eyes. Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga-mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters, which fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the  Intracellular organelles with membranes are characteristic features of eukaryotic cells. The compartmentalization of the cytoplasm enables specific cellular activities occur in restricted regions. The physical contacts between different organelles at defined loci allow both material and information exchange between organelles\u00a0. ER\u2013mitoERMCSs play various roles including phospholipid exchange and calcium flux between ER and mitochondria\u00a0. ERMCSs 2+ channel IP3R are a pair of molecules mediating ERMCSs in mammals\u00a0 which contains the outer membrane mitochondrial proteins Mdm10 and Mdm34, cytosolic protein Mdm12, and integral ER protein Mmm1\u00a0. The ER  mammals\u00a0. Althoug mammals\u00a0. ER prot mammals\u00a0. Recent es ERMCS\u00a0. In addies ERMCS\u00a0, Drp1\u00a0 of Miga1 and Miga2 are viable. Both Miga2 and Miga1/2 KO mice showed reduced body weight and body fat. Under high-fat diet consumption, Miga2 KO mice has minimal lipidosis\u00a0\u00a0. Miga coembranes\u00a0. Loss ofipidosis\u00a0. In a co in pigs\u00a0. In additentials\u00a0. Miga2 Kpression\u00a0. A most pression\u00a0.+/calmodulin-dependent protein kinase II (CaMKII) at multiple sites, which affect the interaction between Miga and Vap33 and fine-tuned Miga activity. The extra ERMCSs caused by overexpression of Miga or other tethering molecules led to severe neurodegeneration, which might shed light on the molecular mechanisms underlying neurodegenerative diseases such as Alzheimer\u2019s disease.In this study, we found that Miga interact with Vap33 to mediate ERMCSs. Miga is phosphorylated by casein kinase I (CKI) and Ca\u00b2GMR-Gal4, a Gal4 driver expressing in the developing eyes, to drive UAS-Miga expression and examined the adult fly eyes at days 1 and 30. The adult eyes with Miga overexpression looked grossly normal from outside. However, when we examine the retina with the transmission electron microscopy (TEM) analysis  in the cultured S2 cells with FLAG-HA tandem tagged overexpressed Miga and examined its binding partner with mass spectrometry . The ER ild type .FM) with the 247th phenylalanine and the 249th serine residues changed to alanine  flies. However, Miga animals . These dThe interaction between Vap33 and Miga increased ERMCSs and led to eye degeneration. We speculated whether the degeneration was caused by the increasing ERMCSs. In mammals, it has been reported that a protein called PTPIP51 could interact with VAPA/B and mediate ERMCSs\u00a0. There iMiga mutant third instar larvae. However, we did not observe any obvious ERMCS decrease in Miga mutants  with mutated FFAT motif to ensure that MigaFM expression levels and patterns mimicked those of endogenous Miga proteins. FMMiga genomic rescue transgene could rescue the Miga mutant fatality, but the life span of the FMMiga rescued flies was shorter than those of the ones with wildtype genomic rescue transgene   . These dWhen we performed the western blot to detect the interaction between Vap33 and Miga, we realized that Miga shows multiple bands and the higher bands have larger molecular weights than the predicted molecular weights . Intereshttp://gps.biocuckoo.cn/)\u00a0(http://www.cbs.dtu.dk/services/NetPhos/)\u00a0\u00a0 and NetPetPhos/)\u00a0 as potenr events\u00a0. We overr events\u00a0. We alsor events\u00a0. These dInterestingly, Ser246 and Ser249 in cluster V were two phospho-Ser residues located inside the FFAT motif. In the classical FFAT motifs, those two positions were the acidic amino acids E or D. We hypothesized that the phosphorylation of these two serine residues is required for the interaction between Miga and Vap33 and the phosphorylation provides an opportunity to regulate the ERMCS formation. Mutating Ser246 and Ser249 to Ala indeed abolished the interaction between Miga and Vap33  and failS246A, S249A when they were overexpressed in S2 cells  through its C-terminal region and is required for adipocyte differentiation. The LD interaction region of MIGA2 is conserved in fly Miga protein. It needs further investigation whether Miga plays a role in lipid metabolism in Drosophila.Although Miga overexpression greatly increased ERMCSs in both fly eyes and fat body tissues, the loss of Miga did not have obvious effects on the ERMCSs in fly fat body tissues. The ERMCSs (10\u201330 nm), especially the tight contacts (about 10 nm), are rare in the wild type fly fat body. It might be the reason for the difficulty to detect ERMCS reduction in this tissue. We introduced a mmatidia\u00a0. Each omFM was overexpressed, there is no increase of ERMCSs and the mitochondria length was also significantly shorter than that in the wildtype Miga overexpressed tissues , V5 was fused to the C terminus of Miga. pAC Miga I-V5 was generated by site-directed mutagenesis to change the 68th, 71st, 77th, 81st Ser residues to Ala. pAC Miga II-V5 was generated by site-directed mutagenesis to change the 84th, 87th, 89th, 93rd Ser residues to Ala. pAC Miga III-V5 was generated by site-directed mutagenesis to change the 98th, 102nd, 105th, and 107th Ser residues to Ala. pAC Miga IV-V5 was generated by site-directed mutagenesis to change the 122nd, 123rd Ser residues to Ala and the 126th, 129th Thr residues to Ala. pAC Miga V-V5 was generated by site-directed mutagenesis to change the 225th, 228th, 230th, 233rd, 237th, 243rd, 246th, and 249th Ser residues to Ala. pAC Miga VI-V5 was generated by site-directed mutagenesis to change the 299th Ser residue to Ala, 300th, 302nd Thr residues to Ala and 301st Asp residue to Ala. pAC Miga VII-V5 was generated by site-directed mutagenesis to change the 423rd, 426th , 427th Thr residues to Ala and 431st Ser residue to Ala. pAC Miga I-IIISA-V5 was generated by site-directed mutagenesis to change the 68th, 71st, 77th, 81st, 84th, 87th, 89th, 93rd, 98th, 102nd, 105th, and 107th Ser residues to Ala. pAC Miga I-IIISE-V5 was generated by site-directed mutagenesis to change the 68th, 71st, 77th, 81st, 84th, 87th, 89th, 93rd, 98th, 102nd, 105th, and 107th Ser residues to Glu. pAC-Miga 6S-A-V5 was generated by site-directed mutagenesis to change the 225th, 228th, 230th, 233rd, 237th, 243rd Ser residues to Ala. pAC-Miga 8S-A-V5 was generated by site-directed mutagenesis to change the 225th, 228th, 230th, 233rd, 237th, 243rd ,246th and 249th Ser residues to Ala. pUASVap33-FLAG, pUAS Vap33P58S-FLAG, pUAS CK1\u03b1 have been described before , a linker sequence (AEAAAKEAAAKEAAAKA), an RFP full length cDNA, and 39 amino acids sequence from FFAT motif region (229aa-267aa) of Miga into pAC-His-V5 vector. pUASattB-Tether was generated by cloning the mitochondrial targeting sequence of human mitochondrial protein Akap1 (MAIQFRSLFPLALPGMLALLGWWWFFSRKK), a linker sequence (AEAAAKEAAAKEAAAKA), an RFP full length cDNA, and 39 amino acids sequence from FFAT motif region (229aa-267aa) of Miga into pUASattB vector. pUASattB Miga-FLAG-HA was generated by cloning the full length Miga cDNA into pUASattB vector with a FLAG tag (DYKDDDDK) and a HA tag (YPYDVPDYA) fused to the C-terminal of Miga. The plasmid pcDNA3.1-MIGA2-HA was generated by cloning Miga2 cDNA into pcDNA3.1 vector with a 3xHA tag fused to the C-terminus. pcDNA3.1-MIGA2 mu-HA was generated by site-directed mutagenesis to change the 293rd, 294th Phe residues of MIGA2 to Ala . The plasmid pcDNA3.1-MIGA2-EGFP was generated by cloning Miga2 cDNA into pcDNA3.1 vector with a C-terminal EGFP tag. The plasmid pcDNA3.1-VAPB-V5 was generated by cloning VAPB cDNA into pcDNA3.1 vector with a C-terminal V5 tag. The plasmid pcDNA3.1-VAPA-HA was generated by cloning VAPA cDNA into pcDNA3.1 vector with a N-terminal 3xHA tag.The plasmid pUASattB-RFP was constructed by inserting cDNA of red fluorescent protein (RFP) into pUASattB vector through exogenous restriction sites of KpnI and XbaI. To generate pUASattB Miga-RFP, sequence . pattB Md before . The plaThe fly strains used in this study were listed in the 2 in Dulbecco\u2019s modified Eagle\u2019s medium . Both the culture mediums were supplemented with 10% fetal bovine serum  and 50 IU/mL penicillin/streptomycin . Plasmids were transfected by using lipo2000  as a transfection reagent. 48 hr after transfection, the cells were harvested and used for immunoprecipitation assay, western blotting or immuno-staining.S2 cells were originally from Invitrogen. HeLa cells and the U2OS cells were originally from ATCC. These cells were recently authenticated and tested for contamination.\u00a0S2 cells were cultured in a 25\u00b0C incubator in Schneider insect cell culture medium . HeLa cells and the U2OS cell lines stable transfected with ERMCS reporters were cultured in a 37\u00b0C incubator with 5% COFor drug treatment, 80 \u03bcM CKI inhibitor D4470  was added into the medium for 48 hr and DMSO was added as a control. For HBSS treatment, cells were cultured in HBSS  for indicated period before harvest. For CCCP  treatment, 10 \u03bcM CCCP was added to the culture medium for indicated period before harvest. For \u03bb-ppase treatment, the cells were harvested and lysed in lysis buffer  and incubated with \u03bb-ppase for 1 hr at 30\u00b0C before western blot analysis.Drosophila RNAi experiments were carried out as previously described. The dsRNA targeting CaMKII was generated by annealing reverse complimented RNA strains generated by in vitro transcription from a template amplified by PCR using following primers: CaMKII-F: 5\u2019-TAATACGACTCACTATAGGGGCAAAGTCCGCTTATTCTCGTTCTT-3\u2019; CaMKII-R: 5\u2019-TAATACGACTCACTATAGGGAATTCTTTGGCTCCCCTCATGC-3\u2019. dsRNA was transfected to the S2 cells with the liposome RNAi max . After 3 days, cells were transfected with pAC-Miga-V5. After 2 more days, cells were collected for real-time quantitative PCR and western blot. The primers used for the real time PCR are: CamKII-F: 5\u2019-ATCCCAACATAGTGCGGCTACATGA-3\u2019; CamKII-R: 5\u2019-AAGTCAGCGAGTTTCACTGCTGCA-3\u2019. RpLP0-F: 5'-CTAAGCTGTCGCACAAATGGC-3', RpLP0-R: 5'-ATCTCCTTGCGCTTCTTGGA-3'.The fat body tissues or the cultured cells were fixed in 4% paraformaldehyde  for 45 min, followed by permeabilizing in PBST ). Samples were incubated with primary antibody at 4\u00b0C overnight. After washing with PBST, samples were then incubated with secondary antibodies for 1 hr at room temperature in dark. After that, samples were mounted in 80% glycerol  with 5 ng/\u03bcL DAPI  followed by confocal microscopy .For the adult fly eyes, fly head was dissected and fixed in EM eye solutions , 4% paraformaldehyde , and 1% Glutaraldehyde , pH7.2.) for more than 48 hr at 4\u00b0C. Samples were then washed five times with Millipore water, and post-fixed with 2% osmium tetroxide  for about 2 hr. After rinsing five times with Millipore water, samples were gradually dehydrated through a graded series of ethanol . After that, samples were dehydrated in propylene oxide (PO)  for three times with 30 min for each time. And then, samples were embedded in Eponate 12 resin, which was made up from Embed 812 , DDSA , NMA  and DMP-30 . The samples were then cured in 65\u00b0C for 48 hr. For the fat body and the muscle tissues, the initial fixation solution is 2.5% glutaraldehyde  and the samples were washed with PBS after fixation. All the other steps were same as those used for the fly eyes. The samples were cut into 50 nm thin sections and stained with 4% uranyl acetate  and 2.5% lead nitrate  for electron microscopy analysis . For the semi-thin sections of muscle tissues, the samples were cut into 1.5 \u03bcm thin sections and stained with toluidine blue  and examine under a light microscopy .3VO4,1% NP-40, 10% glycerol, 1.5 mM EDTA pH 8.0 (for S2 cell)), supplemented with protease inhibitors PMSF, aprotinin, pepstatin, leupeptin, and phosphatase inhibitor PhosSTOP . Samples were centrifuged at 16000 g for 10 min and supernatant were collected and incubated with HA beads  or Flag beads  or V5 antibody followed with Protein A Sepharose 4 Fast Flow beads  for 2\u20134 hr at 4\u00b0C. Spin down the beats at 500 g for 30 s. Then washed with lysis buffer for three times and add SDS running buffer to the beads and proceed to western blot analysis. For western blotting analysis, proteins were separated by SDS\u2013PAGE, and transferred onto a PVDF membrane. The membrane was then blocked with 5% non-fat milk  in TBST buffer and incubated with primary antibodies in TBST with 5% non-fat milk overnight at 4\u00b0C. The membranes were then washed in TBST and incubated with HRP labeled secondary antibodies (1:5000 in TBST with 5% non-fat milk) for 1 hr at RT. The membranes were then washed in TBST and developed with ECL reagents  and exposed. Quantification of protein bands was done with Image J software.Cells were lysed in lysis buffer ; 150 mM NaCl\uff0c50 mM Tris-HCl pH 8.0, 10 mM NaF, 1 mM NaM Phos-tag acrylamide  and 200 \u03bcM MnCl2. After electrophoresis, Phos-tag acrylamide gels were washed with transfer buffer  containing 10 mM EDTA for 20 min and with transfer buffer without EDTA for 10 min. The proteins on the gel was transferred onto PVDF membranes followed with regular western blot procedure.Phos-tag SDS-PAGE was performed with 7% polyacrylamide gels containing 100 \u03bcAnti-HA antibody  was used with 1:1000 dilution in both western blot and immunofluorescence staining. Anti-GFP antibody (MBL: 598) was used with 1:1000 dilution in western blot. Anti-Flag antibody (Sigma: F3165) was used with 1:1000 dilution in western blot. Anti-V5 antibody (Invitrogen: R96025) was used with 1:5000 dilution in western blot and 1:500 dilution in immunofluorescence staining. Miga phospho-specific antibody was generated by GL BioChem Ltd. Phosphorylated peptide GSDPNFDSAE(S)pFA(S)pA was used as an antigen to immune the rabbits. The antibody was tested by ELESA assays and western blot (1:5000).3VO4,10% NP-40, 10% glycerol, 1.5 mM EDTA pH 8.0) and Miga was pulled down by FLAG beads and eluted with FLAG peptide  twice. The eluted fractions then subjected for pull-down assay with HA beads. Then the pull-down products were separated by SDS-PAGE. The gel was dyed with Coomassie brilliant blue for 30 min and then de-colored by de-staining solution . Then the gel was cut and subjected to the LC-MS/MS analysis.To identify Miga\u2019s binding proteins, FLAG-HA tagged Miga was overexpressed in S2 cells for 48 hr. Cells were lysed in lysis buffer . The gel corresponding to the size of Miga (50\u201375 KDa) was cut. In gel digestion was performed with trypsin at 37\u00b0C overnight. Peptides were extracted with 50% acetonitrile/\u00a05% formic acid, followed by 100% acetonitrile. Peptides were dried to completion and resuspended in 2% acetonitrile/\u00a00.1% formic acid. The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column . The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23% to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system.To identify the modifications on Miga protein, V5 tagged Miga was overexpressed in S2 cells for 48 hr. Cells were lysed in lysis buffer and IP with V5 beats. The proteins were separated by SDS-PAGE and dyed by Coomassie brilliant blue for 30 min and then incubated with de-staining solution  in Q Exactive Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 1.3. Tandem mass spectra were searched against Miga protein sequence. Trypsin/P was specified as cleavage enzyme allowing up to two missing cleavages. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Phosphorylation of serine, threonine and tyrosine were specified as fixed modification and oxidation on Met was specified as variable modifications. Peptide confidence was set at high, and peptide ion score was set\u00a0>20.Drosophila of each genotype were collected and housed at a density of 10 flies per vial (n = 100). All flies were kept in a constant temperature and humidity environment with 12 hr on/off light cycle. The number of surviving animals was counted and transferred to fresh food every 2 days.The  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Drosophila is in line with the very recent analogous discovery of the Klemm group in mammals. Moreover, your discovery of the phosphoregulation of this binding, where phosphate group additions compensate for the poorly acidic nature of Miga's \"di-phenylalanine in acidic track\" (FFAT) motif sheds a new light in the regulation of FFAT motifs, which play pleiotropic roles in organelle interactions.Your timely discovery of the interaction between the mitochondrial protein Miga and the endoplasmic reticulum protein Vap33 in Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Benoit Kornmann as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Suzanne Pfeffer as the Senior Editor.Thank you for submitting your article \"Miga mediated endoplasmic reticulum-mitochondria contact sites regulate neuronal homeostasis\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:In the present manuscript, Xu et al. Identify Vap33, an ER protein, as a binding partner for Miga, a mitochondrial protein. They show that vap33 binds Miga through a FFAT motif. When overexpressing Miga, this causes a massive increase in ER mitochondria contacts, which is dependent upon binding to Vap33. This increase in contacts, whether it is caused by Miga overexpression of by more artificial means  is toxic for omatidial development. The authors also show that while loss of miga is lethal, the lethality can be complemented by the expression of a Miga mutant form unable to bind vap33, indicating that Miga's essential role is not via Vap33 binding and the promotion of ER-mitochondria contacts. Moreover, lack of Miga or Vap33 has little impact on existing ER-mitochondria contacts. Finally, the authors show that Miga's FFAT  motif is unconventional, in that it is not really acidic, but can be made acidic through phosphorylation of serine residues, thus tuning affinity to Vap33.Revisions for this paper:As you will read from the attached reviews, the works was deemed interesting, yet insufficiently supported at places. In particular, most of the data here are from overexpression experiments. The interaction of Vap33 and Miga is not shown for proteins expressed at endogenous levels and the physiological implication of overexpressing the proteins are not straightforward. In particular, the ALS causing mutations are thought to reduce vapb binding to FFAT motifs, while the overexpression herein would rather increase it. Moreover, the text is at places incomplete or imprecise. In particular, the study of Freire et al., 2020, which shows interaction with mammalian Miga-2 and VAP and predates the present study by a few months should be much more thoroughly cited.Revisions expected in follow-up work:Pull down should be attempted with endogenously expressed proteins.Reviewer #1This is carefully executed and exciting story but its main problem is that it comes after Freyre et al., 2019, who already demonstrate binding of VAP and miga in cultured mammalian cells. Given the novelty of the Freyre publication and the need for \"scooping protection\", this need not preclude publication. However, it is important that this previous work is properly acknowledged along the whole manuscript and not as a side note in the Introduction. Freyre et al. already demonstrated VAP-Miga binding, the dependency upon the degenerated FFAT motif, the effect in increasing ER-mitochondria contacts by Miga overexpression, the relocalization of VAP to mitochondria upon miga overexpression.Additional point:\u2013 Miga has at least one function that doesn't require Vap33 binding, since lethality of a miga mutant can be rescued by expressing a FFAT-less Miga. What is/are this/ese function(s)? Do they relate to mitochondrial fusion. Is there any genetic interaction between Miga and mitoPLD or Marf to support this? A discussion of the other function(s) of Miga would be useful.Reviewer #2eLife. This reviewer recommends publication after the following points have been addressed.The manuscript by Xu and Wang et al. reports on mitoguardin (MIGA) mediated interaction between the ER and mitochondria and a critical function in maintenance of the retina. The results presented here confirm published work on the mammalian ortholog MIGA2, and add interesting new data on the activation of MIGA by phosphorylation. The function of ERMCs in neuronal homeostasis are also highly relevant, and the insights will be of high interest to the general readership of The novelty of the paper relates to the characterization of Miga function in neurodegeneration , and the characterization of Miga activation by phosphorylation .For a general reader it is not trivial to recognize the cell types and sub-cellular structures described in Figure 1. It would be helpful to segment and label the rhabdomeres, as well as indicate the outline of mitochondria and the ER in all panels that are compared. The areas enlarged in the figure panels called e.g. A', B' etc. should be outlined in the corresponding panels showing lower magnification, e.g. A, B etc. This also applies for the panels in Figure 3.It would be helpful to include an introductory sentence to the kinases studied in Figure 5, spell out CK and CamKII when used first, and explain what is known about their function, possibly even already in the Introduction. While the inhibitor experiments in Figure 5 are very convincing it is not clear why the WT condition in Figure 5C and the untreated condition in Figure 5F (which should be the same conditions) are showing almost the opposite band intensities between non-phosphorylated and phosphorylated Miga-V5. This reviewer is also not confident in agreeing with the claimed effect of CamKII. The data in Figure 5G and H rather indicate that CamKII RNAi increases MIGA phosphorylation. The unphosphorylated bands in the phostag gels seem less intense than in controls. Consider changing the text to explain this figure panels better, or interpret the data differently.The interaction with the VAP proteins was recently reported in work on mammalian MIGA2, and this work should be cited in the section reporting the interaction between Vap33 and Miga. This also applies for the Introduction and the text to Figure 2 and Figure 7. It is a strength of this paper to show that the molecular properties of mitoguardins are evolutionarily conserved and the authors might want to highlight this fact instead of ignoring it.The text to Figure 7A is confusing and does not match the labelling of the figure panels. E.g. the interaction between MIGA2 and VAPA and VAPB is shown in the panel, and the text indicates that MIGA1 is shown. The text jumps to Figure 7N before the rest of the panels are described. Consider the relevant corrections and changes in the text.The reason for the changes in relative abundance of full length MIGA-V5 and p-MIGA shown in Figure 6B are not clear. Are the authors suggesting that phosphorylation of the p-clusters I-III occur independently of starvation and only p-cluster V is modified after starvation? Or is this increase in phosphorylation contributed only from endogenous Miga. It seems important to show that the increase of p-Miga in Figure 6G is inhibited after the addition of the kinase inhibitors.VAP proteins are tail anchored proteins and C-terminal tagging might be problematic in particular when using a FLAG tag which is highly charged. This could explain the issues with the Co-IP experiments reported in Figure 7. Have the authors considered using N-terminally tagged.The Materials and methods section indicates the use of 10% NP-40 in the lysis and IP buffer used for mass spectrometry proteomics. Please double check the amount of detergent and correct the text if needed.Reviewer #3Xu et al., argue that Miga interact with the Vap33 protein through its FFAT-like motif and regulates ERMCSs. They argue that this interaction and elevated ERMCSs can promote photoreceptor degeneration. They also show that Miga can be regulated through phosphorylation and this phosphorylation is required for Vap33 binding as well ERMCSs formation.Overall, this manuscript is well structured with interesting discovery on the function and regulation of Miga in ERMCSs and neurodegeneration. A major concern is that much of the work is based overexpression data but it can be considered for publication after addressing my concerns below:1) Figure 2D-H,a) Please show a separate channel for Figure 2F and H. It is difficult to compare expression patterns with merged channels.b) Figure 2D-F, co-expression of Vap33 and Miga leads to a different expression pattern of both proteins, indicating the interaction/co-localization of the two protein may be due to an artifact from over-expression. Please try IP/IF with endogenous proteins c) If the Miga-Vap33 protein interaction is weak at basal level, then how significant is this interaction in the cell. This is an important question that needs to be addressed.2) The authors argue that Miga changes its expression pattern upon Vap33 co-expression: \"many circular structures appeared\". However, there are already some circular structures with Miga expression alone . More representative images and proper quantification are required to make this statement.3) Figure 2J: The authors argue that: Vap33 overexpression alone increased the mitochondrial proportion that have contacts with ER. However, I don't see a mitochondria-ER contact in this image. Please label where the contact is.4) As Vap33 is linked to ALS, does Vap33-RNAi also suppresses the flight muscle degeneration in Miga over-expression flies?5) Vap33-P58S, is associated with ALS. However, Vap33-P58S variant impairs the interaction with FFAT motif containing proteins (such as OSBP), suggesting that loss of Vap33-FFAT interaction is associated with ALS. However, in this manuscript, the authors argue that increased Vap33-Miga interaction lead to neurodegeneration. They should discuss this. Does overexpression of Vap33-P58S variant modify the photoreceptor loss in Miga over-expressing flies?6) Subsection \u201cMiga was phosphorylated at multiple clusters\u201d. Please explain how CKI inhibitor D4476 is identified.7) In the same section the authors claim that: Several Ser residues in cluster V were predicted as potential CaMKII phosphorylation sites. Please specify how phosphorylation sites prediction is performed.8) As CKI and CaMKII regulates Miga phosphorylation. And the author argue that this phosphorylation is required for ERMCSs formation. Can CKI and CaMKII modify the photoreceptor defect observed in Miga overexpression flies? Revisions for this paper:As you will read from the attached reviews, the works was deemed interesting, yet insufficiently supported at places. In particular, most of the data here are from overexpression experiments. The interaction of Vap33 and Miga is not shown for proteins expressed at endogenous levels and the physiological implication of overexpressing the proteins are not straightforward. In particular, the ALS causing mutations are thought to reduce vapb binding to FFAT motifs, while the overexpression herein would rather increase it. Moreover, the text is at places incomplete or imprecise. In particular, the study of Freire et al., 2020, which shows interaction with mammalian Miga-2 and VAP and predates the present study by a few months should be much more thoroughly cited.Revisions expected in follow-up work:Pull down should be attempted with endogenously expressed proteins.During the revision process, we made several attempts to address this problem. We had two anti-Miga antibodies that we generated in this study, but none of them worked properly in immunoprecipitation. We then expressed a genomic rescue construct of Miga with HA tag fused to its C-terminus, which likely expressed in an endogenous level. We do not have anti- VAP33 antibody and fail to obtain from colleagues in other country because of the outbreak of COVID-19 and shut down of many facilities. we overexpressed Flag tagged VAP33 and performed IP experiments. Since VAP33 overexpression only marginally increased ERMCSs in our experimental settings, we think the overexpression might not be very problematic. We included these data in the revised manuscript . Although the experimental condition is not ideal, that is the best we can do now.Reviewer #1This is carefully executed and exciting story but its main problem is that it comes after Freyre et al., 2019, who already demonstrate binding of VAP and miga in cultured mammalian cells. Given the novelty of the Freyre publication and the need for \"scooping protection\", this need not preclude publication. However, it is important that this previous work is properly acknowledged along the whole manuscript and not as a side note in the Introduction. Freyre et al. already demonstrated VAP-Miga binding, the dependency upon the degenerated FFAT motif, the effect in increasing ER-mitochondria contacts by Miga overexpression, the relocalization of VAP to mitochondria upon miga overexpression.Thanks for the comments. In the revised manuscript, we thoroughly acknowledged theprevious publication.Additional point:\u2013 Miga has at least one function that doesn't require Vap33 binding, since lethality of a miga mutant can be rescued by expressing a FFAT-less Miga. What is/are this/ese function(s)? Do they relate to mitochondrial fusion. Is there any genetic interaction between Miga and mitoPLD or Marf to support this? A discussion of the other function(s) of Miga would be useful.FM was overexpressed, there is no increase of ERMCSs and the mitochondria length was also significantly shorter than that in the wildtype Miga overexpressed tissues . It suggested that Miga functions in mitochondrial fusion might be coupled with its function in mediating ERMCSs. The study in mammalian cells indicated that MIGA2 links mitochondria to lipid droplets (LD) through its C-terminal region and is required for adipocyte differentiation. The LD interaction region of MIGA2 is conserved in fly Miga protein. It needs further investigation whether Miga plays a role in lipid metabolism in Drosophila. We add discussion regarding to this point in the revised manuscript.We did not test the genetic interaction between the FFAT-less Miga and MitoPLD or Marf. In this study, we found that when MigaReviewer #2The manuscript by Xu and Wang et al. reports on mitoguardin (MIGA) mediated interaction between the ER and mitochondria and a critical function in maintenance of the retina. The results presented here confirm published work on the mammalian ortholog MIGA2, and add interesting new data on the activation of MIGA by phosphorylation. The function of ERMCs in neuronal homeostasis are also highly relevant, and the insights will be of high interest to the general readership of eLife. This reviewer recommends publication after the following points have been addressed.The novelty of the paper relates to the characterization of Miga function in neurodegeneration , and the characterization of Miga activation by phosphorylation .For a general reader it is not trivial to recognize the cell types and sub-cellular structures described in Figure 1. It would be helpful to segment and label the rhabdomeres, as well as indicate the outline of mitochondria and the ER in all panels that are compared. The areas enlarged in the figure panels called e.g. A', B' etc. should be outlined in the corresponding panels showing lower magnification, e.g. A, B etc. This also applies for the panels in Figure 3.Thanks for the advice. We made the changes accordingly.It would be helpful to include an introductory sentence to the kinases studied in Figure 5, spell out CK and CamKII when used first, and explain what is known about their function, possibly even already in the Introduction. While the inhibitor experiments in Figure 5 are very convincing it is not clear why the WT condition in Figure 5C and the untreated condition in Figure 5F (which should be the same conditions) are showing almost the opposite band intensities between non-phosphorylated and phosphorylated Miga-V5. This reviewer is also not confident in agreeing with the claimed effect of CamKII. The data in Figure 5G and H rather indicate that CamKII RNAi increases MIGA phosphorylation. The unphosphorylated bands in the phostag gels seem less intense than in controls. Consider changing the text to explain this figure panels better, or interpret the data differently.The introduction of the kinases was added in the revised manuscript. The difference between the original Figure 5C and Figure 5F was because of the different sample preparation procedures. The samples in original 5C were prepared by adding SDS loading buffer directly to the pelleted cells. The samples in original Figure 5F were prepared by using lysis buffer to treat cells for 1hour, centrifuging for 10 minutes with high speed, collecting 20 uL supernatant and then adding SDS loading buffer (the rest of the supernatant was collected for IP experiments). The long preparation procedure for original Figure 5F likely reduced the phosphorylation in the protein samples. Now we changed Figure 5F with a new image in which the samples were prepared in the same way as Figure 5C. Since the phosphatase activity is very high in the lysates and the p-Miga is often de-phosphorylated. It is meaningful to compare the bands intensity and shift for the samples prepared side by side but not between different experiments.Figure 5G and H were phostag gel, the band shift in both images indicating CamKII might contribute to the phosphorylation of Miga. The overall intensity was low in the CamKII RNAi groups. We repeated the experiment several times, the RNAi group always have lower expression of Miga (both higher bands and lower bands). It needs further investigation to know why the expression level of Miga was low in the CaMKII RNAi group. Never the less, the mobility shift of both the lower bands and the upper bands was slower than the controls, indicating CaMKII contributes to the mobility shift in the control cells. We changed the image of Figure 5H with a better exposure time to show both bands of Miga. We also made some changes in the text and included discussions about the kinases in the revised manuscript.The interaction with the VAP proteins was recently reported in work on mammalian MIGA2, and this work should be cited in the section reporting the interaction between Vap33 and Miga. This also applies for the Introduction and the text to Figure 2 and Figure 7. It is a strength of this paper to show that the molecular properties of mitoguardins are evolutionarily conserved and the authors might want to highlight this fact instead of ignoring it.Thanks for the comments. We acknowledged the recent publication of Miga thoroughly in the revised manuscript.The text to Figure 7A is confusing and does not match the labelling of the figure panels. E.g. the interaction between MIGA2 and VAPA and VAPB is shown in the panel, and the text indicates that MIGA1 is shown. The text jumps to Figure 7N before the rest of the panels are described. Consider the relevant corrections and changes in the text.Sorry for the confusion. We added the MIGA1 IP results in the new supplemental figure . We also reorganized the Figure 7 to make the flow better.The reason for the changes in relative abundance of full length MIGA-V5 and p-MIGA shown in Figure 6B are not clear. Are the authors suggesting that phosphorylation of the p-clusters I-III occur independently of starvation and only p-cluster V is modified after starvation? Or is this increase in phosphorylation contributed only from endogenous Miga. It seems important to show that the increase of p-Miga in Figure 6G is inhibited after the addition of the kinase inhibitors.We prefer the first possibility suggested by the reviewer. We tried to detect the phosphorylation of endogenous Miga with this antibody and fail to obtained positive signals with or without HBSS treatments. Therefore, the p-Miga signals detected in MIga-V5 expression samples likely is the phosphorylated Miga-V5 protein. RNAi CAMKII did not change the level of p-Miga significantly. We think CAMKII might phosphorylate cluster V on the other sites rather than the S246 and S249 in the FFAT motif recognized by the phosph-specific antibody of Miga. We believe that there are other kinases also responsible for Miga\u2019s phosphorylation. We discussed this point in the revised manuscript.VAP proteins are tail anchored proteins and C-terminal tagging might be problematic in particular when using a FLAG tag which is highly charged. This could explain the issues with the Co-IP experiments reported in Figure 7. Have the authors considered using N-terminally tagged.It is a good suggestion. The Vap33 vector with Flag tag was tagged at the N-terminus. And the VAPA vector was also tagged at the N-terminus. The VAPB vector used here was tagged with V5 tag and it is tagged at the C-terminus. It has normal ER distribution, forms complex with MIGA2, and increases ERMCSs when co-expressed with MIGA2, suggesting VAPB-V5 functions normal. To prevent the confusion of the tag positions, we re-labeled the VAP proteins as Flag-Vap33, VAPA-HA, and V5-VAPB in the revised figures.The Materials and methods section indicates the use of 10% NP-40 in the lysis and IP buffer used for mass spectrometry proteomics. Please double check the amount of detergent and correct the text if needed.It is not correct. We changed it.Reviewer #3Xu et al., argue that Miga interact with the Vap33 protein through its FFAT-like motif and regulates ERMCSs. They argue that this interaction and elevated ERMCSs can promote photoreceptor degeneration. They also show that Miga can be regulated through phosphorylation and this phosphorylation is required for Vap33 binding as well ERMCSs formation.Overall, this manuscript is well structured with interesting discovery on the function and regulation of Miga in ERMCSs and neurodegeneration. A major concern is that much of the work is based overexpression data but it can be considered for publication after addressing my concerns below:1) Figure 2D-H,a) Please show a separate channel for Figure 2F and H. It is difficult to compare expression patterns with merged channels.Thanks for the comments. We split the channels in the revised manuscript as suggested.b) Figure 2D-F, co-expression of Vap33 and Miga leads to a different expression pattern of both proteins, indicating the interaction/co-localization of the two protein may be due to an artifact from over-expression. Please try IP/IF with endogenous proteins During the revision process, we made several attempts to address this problem. We had two anti-Miga antibodies we generated in this study, but none of them worked properly in immunoprecipitation. We then expressed a genomic rescue construct of Miga with HA tag fused to its C-terminus as suggested by reviewer 2, which likely expressed in an endogenous level. We do not have anti- VAP33 antibody and fail to obtain from colleagues in other country because of the outbreak of COVID-19 and shut down of many facilities. Since VAP33 overexpression only slightly increase ERMCSs in our experimental settings, we overexpressed Flag tagged VAP33 and performed IP experiments. We included these data in the revised manuscript. Although the experimental condition is not ideal, that is the best we can do now.c) If the Miga-Vap33 protein interaction is weak at basal level, then how significant is this interaction in the cell. This is an important question that needs to be addressed.As we showed in Figure 4D-M, a genomic rescue fragment of Miga with FFAT motif mutated cannot fully rescue the Miga mutant phenotypes. The rescued flies have reduced life span and retinal degeneration. These data suggested that the interaction between Miga-Vap33 indeed has physiological significance.2) The authors argue that Miga changes its expression pattern upon Vap33 co-expression: \"many circular structures appeared\". However, there are already some circular structures with Miga expression alone . More representative images and proper quantification are required to make this statement.The circular structures might not be a precise description of the structures we are trying to describe. We removed this statement in the revised manuscript.3) Figure 2J: The authors argue that: Vap33 overexpression alone increased the mitochondrial proportion that have contacts with ER. However, I don't see a mitochondria-ER contact in this image. Please label where the contact is.The increase of the mitochondrial proportion that have contacts with ER was modest when Vap33 was overexpression alone. The conclusion was drawing from statistic measurements and it might be hard to see from a single image. We changed an image with ERMCSs and label it with arrow in the revised manuscript.4) As Vap33 is linked to ALS, does Vap33-RNAi also suppresses the flight muscle degeneration in Miga over-expression flies?We did not perform this experiment due to the shutdown of EM facility on the campus during the epidemics of COVID-19. It has been reported before that the loss of Vap33 cause severe muscle defects. It is difficult to predict whether Vap33-RNAi could rescue Miga-overexpression phenotypes in muscles.5) Vap33-P58S, is associated with ALS. However, Vap33-P58S variant impairs the interaction with FFAT motif containing proteins (such as OSBP), suggesting that loss of Vap33-FFAT interaction is associated with ALS. However, in this manuscript, the authors argue that increased Vap33-Miga interaction lead to neurodegeneration. They should discuss this. Does overexpression of Vap33-P58S variant modify the photoreceptor loss in Miga over-expressing flies?We believe both loss and gain of ERMCS is devastating to the neuronal homeostasis. We discussed this in the manuscript as following \u201cIn ALS patients, a point mutation in the VAPB MSP domain was identified. The resultant mutant VAPB has reduced affinity to the FFAT motif containing proteins ). Indeed, the disease mutation mimic Vap33 had less affinity to Miga than its wild type form. The reduction of organelle contacts including ERMCSs might contribute to the disease conditions in the ALS patients.\u201d In the revised manuscript, we further emphasized that both reduction and increase of ERMCS are devastating to the cellular homeostasis and leads to neurodegeneration.For a similar reason described above, we did not perform the experiments suggested here.6) Subsection \u201cMiga was phosphorylated at multiple clusters\u201d. Please explain how CKI inhibitor D4476 is identified.R kinase inhibitor library (BML-2832 Version 2.2) and found that an inhibitor called 5-Iodotubericidin could inhibit the band shift. However, this inhibitor is not very specific. It inhibits ERK2, adenosine kinase, CKI, CKII, we then tried several other inhibitors including D4476 and found that only this one can inhibit the band shift nicely. During the screening, we also have some other hits that cannot be confirmed by further experiments. It is not very surprising, since most of the inhibitors in this library are not very specific. To simplify the story and make reader easy to follow, we only described D4476 in the manuscript.We did a small-scale kinase inhibitor screening with the screen well7) In the same section the authors claim that: Several Ser residues in cluster V were predicted as potential CaMKII phosphorylation sites. Please specify how phosphorylation sites prediction is performed.http://gps.biocuckoo.cn/) and NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) did the prediction. We add the description in the revised manuscript.We used online program GPS (8) As CKI and CaMKII regulates Miga phosphorylation. And the author argue that this phosphorylation is required for ERMCSs formation. Can CKI and CaMKII modify the photoreceptor defect observed in Miga overexpression flies?Because the shutdown of the EM facility, we could not perform the experiments requested here. It is hard to predict whether the knockdown of these kinases will modify the photoreceptor defects in Miga overexpressed flies because these kinases have many different substrates and CKI is important for several signaling pathways for eye development."}
+{"text": "The non\u2010covalent affinity of photoresponsive molecules to biotargets represents an attractive tool for achieving effective cell photo\u2010stimulation. Here, an amphiphilic azobenzene that preferentially dwells within the plasma membrane is studied. In particular, its isomerization dynamics in different media is investigated. It is found that in molecular aggregates formed in water, the isomerization reaction is hindered, while radiative deactivation is favored. However, once protected by a lipid shell, the photochromic molecule reacquires its ultrafast photoisomerization capacity. This behavior is explained considering collective excited states that may form in aggregates, locking the conformational dynamics and redistributing the oscillator strength. By applying the pump probe technique in different media, an isomerization time in the order of 10 ps is identified and the deactivation in the aggregate in water is also characterized. Finally, it is demonstrated that the reversible modulation of membrane potential of HEK293 cells via illumination with visible light can be indeed related to the recovered trans\u2192cis photoreaction in lipid membrane. These data fully account for the recently reported experiments in neurons, showing that the amphiphilic azobenzenes, once partitioned in the cell membrane, are effective light actuators for the modification of the electrical state of the membrane. The use of light to achieve control of cellular signaling is an important topic in pharmacology. Here, it is shown that an amphiphilic azobenzene performs ultrafast isomerization when partitioned into the cell membrane, while in water its photoisomerization ability is lost because of the formation of excimeric aggregates. This enables fast and reversible modulation of the membrane potential. The precise, fast, and efficient spatiotemporal control of cellular signaling via the use of external triggers has become an important topic in chemical biology[qv: 1] and photopharmacology. In this regard, light\u2010responsive systems offer the possibility to employ light as a clean, non\u2010invasive, and spatio\u2010temporally precise tool for controlling a variety of biological signals both in vitro and in vivo.[qv: 4\u20139] A plethora of photochromic systems allowing modulation of molecular responses in a reversible fashion have been developed. Among them, azobenzenes[qv: 12] are widely employed as versatile photoresponsive element for conferring reversible sensitivity to bio(inspired) targets and drugs, such as artificial membranes,[qv: 13\u201315] peptides, nucleic acids, ion channels, living organisms,[qv: 22] and antibacterial molecules.[qv: 23\u201325] These studies have demonstrated that effective and reversible photomodulation can be achieved both in vitro and in vivo, by the covalent binding of the azobenzene unit to the selected target. Alternatively, one can exploit non\u2010covalent interactions to assist the incorporation of photoactive molecules in bio\u2010systems.[qv: 26\u201330] Although this method is less specific than the covalent approach, the relatively weak non\u2010covalent interactions still allow for driving affinity to various bio\u2010interfaces, while ensuring full reversibility and much lower invasiveness. For instance, the ability of amphiphilic azobenzenes to intercalate spontaneously in model artificial membranes and micelles has been used to guide their internalization into these systems, mostly to study intrinsic membrane properties, to build up photoresponsive cargos for drug delivery applications, or to provide novel antibacterial systems.[qv: 25] Very recently a new amphiphilic azobenzene molecule named ZIAPIN2 has been introduced. ZIAPIN2 predominantly localizes in the plasma membrane and potently modulates neuronal firing in vitro as well in vivo via an opto\u2010mechanical effect.[qv: 34] In order to fully characterize the photostimulation mechanism of ZIAPIN2, here we report a detailed investigation of its photophysics and dynamics in different media. By combining steady\u2010state and time\u2010resolved optical spectroscopies, we observed that ZIAPIN2 in water lends itself to a radiative transition likely mediated by the formation of excimers, whereas it reacquires its conformational dynamics in membrane\u2010mimicking environments. Characteristic time scales and reaction yields are reported, reconciling the fast response (ms) observed in functional studies and the apparent slow ensemble evolution(s) by determining the molecular conversion time in the tens of picosecond range. Finally, we validate this opto\u2010mechanical phenomenon in a non\u2010excitable cell line that has an intrinsically low concentration of ion channels, to single out the effect on the plasma membrane itself.Figuref = 1.33) featuring an n\u2013\u03c0* character with molecular orbitals delocalized over the azobenzene moiety. The cis species shows two vertical dipole active excited states computed at 537 nm (f = 0.18) and 367 nm (f = 0.33), associated with the n\u2013\u03c0* and \u03c0\u2013\u03c0* transitions, respectively. Note that the diamino substitution induces a red shift of the optical gap with respect to the unsubstituted azobenzene that is favorable to biological applications. As discussed in the following sections, the addition of water induces a broadening and a red\u2010shift of the absorption spectrum of the trans species. To a first approximation, we performed DFT/conductor\u2010like polarizable continuum model calculations on the single molecule. Considering water as a solvent, two active states are computed at 456 nm (f = 1.34) and 436 nm (f = 0.15), the former being red\u2010shifted by 26 nm with respect to the active state in the vacuum case. We preliminary speculate that such a shift in the ground state absorption might be related to the solvent cage (in this case water) that can play a role in the molecular conformation. As shown later, however, modeling at the single molecule level is not sufficient to fully account for the observed phenomena.ZIAPIN2 was synthesized by reduction of the commercially available Disperse Orange 3 followed by the substitution of the amines with an excess of 1,6\u2010dibromohexane. Full details about the synthesis can be found in ref. We used UV\u2013vis absorption and photoluminescence (PL) spectroscopies to investigate the behavior of ZIAPIN2 in the following media: i) DMSO that is a good solvent, as a control medium and vector for ZIAPIN2; ii) water that is the main component of extracellular and intracellular media; and iii) micelles of sodium dodecyl sulfate (SDS) to mimic the lipid bilayer environment.[qv: 34\u201336] The latter is useful for steady\u2010state and transient absorption measurements (vide infra) that are difficult to perform in intact cells. We selected SDS as it spontaneously forms mixed micelles upon combination with single\u2010tailed surfactants with oppositely charged head groups. In the UV\u2013vis absorption spectra Figure , we obseFigurem, and water . In water  indicate that the photoinduced PL quenching occurs almost totally in the membrane region, while residual ZIAPIN2 molecules present in the aqueous extracellular buffer do not seem to undergo isomerization. This confirms that lipid environment allows restoration of ZIAPIN2 photoswitching ability.Steady\u2010state absorption measurements of ZIAPIN2 in solution under an actinic light allow the assessment of the ensemble dynamics of isomerization. In r Figure ,d,f. In r Figure ,b, we obr Figure , in goodr Figure ,f, ZIAPIr Figure ,d, howevFigure0\u2192S*, PB); ii) another positive peak at 570\u2013610 nm that can be linked to stimulated emission , and iii) a negative feature at around 700 nm due to photoinduced absorption from excited states . To obtain quantitative insights into the complex convolution of the trans and cis spectra, we employed a global analysis algorithm. We describe our experimental data with two components  and the experimental value (0.12) for ZIAPIN2 in water  confirms the kicking in of a different deactivation mechanism in the aqueous buffer. We assign the breakdown of the Strickler\u2013Berg rule to the formation of an aggregate excited state, possibly an excimer, with higher radiative rate. The findings in SDS are consistent with our steady\u2010state spectroscopic data and suggest that ZIAPIN2 performs ultrafast isomerization reaction in membrane. The tiny changes observed in the ensemble measurements in SDS/cells are likely due to the presence of a mixed population in which only a fraction of ZIAPIN2 molecules is protected by water.In electrophysiology experiments, neurons reacted to short burst of light, between 20 and 200 ms. The prompt response seems in contradiction with the time evolution measured in solution, taking place in seconds. One should however notice that steady\u2010state measurements allow accessing the isomer fraction population in the ensemble. In order to gain insights into the molecular isomerization event  it is necessary to employ ultrafast techniques.[qv: 46] We carry out a transient absorption (TA) experiment where a pump pulse brings population to the excited state, while a broadband probe pulse interrogates this state and tracks its temporal evolution. With this technique, we studied the ZIAPIN2 photodynamics in the three media of choice, namely water, DMSO, and SDS. The TA spectra at early delay\u2010times  in HEK293 cells, which represent a well\u2010established cellular model. Possible cytotoxicity of ZIAPIN2 was evaluated by using the Alamar Blue standard test, which indicates that exposure to ZIAPIN2 does not have an appreciable impact on cell proliferation . We measured the light\u2010induced change in the resting plasma membrane potential (Vm) by applying the patch\u2010clamp technique in current clamp mode (I = 0) . In agreement with what has been found in neuronal cells,[qv: 34] photostimulation of ZIAPIN2\u2010loaded HEK293 cells with either short (20 ms) or long (200 ms) visible light pulses  also generated a significant hyperpolarization of Vm that appeared immediately at the light onset and slowly returned to the physiological resting values after a slight rebound depolarization phase after the light offset . In our previous study, we have assigned this potential modulation to a photoinduced thinning/thickening of the cell membrane leading to an increase/decrease of the membrane capacitance.[qv: 34] To link ultimately such an effect to the ZIAPIN2 ultrafast photoreaction occurring in the membrane environment, we therefore recorded the |\u0394Vm| of hyperpolarization and depolarization as a function of the illumination wavelength for 20 and 200 ms light stimulation  with non\u2010covalent affinity for cell membranes can act as an efficient, fast, and reversible modulator of the membrane potential via visible light. By applying steady\u2010state and time\u2010resolved ultrafast spectroscopies, we note that in water a strong aggregation occurs, effectively hampering photoisomerization and opening up a deactivation path likely mediated by excimer formation. Alternatively, internalization in the cell membrane prevents self\u2010aggregation and restores the intrinsic ultrafast response of the photo switch. This, ultimately allows light\u2010triggering a large hyperpolarization in HEK293 cells whose magnitude follows the absorption profile of the trans state in membrane and, remarkably, permits evoking biological photoinduced effects in vivo.[qv: 34] Such actuators are new tools that could contribute to the development of opto\u2010neuroscience, biophotonics, and photopharmacology.The authors declare no conflict of interest.Supporting informationClick here for additional data file."}
+{"text": "Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70% ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA for molecular biology. Highly concentrated ethanol (95\u2013100%), preferred as a DNA preservative, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies on the correlation between ethanol concentration and specimen preservation are lacking. Here, we tested how preservative ethanol concentration in combination with different sample handling regimes affect the integrity of seven insect species representing four orders, and differing substantially in the level of sclerotization. After preservation and treatments (various levels of disturbance), we counted the number of appendages  that each specimen had lost. Additionally, we assessed the preservation of DNA after long-term storage by comparing the ratio of PCR amplicon copy numbers to an added artificial standard. We found that high ethanol concentrations indeed induce brittleness in insects. However, the magnitude and nature of the effect varied strikingly among species. In general, ethanol concentrations at or above 90% made the insects more brittle, but for species with robust, thicker exoskeletons, this did not translate to an increased loss of appendages. Neither freezing the samples nor drying the insects after immersion in ethanol had a negative effect on the retention of appendages. However, the morphology of the insects was severely damaged if they were allowed to dry. We also found that DNA preserves less well at lower ethanol concentrations when stored at room temperature for an extended period. However, the magnitude of the effect varies among species; the concentrations at which the number of COI amplicon copies relative to the standard was significantly decreased compared to 95% ethanol ranged from 90% to as low as 50%. While higher ethanol concentrations positively affect long-term DNA preservation, there is a clear trade-off between preserving insects for morphological examination and genetic analysis. The optimal ethanol concentration for the latter is detrimental for the former, and vice versa. These trade-offs need to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of collected specimens. The first records of the use of ethanol for the preservation of animal tissue date back to the mid 1600s, when Robert Boyle mentions that he successfully used the \u201cSpirit of Wine\u201d to preserve blood and soft parts of a human body, as well as a fish, for many months . The knoDuring the last two decades, as the research focus has shifted from the analysis of morphological features to molecular work, including DNA sequencing and amplification, ethanol has remained the preferred preservative liquid. Ethanol is an excellent fixative for DNA for three reasons: it kills decomposing microorganisms; it removes water from the tissues, slowing down enzymatic processes; and it denatures both the DNA and DNA-degrading enzymes, preventing further enzymatic degradation . For insThe typical ethanol concentration employed for preserving insects for morphological examination used to be 70% . HoweverIncreasingly, insect biodiversity inventories and ecological assessments are accompanied by or solely rely on DNA sequencing e.g.,  for a coWhile it may be challenging to find a sweet spot that is acceptable for all intended uses, there is a clear need for experimental studies of the nature of the trade-offs between the preservation of insects for DNA sequencing and morphological study. Here, we take a few steps towards filling this knowledge gap. Specifically, we examined the effects of increasing ethanol concentrations on specimen fragility and DNA preservation in seven species of insects, spanning four orders and representing different sizes and levels of sclerotization.Macrolophus pygmaeus (Hemiptera: Miridae), Aphidoletes aphidimyza (Diptera: Cecidomyiidae), Drosophila hydei (Diptera: Drosophilidae), Dacnusa sibirica (Hymenoptera: Braconidae), Calliphora vomitoria , Formica rufa (Hymenoptera: Formicidae) and Dermestes haemorrhoidalis (Coleoptera: Dermestidae). For better readability, only the generic names will be used from now on in the article. Adults of these species represent a wide range of body shapes, cuticle hardness levels, and responses to varying ethanol concentrations and treatments based on anecdotal information and prior observations. Specimens of some species were commercially purchased, and others were manually collected . Specimens of Aphidoletes and Dermestes were killed by placing them in the experimental tubes at the desired concentrations directly. All mock communities consisted of ten individuals of Macrolophus, Aphidoletes, Drosophila and Dacnusa, and two individuals of Calliphora, Formica, and Dermestes. The specimens were kept in 50 mL Falcon tubes with 40 mL of preservative ethanol of different concentrations. Communities were prepared over the course of approx. 2 weeks. Once all communities were ready, the ethanol was replaced with a fresh aliquot of ethanol at the same concentration and kept for a month at room temperature to standardize the incubation before the treatments began.For the experiments, we constructed artificial (mock) communities made up of seven species of insects : Macroloollected . SpecimeWe used these preserved mock communities for three experiments, where we measured the effect of ethanol concentration on the morphological integrity of specimens subjected to different handling regimes, as described below. Insects used for the first of these experiments were subsequently stored long-term at the same ethanol concentration and used for the DNA preservation study.Macrolophus, Aphidoletes, and Dacnusa; head, legs, and wings for Drosophila; legs and wings for Calliphora and Dermestes; and legs and antennae for Formica. Only forewings were considered (elytra in the case of Dermestes).In the first experiment, we analyzed whether short-term preservation in high concentrations of ethanol alone increased the fragility of the insects. For the main experiment , we usedIn the second experiment, we measured if the sensitivity to transport-induced damage was influenced by the ethanol concentration. For this experiment , we usedIn the third experiment, we tested if other types of treatment can magnify the effects of high ethanol concentrations on insect brittleness . For eacDacnusa) or a single insect leg (other species) from each of the five tubes from the \u201cGentle\u201d treatment. We reasoned that in specimens stored at lower ethanol concentrations after extended storage, DNA is likely to be more degraded, containing fewer amplifiable copies of target genes in the same amount of insect tissue. To test this, we quantified differences in PCR template amounts across ethanol concentrations by adding the same amount of an artificial target to each sample of a given species prior to DNA extraction. Subsequently, we used the extracted DNA to prepare cytochrome oxidase I (COI) amplicon libraries that were then sequenced and assessed the ratios of an artificial target to insect COI gene in the resulting amplicon datasets.In the last experiment, we used insect samples from the first experiment to test the effects of ethanol concentration on DNA preservation . After tDacnusa or a single leg for the rest of species) in a two mL tube, containing 190 \u00b5L lysis buffer , 10 \u00b5L of proteinase K and ceramic beads (2.8 mm and 0.5 mm). We homogenized the samples on an Omni Bead Ruptor 24 homogenizer during two 30 s cycles at the speed of 5 m/s and then we incubated them at 56\u00b0 C for 2\u00a0h. From this homogenate, we mixed 40 \u00b5L  with a predefined amount of a linearized spike-in plasmid carrying an artificial COI target. The plasmid contained a 471-base insert developed based on the Calliphora vomitoria barcode region sequence, where outside of the conserved primer regions, approximately 40% of bases were substituted. We determined plasmid quantities were determined for each species during pilot experiments and ranged from 11,000 to 317,000 copies per sample , following a two-step PCR library preparation protocol  additional COI OTUs  were relatively abundant, we repeated the analyses after summing up the reads of all OTUs that were at least 90% identical to the dominant sequence. We then translated the abundance ratios to an estimated number of insect COI copies in each sample and standardized the values relative to the median value for 95% ethanol concentration for that same species.We processed the amplicon data using Mothur v1.44.2 . Reads wglmmTMB from package glmmTMB). As some species did not lose any appendages at all in certain treatments, zero inflation in the model was tested by simulating scaled residuals with the function simulateResiduals (package DHARMa) with the model without considering zero inflation, and checking the presence of zero inflation (function testZeroInflation from package DHARMa). If significant, a term for controlling zero inflation dependent on Concentration was included in the model. Subsequently, we used an analysis of variance (ANOVA) to determine whether Treatment, Concentration, and their interaction had a significant effect on the number of lost appendages (function Anova.glmmTMB from package glmmTMB), and we calculated Tukey-adjusted pairwise differences between treatments (function emmeans from package emmeans).We conducted all analyses in R  using thdf\u00a0=\u00a06, F\u00a0=\u00a088.31, p\u00a0<\u00a00.001; Ethanol concentration: df\u00a0=\u00a07, F\u00a0=\u00a041.66, p\u00a0<\u00a00.001; Species x Ethanol Concentration: df\u00a0=\u00a042, F\u00a0=\u00a03.81, p\u00a0<\u00a00.001), we analyzed data from each species separately. In cases where the estimated number of COI target copies depended significantly on ethanol concentration, contrasts were used to test for differences between each ethanol concentration and 95% ethanol concentration treatment, assumed as the reference. We conducted the same analyses on the second dataset, containing all OTUs at >90% identity to the reference sequence.For the DNA preservation analysis, we considered a linear model was fitted to the data with a logarithm-transformed estimated number of COI target copies (relative to 95% ethanol concentration median) as a function of two factors: Species and Ethanol Concentration, and their interaction. As both factors and their interaction were statistically significant  we used in the transport experiment.In the transport experiment, only three species produced enough data points to fit a model: osophila . For DroMacrolophus and Aphidoletes, specimens carried by a careful  technician sustained less damage than those carried by a reckless (Running) technician or sent by mail (PostNord). When the samples were treated with care , preservative ethanol concentration did not affect the number of appendages lost by either species. However, in those treated with less care, including Running and PostNord treatments, both species sustained significantly less damage when preserved in 70% ethanol.For the remaining two species, Dacnusa, Formica, and Dermestes) had to be excluded from the analyses, as they did not generate enough data to fit a model. In the remaining species, we did not observe a significant treatment effect, that is, drying the specimens or exposing them to repeated cycles of freezing and thawing did not seem to affect their brittleness , we obtained a total of 6,061,251 read pairs. Of these, 5,444,2555 reads, for 276 insect libraries , passed all analysis stages and were used for abundance comparisons . Across Macrolophus). Samples preserved in 97% and 99% ethanol did not differ significantly from the 95% reference. The magnitude of the effect of the ethanol concentration varied among species: for example, in samples preserved at the lowest ethanol concentrations (30 and 50%), the decrease in the number of COI copies relative to the 95% reference ranged from 3 to 440-fold.In all studied species, we found significant differences in the estimated COI target copy number between ethanol concentrations  Fig. 5)Fig. 5). Results obtained from a dataset with all OTUs at >90% identity to reference sequence for that species considered do not differ qualitatively from the ones summarized above .It is surprising that there are so few quantitative studies of the effect of ethanol concentration on the preservation of insects for morphological and molecular study. One possible reason for this is the difficulty of quantifying morphological preservation, but also the preservation of DNA for diverse sequencing-based applications. Our approaches allowed us to address both of these challenges.The criterion we used here to evaluate morphological preservation, the number of lost appendages, has the major advantage that it is fast and easy to measure. However, it captures only one aspect of morphological preservation, namely brittleness. Brittleness is important in many contexts, for instance, when handling, examining or mounting specimens, but it is not an ideal measure of the preservation of fine morphological details, or the status of internal anatomy. For instance, we noted a clear discrepancy between brittleness and morphological preservation with the drying pre-treatment. Nevertheless, we believe our approach is a good starting point for further enquiry into the effect of ethanol concentration on the preservation of insects. Undoubtedly, it would be valuable to keep exploring more sophisticated measures of morphological conservation.To assess DNA preservation, we used a quick measure of one simple aspect of DNA degradation: the absolute abundance of a certain 458-base long DNA fragment, part of the mitochondrial genome. It is clearly not a perfect measure of the overall DNA preservation. However, as DNA degradation happens primarily by the accumulation of random breaks within the DNA strands , it is unlikely that many longer fragments would remain once the abundance of 458-base targets decreases significantly. Thus, our results are of direct relevance to sequencing approaches that do not require high-molecular-weight DNA, but also informative to researchers that sequence long DNA fragments .The use of spike-in plasmids with artificial targets in amplicon sequencing experiments has been gaining popularity as a method for the quantification of microbiomes , while sIn general, our results agree with expectations. They clearly show that ethanol concentration does have an impact on the fragility of insect specimens, even though the effects vary among taxonomic groups. High concentrations tend to increase brittleness, especially in weakly sclerotized insects. We also noted a tendency of more disruptive treatments to have a stronger effect on specimens preserved in high ethanol concentrations. Regarding DNA preservation, the expectations are also met, as we observed a lower proportion of suitable PCR-template DNA fragments in the DNA extracts of those individuals stored at lower ethanol concentrations after only one year.Aphidoletes and Calliphora in particular). As the connective and muscular tissues are dried and made brittle by high ethanol concentrations, it is reasonable that insects with less exoskeletal anchorage in their joints will be more severely affected. However, interestingly, the negative effect of low ethanol concentrations was noticeable also in Dermestes. These beetles were very robust to damage at high ethanol concentrations, but tended to lose elytra \u2013abdomen and head too \u2013at low concentrations. The effect was not significant, but likely primarily because of lower numbers of Dermestes individuals (two per tube) compared to other species. Had we included more specimens, we suspect that this effect would have been significant. It would be interesting to test whether the increased fragility at lower ethanol concentrations is associated with the cuticle hardness that prevents preservative penetration into tissues, or perhaps decomposing microorganisms likely harbored by these carcass-feeding beetles .The effect of ethanol concentrations on morphological preservation was most pronounced in the most weakly sclerotized species  were all quite sensitive to the ethanol concentration, regardless of striking differences in size, body shape, and level of sclerotization. They were also consistently among the insects most affected by the different treatments we exposed them to. Coleoptera was represented by only one taxon in our study (Dermestes), but beetles are generally more sclerotized than other insects, and it is no surprise that Dermestes ranked among the most robust of the insects we studied. Hemiptera were similarly represented by a single taxon in our study (Macrolophus). This species was among the most fragile we studied, but the order does present a wide variety of body types, and future studies will have to show to what extent Macrolophus is representative. Our study did not include any representative of the order Lepidoptera, as these are rarely if ever preserved in ethanol when collected for morphological examination, and thus, the trade-off between morphological and molecular preservation in different concentrations of ethanol is of little relevance.As remarked previously, the effect of ethanol concentration varied strikingly among the different insect species we examined. Although a small set of species cannot be regarded as a comprehensive sample of insect diversity or fragility levels, some results may indicate patterns that apply more broadly to particular taxonomic groups of insects. For instance, it is notable that both hymenopterans we examined, ervation . Howeverervation . The thrDermestes the average number of COI template copies was visibly higher at both of these concentrations. The concentration at which significant degradation of DNA was first observed varied among species. For four of the seven species, the preservation of the DNA was significantly worse from 70\u201380% and down, while for Macrolophus, even a concentration as high as 90% underperformed compared to 95%. On the other hand, the DNA of Dacnusa and Calliphora was not significantly degraded until the concentration was lowered to 50 or 30%. The mechanisms underlying these differences are unknown to us. One of the possible explanations offered above for the good morphological preservation of hymenopterans  is directly at odds with the good preservation of DNA . This suggests that the morphological solidity of hymenopterans is due to other causes. For instance, the body is more distinctly divided into hard sclerites in Hymenoptera than is the case for many other insect orders, like Diptera. Potentially, this could be associated with an decreased tendency to lose appendages.In general, the opposite pattern to the preservation of morphology was observed for the preservation of the DNA: the higher the ethanol concentration, the better preserved the DNA was. For most species, individuals preserved at concentrations of 80% ethanol or lower produced significantly fewer copies of the targeted PCR fragment than those preserved at 95%. This shows that, even when an amplification product can be obtained through PCR, there is, indeed, DNA degradation at these ethanol concentrations. Interestingly, concentrations of 97 and 99% ethanol never differ significantly from 95%, albeit in some species like Regardless of the causes, the variation among taxa in the effects of ethanol concentration on DNA preservation is highly relevant for metabarcoding studies. Metabarcoding involves the amplification of a barcoding gene sequence from DNA templates of many species simultaneously, so differences in the preservation of DNA among taxa could potentially result in significant biases, with the poorly preserved species contributing less than others to the pool of accessible template DNA copies. Minimally, this will cause problems with abundance estimation from metabarcoding data; in the worst case, poorly preserved species might go completely undetected, especially if they are rare.Our study may be the first systematic study of the effect of ethanol concentration on morphological and molecular preservation of insects, albeit limited to only a handful of species. Our results largely confirm the commonly held belief that intermediate concentrations of ethanol, around 70\u201380%, are generally the best for morphological preservation, as high concentrations (above 90%) tend to make specimens fragile, whereas lower concentration in many cases allow some level of degradation. In contrast, the optimal DNA preservation is achieved at ethanol concentrations of >90%. Unfortunately this means that there is a conflict between preserving insects for morphological and for molecular work, as the ethanol concentrations that are ideal for the former purposes result in higher degradation rates of DNA. Is it possible to find a treatment that is ideal for both purposes? For now, we do not have a preservation regimen that is ideal for both morphology and molecules. This must be taken into account when planning large collecting campaigns that aim to preserve material for both morphological and molecular work. For instance, we note that catches from Malaise traps, which are frequently used in insect inventories, contain a large portion of Diptera specimens , a group10.7717/peerj.10799/supp-1Supplemental Information 1Methods for spike-in plasmid relative abundance quantification and bioinformatic processing of the sequencing data. Tables S1\u2013S4, significance tables of the statistical analysis of the appendages experiments.Click here for additional data file.10.7717/peerj.10799/supp-2Supplemental Information 2Tables S5-S8. Raw data of the appendages experiments and sequencing read numbers.Click here for additional data file."}
+{"text": "Arabidopsis thaliana were studied using three different quantitative methods to isolate their effect on root-soil cohesion. We present compelling evidence that micro-scale interactions of root hairs with surrounding soil increase soil cohesion and reduce erosion. Arabidopsis seedlings with root hairs were more difficult to detach from soil, compost and sterile gel media than those with hairless roots, and it was 10-times harder to erode soil from roots with than without hairs. We also developed a model that can consistently predict the impact root hairs make to soil erosion resistance. Our study thus provides new insight into the mechanisms by which roots maintain soil stability.Soil is essential for sustaining life on land. Plant roots play a crucial role in stabilising soil and minimising erosion, although these mechanisms are still not completely understood. Consequently, identifying and breeding for plant traits to enhance erosion resistance is challenging. Root hair mutants in  Sarah De Baets et al. investigate the effect of root hair abundance on root-substrate cohesion and experimental soil erosion in Arabidopsis. They find that plants with root hairs are more difficult to detach from soil and are better able to prevent soil erosion than root hairless plants and present a model to predict the impact of root hairs on soil erosion resistance. Soil erosion is likely to worsen as the global population grows, average calorific intake increases and climate changes. Plants limit erosion by sheltering soil from erosive forces with their aerial parts and binding soils with their roots, both of which help to retain soil on slopes and anchor plants in the ground5. Plant root system architecture develops in response to local nutrient concentrations and precision nutrient placement has been mooted as a means of controlling soil erosion6. The selection of cultivars best suited to resisting erosion could be part of future sustainable soil management; however, this requires the identification of erosion-resistant traits. While the importance of meso-scale root properties of plant species  that support soil erosion resistance has been well studied experimentally and through modelling8 the understanding of the potential role of root micro-scale properties  in controlling emergent soil properties like soil erosion resistance is limited.Soil erosion rates associated with agricultural intensification and expansion are 100\u20131000 times higher than natural background erosion and much higher than soil formation, posing a serious threat to sustainable agriculture, food and environmental security10. Likewise, root hairs have been linked to phosphate uptake, rhizosphere soil structure formation11, root penetration12, water uptake13 and rhizosheath (i.e. the weight of soil adhering strongly to roots upon excavation) formation in crop plants14. Plant roots also secrete compounds (exudates) that have been shown to promote soil aggregation15, supporting a composite-like medium consisting of soil particles, plant roots, and plant- and microbe-derived compounds that all contribute to mutual cohesive interactions. Nevertheless, there is no current convincing evidence for micro-scale root properties such as root hairs in soil cohesion. Indeed, the presence of root hairs is required for rhizosheath formation, but the effect of root hair length on rhizosheath strength and size has not been detected14.There is a growing awareness that micro-scale processes at the root\u2013soil interface (rhizosphere) are important for determining the properties and functions of soils and ecosystems that support sustainable agricultural land use and management. Symbiosis between roots and mycorrhizal fungi, for example, positively affect water balance, energy balance, nutrient/element cycling and soil hydrophobicity5. The technical term \u2018cohesion\u2019 refers to the tendency of the \u2018root\u2013soil matrix\u2019  to maintain mechanical integrity16. Thus, root\u2013soil cohesion includes both roots adhering to soil as well as soil particles sticking to one another as an effect of root exudation or plant root\u2013microbe interactions.Previous studies have explored the mechanisms of adhesion of roots to soil and cohesion of the root-soil composite by comparing species with different root architecture to evaluate how thick, deep roots; thin roots; and dense, fine roots change soil erosion resistanceArabidopsis thaliana and novel root\u2013gel attachment and uprooting resistance assays, as well as an established soil erosion assay in conjunction with a mathematical model to quantify the soil cohesion effects of root hairs. Our work advances the quantitative understanding of how root hairs affect root\u2013soil cohesion and have a measurable effect on soil erosion resistance.The explanatory power of prior studies is limited because root\u2013soil cohesion may be influenced by inter-species differences other than those selected, especially differences in root micro-architecture. We overcome these limitations by using mutants and transgenic lines of the model plant 19. Transgenic 35S::RSL4 plants have longer root hairs18 and wer myb23 seedlings produce more root hairs than wild-type seedlings20, while the rsl4-1 mutant seedlings have a decreased number of short roots hairs18 and cpc try mutant seedlings do not produce root hairs21. In soil, the cpc try roots had 97% less dense network of root hairs compared to wild type, whereas the root hair density was 1.6 times higher for wer myb23 compared to wild type  and root hairless or root hair overproducing mutant lines22 and report the P value of the Wald statistic (z), the hazard ratio and the lower and upper bound confidence intervals of the hazard ratio. Using this assay, we observed that the 35S::RSL4 and wer myb23 lines were more resistant to detachment from the gel medium than wild-type plants . Conversely, the risk of detachment for rsl4-1 and cpc try mutants was 5 and 5.4 times more relative to wild-type plants , respectively  are more difficult to detach from sterile gel than seedlings that have no (cpc try) or a decreased root hair number and length (rsl4-1). Therefore, root hairs directly contribute to plant adhesion during plant growth on solid gel medium.We developed a centrifugal assay that measures the strength of root\u2013gel adhesion in Arabidopsis seedlings with and without root hairs Fig.\u00a0. Seedlinnts Fig.\u00a0, with a ely Fig.\u00a0. These r\u22123) of each plant was calculated. Since the root\u2013soil system responds to the uprooting force by a combination of deformation and damage, the maximum force and total energy expended to dislodge the plant from its substrate are macroscopic measures of the strength of root\u2013soil cohesion  and maximum pulling resistance (kg\u2009m\u2009s\u22122\u2009m\u22121 root) required to uproot wild type and mutant plants  and compost  compared to wild-type plants  compared to wild type  for 4\u20136 weeks. After removing the aerial plant tissue, 150\u2009L of water were flowed over the soil\u2013root blocks for a maximum of 110\u2009s to simulate an overland flow event  of six common cover crop species measured in field conditions23. We observed that soil\u2013root blocks that contained root length densities >19\u2009km\u2009m\u22123 of either wild type or wer myb23 roots reduced erosion rates to almost zero or 0.27 times that of the bare soil controls, respectively , \u22120.069\u2009\u00b1\u20090.007 (R2\u2009=\u20090.57) and \u22120.066\u2009\u00b1\u20090.008 (R2\u2009=\u20090.62), respectively.\u00a0At RLD\u2009>\u200919\u2009km\u2009m\u22123 that corresponded with plant densities >850 plants m\u22122, hairless cpc try was best modelled using a linear regression with the constant term 0.268\u2009\u00b1\u20090.033, which indicated that there was no further erosion reduction with RLD\u2009>\u200919\u2009km\u2009m\u22123 for plants without root hairs , especially for wild-type plants Fig.\u00a0. Despite\u22121) indicated that a 3\u2009kPa soil cohesion increase due to the presence of Lolium perenne roots could reduce soil loss to 0.015 that of bare soil7. Therefore, we determined soil reinforcement values in the presence of roots with different root hair densities/phenotypes using the same method employed previously7 for each soil\u2013root block  is represented by M1/M2. Using the model to simulate our experimental observations, we deduced that the maximum effectiveness of root hair enhancement in the lines tested would be wild type\u2009>\u2009wer myb23\u2009>\u2009cpc try . While cohesion enhancement in wer myb23 and wild type were comparable, there was clearly a much lower value for cpc try plants  is converted by soil microorganisms into compounds that increase soil cohesion33 used a novel assay to identify polysaccharides important for soil cohesion, including chitosan, \u03b2-1,3-glucan, gum tragacanth, xanthan and xyloglucan. Similarly, Galloway et al.15 found that xyloglucan, a component secreted by a wide range of angiosperm roots, can increase soil particle aggregation. Building on these recent studies and our current results, we postulate three potential mechanisms by which plant root hairs might reinforce soil: (i) substrate components such as gel molecules or soil aggregates bind directly to root hair surfaces; (ii) root hairs release exudates that reinforce soil; and (iii) root hairs release exudates that are processed into material that reinforces soil by microbes. The approaches applied in this study can quantify relative contributions of other root traits to soil cohesion/erosion. The application of our findings and experimental approach will inform the selection or modification of root properties that could reduce soil erosion in agricultural, recreational and civil engineering contexts. Understanding how root hairs specifically affect plant\u2013soil interactions improves our investigation of plant biology and has applications in soil and land preservation and maintenance under changing climate conditions.Future work will explore the physical and biochemical aspects unique to root hairs that contribute to their soil\u2013root binding abilities. In this respect, it is interesting to note that Akhtar et al.Arabidopsis thaliana (L). Heynh mutants were in the Columbia (Col-0) background except cpc try, which was produced from a Col-0\u2009\u00d7\u2009Wassilewskija cross and repeatedly backcrossed to Col-0. Plants were grown in controlled environment rooms at 20\u201322\u2009\u00b0C, 60% humidity and 16\u2009h light cycle . Plants for the centrifugal root\u2013gel attachment assay were sown onto the surface of gel medium. Plants for uprooting assays were grown on sieved (7\u2009mm), clay soil , or a sieved (7\u2009mm) compost/sand mix . Plants for erosion assays were grown on sieved (7\u2009mm) clay\u2013loam soil . Gravimetric soil moisture content prior to the erosion tests ranged from 26% to 29%. Soil composition was determined using a sedigraph with a hexametaphosphate pre-treatment and ultrasonic bath. The United States Department of Agriculture standard was used to define soil textural description34. All soil was frozen at \u221250\u2009\u00b0C to limit microbes and insects before use.All 35. Ten sterile seeds were sown in two horizontal rows onto 90\u2009mm Petri plates (Thermo Scientific RC2260) containing 1/2 Murashige and Skoog basal medium (Sigma M5519) with 1% (w/v) sucrose and 1% (w/v) agar (Sigma A1296), pH 5.7 and grown vertically for 5 days. Seedlings were spaced 1\u2009cm apart and any seedlings touching each other were excluded during experimental reporting. Plates were placed inverted into a hanging basket centrifuge (Beckman Coulter Allegra X-30R Centrifuge) and subjected to 1-min incremental increases in centrifugal force of 720, 1018, 1247, 1440 and 1611 RPM . The proportion of seedlings that detached from the gel surface was determined between each speed. Data were collected for 87 wild type (Col-0), 87 wer myb23, 91 35S::RSL4, 94 rsl4 and 88 cpc try seedlings. We report the results of a single experiment, which are representative of at least two independent experiments.Seeds were surface sterilised in 10% bleach, 0.05% Triton X-100 for 15\u2009min, washed five times with sterile water and stratified at 4\u2009\u00b0C in the dark for 48\u2009hl\u00a0from its attachment point. We assigned m as the mass of the bucket and plate, \u03c9 as the angular velocity (in radians per second) and \u03b8 as the inclination of the bucket to the vertical. The centrifugal force acting on the bucket was at least 0.07m\u03c92 Newton, and the gravitational force is mg Newton. To define the balancing moments about the attachment point:We determined the perpendicular plane of rotation of the hanging buckets within the enclosed centrifuge mathematically. The bucket is attached 70\u2009mm from the axis of rotation, which also corresponds approximately to the surface of the gel in the plate and is free to swing about the axis. The centre of the bucket and plate mass lies on the axis of the bucket at a distance\u00a0\u03c9 is 720 \u221an rpm where n (i.e. the speed setting) is 1, 2, \u2026, 9. We calculated that \u03b8 at the slowest rotation setting is less than 1.41\u00b0 and, therefore, assumed that the bucket quickly swings out during centrifugation so that the Petri plates are orientated perpendicular to the plane of rotation. Hence, the seedlings experience a centrifugal force that can peel them away from the gel.Thus, \u03c9) and diameter of the centrifuge ) that was experienced by each seedling ) at each centrifugal speed:The maximum force resisted by the seedlings was used as a measure of root\u2013gel adhesion. The angular velocity  and the hazard ratio with the upper and lower bound confidence intervals. Since the hazard ratio is an exponential coefficient that compares the risk of seedling detachment between root hair lines relative to wild type plants, the hazard ratio has been used as a measure of effect size37. Wild-type plants have a hazard ratio of one; root hair lines with a higher risk of detachment will have a hazard ratio above one, while lines with a lower risk of detachment will have a hazard ratio below one.For each regression model run, we report 3 pots containing the same amount of soil. A polytetrafluoroethylene-coated (hydrophobic Tectane) aluminium washer was placed over the seedling within 3\u20134 days of germination. The plants were grown through the centre of the washer for 3\u20134 weeks until the plants had mature rosettes but were not yet reproductive. Therefore, the rosette anchored the washer around the plant so that the cables of the tensile machine could be attached to the washer for uprooting force measurement. Pots were saturated overnight in 3\u2009cm water and plants were uprooted from either a compost\u2013sand mixture  or a clay soil . Plants were pulled vertically from the soil using a tensile testing machine (Instron 3343 with a 10 Newton load cell 2519-201) at a constant speed of 5\u2009mm\u2009min\u22121 (refs. 41). Force traces were analysed to obtain total energy expended (area under the curve), peak force (maximum force reached) and the magnitude of the incremental force drops. The Instron 10 Newton load cell is accurate to 0.25% from 0.05\u2009kg\u2009m\u2009s\u22122 and the mean of force above this threshold was calculated. The significance of the difference was also tested with limits at 0.1 and 0.035\u2009kg\u2009m\u2009s\u22122, which satisfied P\u2009 <\u20090.05.Individual plants from multiple transgenic lines were grown and uprooted at the same time as control for run effects. Single, centrally placed plants were grown in 375\u2009cm\u22123) recorded. RLD is a root trait frequently used to estimate the erosion-reducing potential of plant species and select the most suitable species for controlling soil erosion processes45. To determine RLD, the soil and root complex was washed thoroughly over a 0.7\u2009mm sieve before manually separating the roots from the soil. The dry weights of these roots were used to determine plant RLDs by converting the root masses into root lengths using specific root length (i.e. root length per unit mass) values for each genotype. To obtain root specific length values, at least 10\u2009m of root per genotype from at least three representative plants were separated into single strands on a high contrast background, photographed, measured in ImageJ46, dried and weighed. The root specific length values (m mg\u22121 root\u2009\u00b1\u20091 standard deviation) for wild type, cpc try and wer myb23 were 0.63\u2009\u00b1\u20090.04, 0.43\u2009\u00b1\u20090.07 and 1.02\u2009\u00b1\u20090.31\u2009m\u2009mg\u22121 in clay soil and 0.51\u2009\u00b1\u20090.03, 0.39\u2009\u00b1\u20090.02 and 0.53\u2009\u00b1\u20090.02\u2009m\u2009mg\u22121 in compost, respectively.After uprooting, plant material was recovered and RLD  to investigate the variable-under-investigation/RLD relationship and how it is affected by the mutant background (Supplementary Table\u00a0f (kg\u2009m\u2009s\u22122) be the uprooting force corresponding to a deformation x (m). Allowing for damage we may write generally that:0\u2009<\u2009x\u2009<\u2009xp, where the function k(x)\u2009>\u20090 is a macroscopic elastic modulus and xp is the deformation corresponding to the peak force.Since the root\u2013soil system responds to the uprooting force by a combination of deformation and damage, the peak force and total energy expended are macroscopic measures of root\u2013soil cohesion. Let xp\u2009<\u2009x\u2009<\u2009xu where h(x)\u2009<\u20090 and xu is the deformation corresponding to uprooting.At the peak force, f from 0 to xu.At uprooting, the force decreases to 0 andk(x) and h(x) can vary from plant to plant and determine the mechanical properties of the system. We chose the peak force f(xp) and total energy expended, E (kg\u2009m2\u2009s\u22122), as a measurement of the mechanical resistance as they are both functions of k and h and allow for the comparison of the mechanical resistance of the different mutants.The (possibly non-differentiable) functions f(xp) and E, which imply statistically significant differences for the functions k and h between mutants.From our measurements, we found statistically significant differences between the mutants for \u22123) in boxes with inner dimensions 250\u2009\u00d7\u2009250\u2009\u00d7\u2009150\u2009mm fitted with a weed suppression mat. Different plant densities of 9, 16, 32, 49, 81 and 100 plants in each box box (i.e. a plant density range of 144\u20131600 plants m\u22122) were established, which corresponded to shoot densities between 0.15 and 2.37, 0.37 and 1.72, and 0.57 and 2.66\u2009kg\u2009m\u22122 for wild type, cpc try and wer myb23, respectively. All boxes were tested for erosion resistance at the same developmental stage after about 5 weeks growth (i.e. shortly after bolting). Data were collected from 18 boxes with wild-type roots, 17 with cpc try roots or 27 with wer myb23 roots.Plants were grown in sieved clay\u2013loam soil  for each collection interval. Soil detachment rates were averaged out per sample and normalised using the extrapolated soil detachment value for a root density of zero.Immediately prior to erosion, the boxes were saturated by capillary rise, photographed and the aerial tissue and weed mats were removed. Gravimetric soil moisture content before erosion tests was between 0.26 and 0.29\u2009g\u2009g50. The recovered roots were washed and weighed so that RLD (km\u2009m\u22123) could be calculated using specific root length (root length/unit mass) measured from 10 wild type, cpc try and wer myb23 root samples, which were 0.44\u2009\u00b1\u20090.05, 0.52\u2009\u00b1\u20090.10 and 0.58\u2009\u00b1\u20090.15\u2009m\u2009mg\u22121, respectively , flow and sediment properties:\u03c1s is density of the detached sediments (g\u2009cm\u22123) where 2.65\u2009g\u2009cm\u22123 was used52, \u03c1w (g\u2009cm\u22123) is density of water, g is gravity acceleration, d50 (\u03bcm) is median grain-size diameter , and CD the drag coefficient calculated from a formula using the grain Reynolds number that is calculated from the flow characteristics of the experimental runs and from the average grain size of the soil.Root reinforcement was defined as the difference between bare soil cohesion and the cohesion of soil containing roots. Soil cohesion values for bare and root-containing soils were established by back calculation of transport capacity efficiencies and corresponding soil cohesion values. Measured soil detachment rates were set equal to modelled soil detachment rates using the EUROSEM\u03b2 value was calculated using the following empirically derived equation54:The value of soil cohesion that corresponds to this 7.The full method for back calculation of corresponding soil cohesion when soil detachment and flow characteristics were measured was as described57. The erosion depth increases over the course of the experiment and reaches a maximum value R.For each mutant, the enhancement of soil cohesion with increasing RLD was quantified. We first determined the volume occupied by the Arabidopsis root system. From the flow rate and velocity, we deduced the shear force acting on the soil surface. This force is resisted by the cohesion of root-reinforced soil, but not in a homogeneous manner because resistance is strongest along the primary root, decreases radially outwards, and depends on depth; we use knowledge of root architecture to model this Fig.\u00a0. ErosionQ, m3\u2009s\u22121) and surface velocity  were experimentally measured. Since flow profile is assumed parabolic, the shear stress acting on the soil surface can be calculated asW\u2009=\u2009360\u2009mm is the width of the flume and k is the bare soil permeability, for which we used the representative value 0.2273\u2009\u03bcm2\u200958.The water volume flow rate  reaches a critical value determined by the Coulomb criterion\u00b5 is a friction coefficient, c is the cohesion of soil containing plant roots and N is the normal stress. The cohesion of soil containing plant roots depends on RLD. The maximum cohesion, cMax, that occurs at the tap root iscBare is the cohesion of bare soil and \u03b3(RLDT), in mm\u22121, is the increased depth-integrated soil cohesion due to roots, which is a function of RLDT as the true root length density, or the total root length divided by the volume of the regions occupied by the root. Similarly, the minimum soil cohesion occurs furthermost from the tap root and isSoil is modelled as an isotropic nonlinear elastic material which has limiting behaviour that tends to that of a material described by Mohr\u2013Coulomb theory as a brittle material. We call this an isotropic \u2018elastic-Coulomb' material that is eroded when the shear stress (r and R represent the erosion parameters defined in the previous paragraph. The function \u03b3(x) has two properties that are approximately linear for small values of x and saturates to a constant value at large x . We chose a simple function that has these properties:In Eq.  r and R Mmax, since tan h takes values no larger than 1. For very low root length densities x, T is given by M1 because tan h (x)\u2009\u2248\u2009x for small x. For analysis is it useful to define the new parameter, M2\u2009=\u2009 M1/Mmax, which is the ratio of the initial enhancement rate to the maximum enhancement. M1, M2 are thus parameters describing the reinforcement plant roots provide and depend on the micro-scale properties of plant roots. To model the regular periodic array of plants, the cohesion c in Eq.  and function \u03b3 in Eq.\u00a0, pH 5.7, and sealed with Parafilm . Plates were incubated vertically for 10\u201311 days, when the root tips of wild-type plants reached within 1\u2009cm of the bottom of the plate. For each genotype, 20 single seeded plates and 5 plates with 5 seeds were used to measure and compare the growth of single and grouped plants. Each set was compared with a wild-type control. \u2018Root depth\u2019 was the vertical distance the root tip had progressed down the plate. Root-hair counts were taken using dark field lighting on a Leica MZ FLIII microscope. A Nikon D50 camera with a polarising filter and SPOT image capture software (SPOTIMAGING) was used to capture microscope images. Image analysis was conducted with a combination of ImageJ42 and RootNav software61. Plants for X-raying were grown in 200\u2009\u03bcl pipette tips filled with sieved clay soil for 5\u20137 days. Tips were scanned with a Nikon XT H 225 ST CT scanner  exposure 1\u2009s, 5 frames averaged per projection, voxel size\u2009=\u20090.00278056).Surface sterilised seeds were stratified at 4\u2009\u00b0C for 48\u2009h then grown on square 12\u2009cmFor all experiments, biological replicates for each root hair line were randomly selected from pools of seed containing genetically identical individuals for the trait of interest.n\u2009=\u200970 for each line. To account for potential heterogeneity in gel thickness and composition between Petri plates, angular rotation that each seedling experienced and spin number were incorporated as covariates in our analysis. The Petri plates were oriented vertically at approximately 80\u00b0, in stacks of five in a controlled growth room using a Latin Square design. Statistical analysis was performed in R with a Cox hazard function regression that included all covariates listed above in our statistical model and were removed only if they had no significant effect. The reported effect size is the hazard ratio, which includes lower and upper bound confidence intervals. P values were calculated from the Wald Statistic (z) with a significance level of 0.05. This study was conducted blind. The results for each line presented in this paper are representative of at least two independent experiments, although we have observed similar results in at least five independent trials, each run by different lab members.Ten seedlings of each line were sown onto a single Petri plate containing 30\u2009ml gel medium. For an individual experiment, there was a replicate size of over R. The number of samples tested per line was sufficient for linear regression and pairwise comparisons of regression parameters for all genotypes. A significance level of 0.05 was used for all regression parameters.Pots containing single plants were grown in trays containing six pots, organised in a Latin square design in a temperature and light-controlled growth room, and were rotated every 2 days to prevent edge effects. The tensile testing machine used to uproot plants was tested prior to conducting an experiment. The night before an experiment, pots were placed in 3\u2009cm water to allow saturation to ensure a consistent soil moisture level. Uprooting experiments were conducted blind to genotype. Between 13 and 17 individual plants were uprooted for each genotype. Pairwise comparisons of peak force, work done and force drop magnitude were conducted on wild-type plants relative to root hair mutants using the lm function in cpc try) and 27 (wer myb23) soil boxes were tested. The number of soil boxes tested per line was sufficient to conduct nonlinear regression models and the comparison of regression parameters for different lines. Statistical analysis was performed in IBM SPSS Statistics 25 using the nonlinear regression function and in MATLAB R2014a  for computing the error bounds on the modelled regressions. To compute the regression error bounds, we perturbed the parameter estimates 10,000 times from a set of parameter values randomly chosen from a normal probability distribution with mean and standard deviation equal to the estimated value and its standard error, respectively. Therefore, when the modelled curves do not fall within another curve\u2019s uncertainty bound, this indicates a significant difference between lines at P\u2009<\u20090.05.We grew 9, 16, 32, 49, 81 or 100 plants per box in a sieved clay-loam soil medium. For each experimental replicate, plants from all three lines were grown simultaneously in a controlled environment growth room to keep growing conditions as homogeneous as possible. On each experimental day at least five boxes were tested from at least two different lines. Different planting densities were used to obtain variation in root density. In total, 18 (wild type), 17 (Further information on research design is available in the\u00a0Supplementary Movie 1Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesPeer Review File"}
+{"text": "Long-term and widespread cotton production in Xinjiang, China, has resulted in significant soil degradation, thereby leading to continuous cropping obstacles; cotton stalk biochar (CSB) addition may be an effective countermeasure to this issue, with effects that are felt immediately by root systems in direct contact with the soil. In this study, we assess the effects of different CSB application rates on soil nutrient contents, root morphology, and root physiology in two soil types commonly used for cotton production in the region. Compared with CK (no CSB addition), a 1% CSB addition increased total nitrogen (TN), available phosphorus (AP), and organic matter (OM) by 13.3%, 7.2%, and 50% in grey desert soil, respectively , and 36.5%, 19.9%, and 176.4%, respectively, in aeolian sandy soil. A 3% CSB addition increased TN, AP, and OM by 38.8%, 23.8%, and 208.1%, respectively, in grey desert soil, and 36%, 13%, and 183.2%, respectively, in aeolian sandy soil. Compared with the aeolian sandy soil, a 1% CSB addition increased TN, OM, and AP by 95%, 94.8%, and 33.3%, respectively, in the grey desert soil , while in the same soil 3% CSB addition increased TN, OM, and AP by 108%, 21.1%, and 73.9%, respectively. In the grey desert soil, compared with CK, a 1% CSB application increased the root length (RL) (34%), specific root length (SRL) (27.9%), and root volume (RV) (32.6%) during the bud stage, increased glutamine synthetase (GS) (13.9%) and nitrate reductase (NR) activities (237%), decreased the RV (34%) and average root diameter (ARD) (36.2%) during the harvesting stage. A 3% CSB addition increased the RL (44%), SRL (20%), and RV (41.2%) during the bud stage and decreased the RV (29%) and ARD (27%) during the harvesting stage. In the aeolian sandy soil, 1% CSB increased the RL (38.3%), SRL (73.7%), and RV (17%), while a 3% caused a greater increase in the RL (55%), SRL (89%), RV (28%), soluble sugar content (128%), and underground biomass (33.8%). Compared with the grey desert soil, a 1% CSB addition increased the RL (48.6%), SRL (58%), and RV (18.6%) in the aeolian sandy soil, while a 3% further increased the RL (54.8%), SRL (84.2%), RV (21.9%), and soluble sugar content (233%). The mechanisms by which CSB addition improves the two soils differ: root morphology changed from coarse and short to fine and long in the grey desert soil, and from fine and long to longer in the aeolian sandy soil. Overall, a 3% CSB addition may be a promising and sustainable strategy for maintaining cotton productivity in aeolian sandy soil in the Xinjiang region. Howevers total) . It alsos total) . These cs total) , which us total) . TherefoIn order to alleviate the continuous cotton cropping obstacles, crop rotation, treatment with organic fertiliser and biochar, and other mitigation measures  have beeCrop straw biochar is mostly used in farmland . In XinjSoil improvement is manifested in plants through the root system, which is the first to perceive changes in the soil microenvironment, and is the touchstone for determining the effect of soil improvements . The rooIn this study, the effects of different CSB application rates on soil nutrients, root growth, cotton biomass, and cotton yields are investigated in two soils to: (1) determine the effects of CSB on different types of soil in which cotton is grown; (2) determine the effects of biochar application on cotton roots in different soil types, thereby allowing for the biological potential of roots to be fully utilised; (3) provide a scientific basis for the utilization of straw resources. We hypothesise that (i) CSB addition would increase the soil nutrient contents, and that this improvement would be larger in grey desert soil than aeolian sandy soil, and (ii) CSB addition would improve root physiology and morphology, as well as seed cotton yields, in both soil types, and would be correlated with the CSB addition rate.This study was conducted at the Shihezi University Test Site , part of the Xinjiang Production and Construction Corps of Northwest China. This location has a temperate continental climate, with an average annual temperature of 7.0\u00a0\u00b0C. From April to October, the mean maximum and minimum temperatures are 26\u00a0\u00b0C and 11\u00a0\u00b0C, respectively. Annual evaporation, sunshine duration, and precipitation are 1664.1\u00a0mm, and 2861.2 h, 210.6\u00a0mm, respectively. The frost-free period usually lasts 170 d.The tested grey desert and aeolian sandy soils were obtained from the cotton fields at the test site and from Jiahezi Township, Wusu City, Xinjiang, respectively. The soils were cultivated for 28 consecutive years and experienced significant degradation. The parent material of the grey desert soil was loess-like diluvial\u2013alluvial, partly aeolian, and slope sediments, yielding a soil type with a low gravel abundance and fine grains . The aeoGossypium hirsutum L. cv. Xinluzao 45 used in this study is a cotton cultivar planted throughout Xinjiang. The experiment was conducted using a polyvinyl chloride (PVC) pipe with a height of 40 cm and an inner diameter of 20 cm. A shovel was used to dig a pit 45 cm long and 25 cm wide, into which the PVC pipe was placed vertically with its mouth five cm above ground. The excavated soil was passed through the PVC pipe and filtered using a 20 mm sieve, while the remaining large soil particles after sieving were backfilled. Then, 15 kg of the sieved and air-dried soil was inserted into the PVC pipe. The added CSB was calculated as 1% and 3% of the dry soil weight per PVC pipe . Thereafter, 1% and 3% CSB were mixed with the sieved air-dried soil at ratios of 1:100 and 3:100, respectively. Finally, the mixture was filled into different PVC pipes in the corresponding order of the excavated in situ soil.The The experiment was conducted using a randomised complete block design based on soil type and CSB addition rate (0% CSB (CK), 1% CSB, and 3% CSB). Six treatments were used, each of which had four replicates . The two experimental plots were placed 90 cm apart with 30 cm between the first and second rows and 20 cm between adjacent PVC pipes .CSB was purchased from the School of Agriculture at Shihezi University, which was produced from cotton straw with initial total nitrogen (TN), total potassium (TK), total phosphorous (TP), and organic carbon contents of 8.9%, 86%, 69%, and 62.5%, respectively, and a pH of 10.5. The pyrolysis temperature and time were 450\u00a0\u00b0C and 6 h, respectively. The resulting material was then passed through a two mm sieve.\u22122 N, 420 kg hm\u22122 P2O5, and 270 kg hm\u22122 K2O. In addition, 20% N, 70% P2O5, and 100% K2O were applied once as a basal fertiliser. Fertiliser was applied as topdressing via drip irrigation five times during the cotton growth period (monthly), accounting for 15%, 15%, 20%, 20%, and 10% of the total amount of fertiliser applied, respectively. Irrigation was applied nine times during the growth period, for a total of 520 mm. The cotton was sown on 30 April 2018, using 10 seeds per PVC tube. Two healthy seedlings with similar growth were retained in each pot when only having two leaves and one stem. Weed and insect control were conducted according to normal management of high-yield fields in this region  and stored in a refrigerator at 4\u00a0\u00b0C. Root activity, glutamine synthetase (GS) activity, nitrate reductase (NR) activity, and soluble sugar content were determined according to previous methods .Data of root morphology were collected as previously described by our group . Root voSeed cotton was picked manually during the harvesting stage. Aboveground and underground parts were analysed after drying at 105\u00a0\u00b0C in a blast dryer for 30 min to kill all green material, and then at 75\u00a0\u00b0C until a constant weight is obtained. The aboveground, underground, and seed cotton parts were weighed and recorded, after which the seed cotton yields, specific root lengths (SRL), and specific root surface areas (SRA) were calculated as follows: \u22121) were calculated as root length (cm)/root dry mass (g), while the specific root surface areas (cm2 g\u22121) were calculated as root surface area (cm2)/root dry mass (g).The specific root lengths  was performed to examine the effects of soil types, BC addition rate and their interactions on all variables in two stages. The fixed factors were soil types and BC addition rate treatment. The differences of all variables between BC addition and control plots in the different soil type were analysed using one-way ANOVA in same stage. Duncan analysis was used for multiple comparisons as a post hoc test in all ANOVAs. All data was analysed using the SPSS 25.0 software package . The Origin 2018 software  was used to create the figures.Soil properties were significantly affected by soil type, CSB rate, as well as TN, AN, AP, AK, OM, and pH . CompareThe RL, SRL, RSA, and SRA significantly varied with soil type during both stages, as did RV during the bud stage and ARD during the harvesting stage. CSB addition caused significant differences in RL, SRL, and RV during the bud stage . CompareRoot soluble sugar contents differed substantially by soil type during both stages, as were root and GS activities during the harvesting stage. The CSB rate had a significant effect on the root soluble sugar contents, and GS and NR activities during the bud stage. Interactions between the soil type and CSB rate had significant effects on root soluble sugar contents and GS activity during both stages, and on NR during the harvesting stage . GS actiSoil type had a significant effect on the underground biomass during the bud stage, and both aboveground and underground biomasses during the harvesting stage. The CSB rate had a significant effect on the underground biomass during the bud stage. Their interactions had significant effects on underground biomass during the bud stage and on underground biomass and seed cotton yield during the harvesting stage . CSB addThe basic nutrient levels in the grey desert soil were higher compared with those in the aeolian sandy soil, while the positive effect of CSB addition was larger in the former. The AP increased by 44% in the aeolian sandy soil after 3% CSB addition, similar to the 45% increase in available P observed in a previous study . One reaChanges in the soil environment allow root systems to optimise water and nutrient absorption by adapting their morphological and physiological characteristics , which aCSB addition significantly promoted RL, RSA, RV, and other root morphological traits, possibly by improving the soil\u2019s physical structure , acting In the grey desert soil, CSB addition increased soil TN through the mineralisation of microbial nitrogen, which may result in a larger nutrient deficiency for plant growth . HoweverIn the aeolian sandy soil with 3% CSB addition, changes in RL were more pronounced than those of underground biomass. Root changes were also more pronounced in the underground biomass than in the ARD, indicating that cotton preferred root elongation, particularly distal fine root proliferation (root diameters up to 0.5 mm). Previous studies have shown that high production of distal fine roots increased the possibility of growing into biochar pores for access to water  and nutrSoil fertility, nutrient absorption, and utilisation efficiency are the main factors limiting the physiological growth of crops, and effective nutrient absorption . CSB addthe soil . NR is amilation . The subA) cycle . In the A) cycle ), are reA) cycle . Second,A) cycle , which iA) cycle , and is A) cycle . In the A) cycle  and promA) cycle .CSB addition did not have an effect on the aboveground or underground biomasses during the bud stage in the grey desert soil, indicating that the distribution of biomass did not change; thus, root and stem growth were coordinated . The addImproving cotton fields using CSB is currently being investigated for biochar preparation , soil phBiochar addition, which can increase soil TN, OM, AP, and AN, had a greater impact on grey desert soil than on aeolian sandy soil, although the root change mechanisms differed. In the grey desert soil, CSB mineralised more soil nitrogen, while the roots changed from coarse and short to fine and long. In the aeolian sandy soil, both the initial soil nutrients and those obtained after CSB addition were lower than those in the grey desert soil, resulting in further root elongation. The addition of 3% CSB may be a promising sustainable strategy for improving soil nutrients, promoting root growth, and maintaining crop productivity in aeolian sandy soil. However, a longer study period is warranted to verify these effects. The development and utilization of CSB should be increased and its reproducibility promoted to enable mass production. Maximizing the use of biochar can reduce pollution and resource waste, improve cotton field soil, and promote cotton growth to alleviate continuous cropping obstacles.10.7717/peerj.12928/supp-1Table S1Note: CK: control, 1% BC: 1% biochar addition, 3% BC: 3% biochar addition; Root SP content: Root soluble protein content, Root SS content: Root soluble sugar content, Root NR activity: Root nitrate reductase activity, Root GS activity: Root glutamine synthetase; RL: Root length, SRL: Specific root length, RSA: Root surface area, SRA: Specific root surface area, RV: Root volume, ARD: Average root diameterClick here for additional data file."}
+{"text": "More long-term follow-up studies beyond 10\u00a0years after secondary sphincteroplasty for obstetric damage are warranted. This prospective study aimed to compare reported data on incontinence and satisfaction in a cohort of such patients examined at short-, long-, and very long-term follow-up.Twenty out of 33 obstetric patients (61%) operated with secondary anterior overlapping sphincteroplasty during February 1996 to April 2004 were evaluated preoperatively and at short-, long-, and very long-term follow-up. Anal incontinence was scored by a combination of Wexner\u2019s and St. Mark\u2019s incontinence scores. The patients also reported degree of treatment satisfaction.p\u2009<\u20090.01), 10.0 (0\u201318) (p\u2009>\u20090.05), and 12.0. (1\u201318) (p\u2009>\u20090.05). With increasing follow-up times, patients reporting a better outcome were 75%, 65%, and 45%. At very long-term follow-up patients, reports were more dismal than expected in those also reporting improved incontinence cores. Incontinence scores did not improve in patients with neuropathy (n\u2009=\u20095) or patients (n\u2009=\u20095) with more than 10\u00a0years of symptoms.Twenty patients were examined preoperatively and after a median (range) of 5 (2\u201362), 102 (64\u2013162), and 220 (183\u2013278) months. Corresponding incontinence scores were 11.5 (5\u201318), 5.5 (1\u201317) (Initial improvement of anal incontinence attenuated with time, in particular from short- to long-term follow-up. Patients with neuropathy experienced no improvement of incontinence. Beyond stoma formation, in compliant patients, one should consider other treatment options like sacral nerve stimulation and neosphincter formation. The reported incidence of overt obstetric anal sphincter injuries in the Nordic countries varies from 0.6 to 4.2% . HoweverIn 2010 we reported short- and long-term outcomes (102\u00a0months) in 33 obstetric women following treatment of anal incontinence with secondary anterior sphincteroplasty . This stTwenty of the thirty-three patients (61%) reported in 2010 were eligible for very long-term follow-up in March 2019. Three patients were deceased. Ten patients were excluded because of not responding in seven and needing a stoma in three.n\u2009=\u200920), recording of incontinence scores (n\u2009=\u200920), manometry (n\u2009=\u200918), ultrasonography (n\u2009=\u200917), pudendal nerve terminal motor latency (PNTML) (n\u2009=\u20099), and needle electromyography (EMG) (n\u2009=\u20099). A PNTML\u2009\u2265\u20092.3\u00a0ms. and needle EMG showing polyphasic potentials and signs of denervation and reinnervation were compatible with neuropathy. At short-term follow-up, patients were scored for incontinence and patient satisfaction  in collaboration with the physician at the ambulatory unit. At long term and very long term, patients were evaluated by phone interview and by completing a questionnaire returned by mail. Incontinence was evaluated using a combination of Wexner\u2019s and St. Mark\u2019s scores. Parameters reported at all follow-ups were leakage of air, leakage of liquid stool, solid stool, and lifestyle alteration expressed as seldom (1 point), monthly (2 points), weekly (3 points), and daily (4 points). Use of pad expressed as yes (2 points) and no (0 points); complete incontinence giving a maximum score of 18.Preoperative ambulatory workup included clinical evaluation (n\u2009=\u20091), 3b (n\u2009=\u200911), 3c (n\u2009=\u20096), or 4 (n\u2009=\u20092).The median number of vaginal births was 2 (range 1\u20134). Eleven had an overt rupture of the anal sphincters. Of these, three had delivery by vacuum extraction and breech birth in one. Three had episiotomy performed during delivery. Judged by peroperative findings and anal ultrasonography, all patients had third or fourth-degree sphincter tear: grade 3a . We used Graphpad Instat version 3.0 for Windows , and p\u2009<\u20090.01), 10.0 (0\u201318) (p\u2009>\u20090.05), and 12.0. (1\u201318) (p\u2009>\u20090.05).At initial sphincteroplasty median (range) age and duration of symptoms for the 20 patients were 36\u00a0years (29\u201359) and 3 (1\u201320) years. They were examined at short-, long-, and very long-term follow-up after median (range) 5 (2\u201362), 102 (64\u2013162), and 220 (183\u2013278) months. Median (range) incontinence scores preoperatively, after short-, long-, and very long-term evaluation, were 11.5 (5\u201318), 5.5 (1\u201317) , 8 (40%), 11 (55%), and 10 (50%) patients wore a pad before operation and after the respective times of follow-up. Corresponding figures for patients using constipating medicines were 5%, 5%, 15%, and 10%. After long- and very long-term follow-up, 11 (55%) and 10 (50%) of the patients could defer defecation for 15\u00a0min or more.Results concerning the degree of incontinence and lifestyle alteration preoperatively and at follow-up are shown in Fig.\u00a0n\u2009=\u20091) had incontinence score of median (range) 17 preoperatively, 17 at short term, 13 at long term, and 15 at very long term. Grade 3b (n = 11), 11 (6\u201317), 8 (1\u201315), 12 (1\u201316), 12 (1\u201317). 3c\u00a0(n = 6), 11 (5\u201316), 5,5 (1\u201313), 7 (2\u201314), 10,5 (2\u201318). Grade 4 (n\u2009=\u20092): 13,5 (12\u201315), 8 (2\u201314), 11,5 (6\u201317), 11 (8\u201314).The patients were analyzed for incontinence scores related to the degree of sphincter tear without revealing striking differences in incontinence. Grade 3a , proved to have little effect of operation. Respective scores were 14 (11\u201319), 13 (7\u201316), 15 (6\u201318), and 17 (12\u201320).n\u2009=\u200915), the preoperative and post-operative incontinence scores with increasing follow-up times were 11 (5\u201316), 5 (1\u201313) (p\u2009<\u20090.05), 6 (0\u201315) (p\u2009>\u20090.05), and 9 (1\u201318) (p\u2009>\u20090.05), respectively. In patients (n\u2009=\u20095) with neuropathy, corresponding scores were 16 (13\u201318), 13 (8\u201317) (p\u2009>\u20090.05), 15 (14\u201318) (p\u2009>\u20090.05), and 14 (12\u201317) (p\u2009>\u20090.05).In patients without known neuropathy  versus long- to very long-time (2 points) follow-up.However, in the 15 patients without neuropathy, although not significant, there probably was a treatment effect with improved continence after both 8.5\u00a0years (5 points) and 18\u00a0years (2 points) of follow-up. The pattern of reduced continence as a function of observation times, mostly from 10 to 120\u00a0months, but also after 18\u00a0years, was consistent with other reports , the preThe five patients with neuropathy based on delayed PNTML and needle EMG had no definitive improvement of continence. Still, the scores remained stable at long and very long-term follow-up, supporting that sphincteroplasty per se consolidated the sphincter.The discrepancy at very long-term evaluation reported between reduced incontinence score in 65% and improved treatment results in only 45% of the patients could partly be owing to a negative psychological effect because of many years with persisting incontinence. However, at the ultimate evaluation, only half of the patients wore a pad, comparable with both before operation and after long time evaluation. This corresponds with findings that the major decline of incontinence took place from short- to long-time follow-up Fig.\u00a0. HoweverWe found that degree of sphincter tear did not have any crucial influence on the treatment results in this cohort of 20 patients. On the contrary, five patients with long symptom duration without neuropathy had no symptomatic benefit of sphincteroplasty. This is consistent with other reports .Reasons for early failure of a sphincteroplasty can be lack of a tension-free repair and complications from hematoma, wound infection, and fecal impaction. Crucial factors for long-term failure are less evident but could be denervation of the external sphincter muscle during dissection  and timeFurther treatment options, particularly for older women with progressing intolerable long-term incontinence, could be placement of a stoma. Alternatively, in younger and compliant patients\u2019 sacral nerve, stimulation or formation of a neosphincter by dynamic graciloplasty or artificial bowel sphincter is potential but also complex treatment options with inherent complications and low success rate , 11.In conclusion, even after 18\u00a0years, 20 out of 33 patients (61%) reported data after secondary sphincteroplasty. The incontinence and lifestyle alteration results most strongly declined from short- to long-term follow-up. The degree of incontinence reached the preoperative level at the hitherto almost unparalleled very long-term follow-up of 18\u00a0years. Although not statistically significant, patients without neuropathy still had an improved result at ultimate follow-up. Thus, secondary sphincteroplasty for obstetric damage is worthwhile, but the effect deteriorates, and one should consider additional treatment options in selected patients."}
+{"text": "Bacillus pacificus strain was isolated, and the whole-genome sequence is reported in this paper. The draft genome sequence of Bacillus pacificus KVCMST-8A-12 constitutes 2.4 Gbp of raw reads, with a GC content of 35.24%. In total, 5,661 protein-coding genes, 64 tRNA genes, and 4 rRNA genes were predicted.A DNase-producing  Bacillus pacificus KVCMST-8A-12 was isolated from a sediment sample from the coastal region in Kanyakumari, India  and following the manufacturer\u2019s instructions. DNA quantity and quality were assessed using NanoDrop measurements at 260 nm and 280 nm and resolution in a 0.8% agarose gel, respectively. The library was prepared following the workflow of the TruSeq DNA library preparation kit (Illumina) , with a GC content of 35.24% and 100\u00d7 coverage, was obtained for our strain. The online RAST gene annotation pipeline (A total of 2.4\u2009Gb of raw data was generated from 8,053,402 raw reads for  v3.14.1 . A genompipeline , 12. Thipipeline  and annoB. pacificus KVCMST-8A-12 has been deposited in GenBank under BioProject accession number PRJNA656513 with BioSample accession number SAMN16200492, and raw reads can be found under SRA accession number SRX9149524.The whole-genome sequence of"}
+{"text": "An axial flux permanent magnet single-rotor generator has good potential in various applications that require high efficiency, prolonged service life, as well as low mass and dimensions. However, the effect of cogging torque diminishes generator efficiency and flexibility of functionality. The effect of cogging torque arises because of a small air gap between the stator teeth and the rotor. In this article, we suggest that shifting the opposite teeth of the stator to the optimal angle can reduce the effect of cogging torque. A special axial flux permanent magnet generator is developed to choose the optimal disposition of the permanent magnet and stator teeth in the frame. The impact of the optimal angle on the cogging torque, output power, and generator efficiency is investigated. This analytical study with experimental testing proves that the optimal angle between opposite teeth can significantly decrease cogging torque and improve output power and efficiency. The results show that cogging torque decreases significantly (4\u20135 times) at an optimal angle of 7.5\u00b0 as compared with that of other angles, although magnetic flux and output power decline slightly but efficiency increases. Recently, axial flux permanent magnet (AFPM) electrical machines have represented the most potential class of energy transforming appliances, which provide the best power/weight ratio and excellent efficiency for different applications ,2,3,4. THowever, the use of PMs produces the effect of cogging torque, which arises due to the interactions between the PMs and stator slots. The tendency of PM rotors to align in a few stable positions results in an alternating pulsating force ; cogging torque does not contribute to the average torque ,6,7. ThePreviously, several methods have been proposed and analyzed for reduce the effect of cogging torque ,19,20,21In this study, we provide a comprehensive experimental analysis of the cogging effect in a machine with a double-stator design. The torque deviations because of the cogging effect are studied through the rotor and stator rotation angle. The effect of the angle between opposite double-stator teeth on the cogging magnitudes and the output power is also studied.We experimentally tested and analyzed a specially developed axial flux double-stator generator with PMs attached in the rotor. There were several reasons for the choice of this type of generator design. First, the magnetic flux penetrating the rotor placed inside two opposite halves of stators is higher than that in a generator with a double rotor; thus, it can effectively work in lower rotating velocities and produce more power. Second, a single rotor includes half of the PM segments as compared with a double-rotor frame; this circumstance decreases the cost of the machine, since the PMs determine a significant portion of the expenses. The third reason is related to cooling possibilities; the main heat flux is produced in the winding part of the machine, which is the stators located outside of a disk rotor equipped with permanent magnets, and therefore the ventilation cooling can be more efficient.i.Stator teeth should consider the dimensions of the PMs.ii.The teeth dimensions should induce a maximal amount of torque between the stators and the PMs. For this aim, the teeth width should be slightly narrower than the width of a PM.iii.The stator teeth shoes were made with the optimal dimensions for absorbing maximum magnetic flux.The stator cores were made using cold-rolled grain-oriented silicon steel that was insulated with a special heat resistant coating. The material was designed to substantially reduce core losses and to produce certain magnetic properties and high permeability. The thickness of the core laminate was 0.05 mm, with a permeability of i.High magnetic flux density relative to weight and size;ii.Temperature resistant;iii.Availability and costs;iv.Stator core size compatibility.In each stator, 24 slots were created with a width of 6 mm and a height of 25 mm for each tooth. A CNC wire cutting machine was used due to the required complex geometry of the stators a. Each si.Non-metallic isolating material to support (frame) the PMs and eliminate loss due to conductivity of the backing material;ii.An even number of PMs;iii.Minimal thickness to reduce the air gap between the stators and the rotor;iv.Reliable and strong enough to be machined while maintaining tolerances.The PMs chosen for the system were neodymium (N45M) magnets with a composition of A 12-layer carbon fiber fabric reinforced with epoxy composite material (3 mm thickness) was chosen for the rotor. The material was cut using a CNC machine and attached to an iron axle using screws, shims, and attach plates for precise locating with an even distance from both stators to ensure an air gap of 0.5 mm b.Each coil group terminals were connected to a Schottky diode bridge rectifier for DC current conversion. All rectifier outputs in both halves of the stators were serially connected to provide the desired voltage level and to compensate for possible phase misalignment when opposite teeth shifted at some angles. The main parameters  of the proposed AFPM generator are shown in The cogging effect is manifested due to the attractive forces acting between permanent magnets of a rotor and stator teeth. The cogging effect produces an alternating torque, which disturbs even rotor movement especially perceptible at low velocities, and therefore causes technical difficulties in AFPM machines. In this study, we aimed at finding a relatively simple way to diminish the magnitude cogging effect. This method was based on the optimal angle shifting between opposite teeth of a double stator. To ensure this approach, estimation of the produced interactive forces (torque) between a rotor and stator must be carried out for the different stator\u2013rotor relative dispositions. The cogging torque value can be obtained as the derivative of the total magnetic field energy The total energy is a result of the interaction between stator teeth with all PMs in the rotor. The system of partial differential equations (PDE) must be solved to find the total energy analytically. The theoretical decision of such a PDE system for a complex ferromagnetic stator tooth-PM configuration seems practically impossible. Therefore, we developed a semi-analytical approach based on numerical calculations of magnetic field energy for different rotating angles with the following analytical approximation of obtained values using a polynomial function and its differentiation. The numerical assessment of magnetic field flux and energy was carried out using COMSOL software . The cogThe output characteristics are used to analyze the AFPM generator. These characteristics are output voltage, power, and efficiency as a function of DC load current. These parameters are calculated based on the energy flow, as shown in Mechanical and iron power losses ( (\u03c9mech)  as folloThe copper losses are taken into consideration as a result of the stator winding resistance  and \u03c9 (rad/s) are the rotation angle and velocity, and \u03c6 (Wb) is the magnetic flux penetrating every single coil.Special attention should be applied to the assessment of the output voltage, which is calculated according to electromotive force (EMF) produced by serially connected stator windings. The EMF of a single coil  of the generator were obtained. The output characteristics included voltage (V), current (I), torque (\u03c4), and velocity (n). The primary motor was operated at the required rotating velocity, and the velocity was changed for each set of experiments . The generator was loaded with an electronic load  [The experimental tests were conducted according to /1200 W)  for eachThe cogging torque was calculated for the varying rotation angles. The torque sensor generated time-dependent data from the scope. Knowing the distance between pulses allowed us to translate time-dependent to an angle-dependent torque signal. Furthermore, juxtaposing the torque and angle data allowed us to determine the function of the cogging torque vs. the rotation angle. On the basis of this function, the average cogging torque was estimated as follows:The integration was solved numerically by summating the current values of a torque multiplied on the elemental growths of the angle propagation.Cogging torque and the performance of the generator were measured at different angles, i.e., 0\u00b0, 7.5\u00b0, and 11.25\u00b0. The performance of the generator was also checked at various velocities and under varying loads. The cogging torque values were measured using a torque meter. The cogging torque values as a function of rotation angles for different angles  are shown in Here, we also want to discuss the performance of the generator at a low cogging torque configuration. This study also verified that any configuration change would not damage the performance capability of the generator. The different configurations  were tested at a speed range of 1200\u20131700 rpm and varying loads of 0\u20132 A. The current-voltage (I\u2013V) curves of the AFPM generator at 1500 rpm are shown in The efficiency and power of the generator for different shifting angles and rotating velocities are represented in This study investigated cogging torque reduction in AFPM machines using stator to stator angle displacement. The machine topology allowed us to adjust its geometry to investigate and achieve the target. The experimental and theoretical analysis proved that the 7.5\u00b0 displacement exhibited a significant reduction in cogging torque (four to five times) and good performance. However, shifting to the optimal angle was accompanied by a slight decrease in the magnetic flux and output power (~5\u201310%). More importantly, it led to improved efficiency at high rotational speeds. This study showed the superiority of AFPM generators over conventional radial flux types regarding the achieved output power versus generator weight. This advantage is especially noticeable at low rotational speeds."}
+{"text": "Specific markers signing RBC senescence were quantified after the incubation with SCD plasma samples. The physiologic in-flow adhesion was investigated on senescent RBCs, an in vitro technic into biochips that mimic adherence of RBCs during the occlusive events of SCD. RESULTS: Senescence markers on AA RBCs, together with their in-flow adhesion to the plasma-bridging protein thrombospondin, were associated with the clinical status of the SCD patients from whom plasma was obtained. In these experiments, the highest values were obtained for SCD acute plasma samples. Adhesion of senescent RBCs into biochips, which is not reversed by a pre-treatment with recombinant Annexin V, can be reproduced with the use of chemical agents acting on RBC membrane channels that regulate either Ca2+ entry or modulating RBC hydration. CONCLUSION: We found that markers on red cells are correlated, and that the senescence induced by SCD plasma provokes the adhesion of RBCs to the vessel wall protein thrombospondin. In-flow adhesion of senescent red cells after plasma co-incubations can be reproduced with the use of modulators of RBC membrane channels; activating the Piezo1 Ca2+ mechanosensitive channel provokes RBC adhesion of normal (non-senescent) RBCs, while blocking the Ca2+-dependent K+ Gardos channel, can reverse it. Clinically modulating the RBC adhesion to vascular wall proteins might be a promising avenue for the treatment of painful occlusive events in SCD. BACKGROUND: Blood transfusion remains a key treatment for managing occlusive episodes and painful crises in sickle-cell disease (SCD). In that clinical context, red blood cells (RBCs) from donors and transfused to patients, may be affected by plasma components in the recipients\u2019 blood. Senescence lesion markers appear on the red cells after transfusion, shortening the RBC lifespan in circulation. In the specific context of SCD, senescence signals can also trigger the occlusive painful events, typical of the disease. This work follows through our previous data that described a RBC senescence process, rapidly detected after challenge with SCD pathological plasmas. In this clinical context, we wanted here to further explore the characteristics and physiologic consequences of AA RBC lesions associated with senescence, as lesions caused by RBCs after transfusion may have adverse consequences for SCD patients. METHODS: Plasma samples from SCD patients, with acute symptoms ( Blood transfusion is part of the therapeutic arsenal in various diseases, for increasing circulating hemoglobin (Hb) levels and improving oxygen delivery to tissues . To assu2+ increase  occurring in altered or physiologically aged RBCs seems to be a central event leading to RBC \u201ceryptotic\u201d senescence or ageing. Calcium increase into red cells modifies the physical properties of RBCs by regulating cell volume [+ Gardos/KCa3.1 channels, modulating RBC hydration and leading to a decrease in size [The intracellular Cal volume ,17; this in size ,17,18. T in size ,20 or ph in size ,22. With the use of stored RBC units selected on the basis of their relative age (\u201cfresh\u201d (5 to 8 days) versus \u201cold\u201d (\u226538 days) units), we previously demonstrated that SCD plasmas can adversely affect RBCs from AA donor units in co-incubations at +37 \u00b0C  with theThe transfusion of blood units is often required in patients with homozygous (SS) sickle-cell disease (SCD), a monogenic red blood cell disorder that provokes the formation of an abnormal polymerizing hemoglobin, named HbS ,9,26. InAnother characteristic and specificity of SCD patients is the detection of abnormally dense RBCs (density\u2009 >1.11) in circulation, that comprise a substantial number of irreversibly sickled cells with HbS polymer. They have high mean corpuscular Hb concentrations due to dehydration ,29. In oIn this work, we wanted to shed additional light on red cell senescence parameters, previously detected in our team after challenge with plasmas and further explored the characteristics and behavior of senescent RBCs due to contact with sickle-cell plasmas, as lesions caused to donor-transfused AA RBCs may have adverse consequences in the transfusion context of patients with SCD. Notably, we investigated the correlations existing between the markers appearing on senescent red cells, which have never been shown before, and the possible contribution of senescent AA RBCs to the adverse consequences of RBC adherence that occur in sickle painful crisis. Full details regarding the recruitment of patients and controls, the selection of RBC units, and the methods used are provided in the g for 10 min at room temperature. Processing the RBC units from whole blood of (AA) donors was carried out at EFS Preparation  according to the European guidelines ; the RBCn = 31) or during hospitalization for acute symptoms . The selected SCD plasma were thawed and incubated with RBCs at an 8% Hct drawn from stored blood units, for 24 or 48 h at +37 \u00b0C, as previously described [Plasma samples were obtained from 54 randomly selected adult patients with a definitive diagnosis of SCD  from Henri-Mondor Hospital . Patients were followed for routine explorations outside of painful episodes  to diluted AA RBCs (0.5% Hct) was carried out for a 10 min incubation at +4 \u00b0C before processing the in-flow adhesion assays. Where indicated, fresh native RBCs from blood units were treated with the Piezo1/TRPC5 channel agonist Yoda1 (50 mM) for 30 min at +37 \u00b0C, with or without the Gardos/KCa3.1 inhibitors Senicapoc or TRAM-34 (10 mM each) as pretreatments for 10 min at +37 \u00b0C before Yoda-1 addition. At the end of these treatments, diluted AA RBCs (0.5% Hct in HBSS /BSA) were processed the flowing adhesion assays. t test (Wilcoxon signed-rank tests), as appropriate, with the Prism software (GraphPad Inc). Values of p < 0.05 were considered significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Linear regressions were made to test the correlations between the senescence markers for the patients\u2019 plasma, tested for whom we collected results for the three different markers (n = 20) using the Stata software (StataCorp).Statistical analysis was performed with Kruskal\u2013Wallis, Mann\u2013Whitney U, or two-tailed Student\u2019s First, we confirmed here the previous findings obtained from our team and from other groups ,19 showip < 0.0001; Wilcoxon test). We found that the PS-positive RBC percentage (PS+ RBC%) is dependent and correlated with the time of in vitro contact at +37 \u00b0C between AA RBCs from units and sickle plasmas see: : the PS+p = 0.2999). After 48 h, the PS+ RBC population was significantly larger for plasma samples collected in painful conditions  than in those collected in basal \u201csteady-state\u201d conditions (p < 0.0001)  and basal \u201csteady-state\u201d plasmas (collected from patients out of the context of painful episodes or transfusions) . A weak p < 0001) on PS-exposure quantification. The duration of the RBC/plasma contact (T 24 h and T 48 h) had a less but also significant effect (p = 0001) and a significant interaction between the two variables is observed (p < 0001)  had a highly significant effect (n = 54 plasmas), we needed to show the results for the three senescence markers in parallel. For this, we detailed in three graphs the results obtained for 20 individual SCD plasma samples for whom we had collected data, at T 24 h and T 48 h co-incubation, for the three RBC markers: PS-exposure on red cells, calcium influx into red cells and reduction in RBC size (2+ influx positive red cells (%) were near zero for both markers. After these first results concerning the PS-exposure of senescent AA RBCs after plasma challenge, at 24 and 48 h co-incubations . Similarly, no correlation was found between PS-exposure (Annexin V-FITC positive staining) and calcium influx (p = 0.4765). Only the changes quantified between PS-exposure and RBC size were found to be inversely correlated . Following the incubation of the AA RBCs for 48 h, all the markers of RBC senescence were found to be well correlated (Following co-incubations for 24 h with the plasma samples (n = 5) or experiencing a painful acute-phase episode  at the time of sample collection. As a control, plasma samples from healthy adult donors  were also tested . Numbers in percentage indicate on the graphs the respective changes obtained for each category of plasma between the 0.5 Dyn and the 5 Dyn shear stress applied in the test.2, respectively).If PS exposure on senescent AA RBCs is mechanistically involved in the in-flow adhesion of those particular RBCs to TSP, then the blockade of PS with Annexin V should significantly decrease the level of adhesion observed in adhesion assays to TSP . PreviouCalcium influx into red cells is a central event that lead to the RBC senescence mechanism. Furthermore, the stimulation of calcium influx by acting on RBC channels with a calcium ionophore can rapidly modify the adhesion properties of normal RBCs to vascular vessel wall proteins ,30.In parallel, dense dehydrated RBCs observed in SCD patients have the ability to adhere to vessel wall proteins . ConsideWhen native AA RBCs from units showed only slight adhesion to TSP (12.6 \u00b1 1.1%), the Yoda1 treatment (10 and 25 \u00b5M) induced a dose-dependent increase in RBC adhesion to TSP in that physiologic assay, up to 66.9 \u00b1 28.3%. When RBCs stimulated to adhere with Yoda1 (25 \u00b5M) were pretreated with the specific inhibitors of the Gardos/KCa3.1 channel (Senicapoc and TRAM-34), their adhesion to TSP decreased to reach the values of 34.2 \u00b1 6.7 and 27.2 \u00b1 2.1%, respectively. Such a result clearly shows that abnormal activation of Piezo1 and Gardos ion channels in RBC membranes can modulate the adhesion of normal RBCs to the vascular wall-associated proteins.We confirmed here that pathological plasma samples from SCD patients can directly affect ex vivo the integrity of (HbA) RBCs from blood units, when in contact with them. The deleterious impact of plasmas can alter several properties of RBC membranes, including cation permeability , dehydration (cell shrinkage or reduction in RBC size) and phospholipid scrambling (exposure of PS on the outer membrane)n = 54) as we describe here.Previous findings reported by Lang et al. ,22,31 orFrom a mechanistic point of view, we concluded that the strong proinflammatory environment found in plasmas cannot by itself promote rapid alterations to RBCs . InsteadOne of the key features demonstrated here is the relationship existing between the markers of RBC senescence. The three markers quantified after challenge with plasma samples , that characterize senescence, were found to be closely correlated after a T 48 h plasma contact. Although this result was not unexpected, it has never been demonstrated before. Our results particularly focused on the phospholipid PS easily detected on senescent AA RBCs.PS-exposure can provoke the destruction of immunoglobulin-sensitized RBCs by macrophages or activated complement ,33; it mInstead of being impacted by sPLA2, we rather imagine that free Hb or free heme byproducts of RBC hemolysis, present in the SCD plasma samples used in co-incubation experiments, are the factors that may provoke the senescence of AA RBCs, through oxidative stress, in our model. Gatidis et al.  reported2+-dependent K+ efflux erythrocyte senescence driven by intracellular Ca2+ activating the Gardos channel. In their context, the mechanism is also due to Piezo1-mediated calcium permeation, a phenomenon that can be described on red cells physiologically aged in circulation, on pathological sickle erythrocytes, or RBCs extensively stored for transfusion or artificially dehydrated [Recent works from Klei et al. on Gardos and Piezo1 relative activities on RBC removal, by way of acting on RBC dehydration and deformability, have also shed new insights ,40 on thhydrated . Wadud ehydrated .In parallel to the implication of Piezo1 we tested in that study, the mechanistic insights about calcium influx-related sickle RBC dehydration are much larger and more multifactorial . CalciumThe thrombospondin (TSP) vessel matrix protein was chosen here as the coated molecule into Vena8 biochips for in-flow adhesion tests. We previously verified that senescent RBCs after SCD plasma contact did not lead to adhesion with the two most common extracellular matrix (ECM) proteins Laminin or Fibronectin ,46  , the mol2+ influx into red cells [The TSP\u2013CD47 signaling pathway can mediate RBC survival and deformability by in mediating Caed cells , and a Ted cells . Here inWe showed here that red cells from units can suffer stress after exposure to plasma samples from SCD patients. Nevertheless, some limitations in the study and our results can be pointed out.One fact that limits the study\u2019s originality is that we are not the first to describe this ex vivo deleterious effect of pathological plasma obtained from patients on RBCs from units, as reported by Lang et al. or Kempe et al.; also in our laboratory, we already described that the acute plasmas from various pathological contexts can have such an impact on red cells leading to senescence.Another limitation, in the way to definitively prove the mechanistic scenario of calcium-induced adhesion, could be the absence of control experiments in the adhesion tests with the sickle plasma-treated AA-RBCs, pre-treated or not with Gardos inhibitors. Furthermore, we need also to increase the number of plasma samples from SCD patients (i) to implement results concerning the in-flow physiological adhesion tests in biochips and (ii) the in vitro effects of chemical effectors of RBC channels on adhesion to upgrade the significance of our results.In the experiments with recombinant Annexin V, a bias could be that Annexin V added did not totally bind to the large amount of PS-exposed on senescent RBCs. In further experiments, it could be important to verify this fact with rapid FACS analysis of PS-positive senescent RBCs pre-incubated with similar amounts of Annexin V.Calcium influx in RBCs can be considered multi-faceted, as it is not only regulated by the only Piezo1 channel, with TSP and CD47 directly implicated in it. The fact that the TSP\u2013CD47 signaling pathway wasn\u2019t explored in our work could be considered as a weak point.Finally, further biochemical studies will be required now to clearly identify the mediators in the plasma that potentiate the transfusion-driven alterations to AA RBCs.We showed that AA red cells from blood units can suffer stress after exposure to plasma samples from SCD patients. Nevertheless, at this time point, how TSP and senescent AA RBC interacts is yet to be determined. It will be now of importance to better understand the in vivo fate of transfused RBCs in such pathological contexts, collecting and testing a higher number of SCD samples, as this phenomenon can have adverse clinical consequences for the patients.We think that new insights on RBC senescence and their connection to red cell adhesion to the vessels, will give an easy way to overcome, in the future, the deleterious occlusive events in SCD. Clinical studies might be necessary to confirm these results obtained ex vivo along with mechanistic studies on the byproducts of RBC destruction, factors which may play a role in the RBC damage. How to overcome senescence may now be the key to discovering successful future treatments of anemia or hemolytic transfusion reactions in SCD. The capability we demonstrated here, to easily modulate the adhesion of AA RBCs to vascular-wall proteins, might represent a promising target for the treatment of occlusive events in that disease."}
+{"text": "Dysmorphic pulmonary vascular growth and abnormal endothelial cell (EC) proliferation are paradoxically observed in premature infants with bronchopulmonary dysplasia (BPD), despite vascular pruning. The pentose phosphate pathway (PPP), a metabolic pathway parallel to glycolysis, generates NADPH as a reducing equivalent and ribose 5-phosphate for nucleotide synthesis. It is unknown whether hyperoxia, a known mediator of BPD in rodent models, alters glycolysis and the PPP in lung ECs. We hypothesized that hyperoxia increases glycolysis and the PPP, resulting in abnormal EC proliferation and dysmorphic angiogenesis in neonatal mice. To test this hypothesis, lung ECs and newborn mice were exposed to hyperoxia and allowed to recover in air. Hyperoxia increased glycolysis and the PPP. Increased PPP, but not glycolysis, caused hyperoxia-induced abnormal EC proliferation. Blocking the PPP reduced hyperoxia-induced glucose\u2013derived deoxynucleotide synthesis in cultured ECs. In neonatal mice, hyperoxia-induced abnormal EC proliferation, dysmorphic angiogenesis, and alveolar simplification were augmented by nanoparticle-mediated endothelial overexpression of phosphogluconate dehydrogenase, the second enzyme in the PPP. These effects were attenuated by inhibitors of the PPP. Neonatal hyperoxia augments the PPP, causing abnormal lung EC proliferation, dysmorphic vascular development, and alveolar simplification. These observations provide mechanisms and potential metabolic targets to prevent BPD-associated vascular dysgenesis.  Babies born prematurely have underdeveloped lungs that do not facilitate proper gas exchange; therefore, they need respiratory support, including supplemental oxygen and/or mechanical ventilation, as lifesaving measures. Unfortunately, these therapies can cause bronchopulmonary dysplasia (BPD), a chronic lung disease, which affects 10,000\u201315,000 premature infants annually in the USA. Although most BPD survivors eventually are weaned off oxygen, they may show evidence of persistent pulmonary injury and lung function decline as adolescents and adults . Lung paECs are highly glycolytic, generating more than 80% of their ATP through this pathway , 11. Gly2/5% CO2) for 24 hours, followed by air recovery (21% O2/5% CO2) for 24 hours. Exposure to hyperoxia significantly increased ECAR, which was reflected by augmented basal glycolysis and glycolytic capacity (2 derived from the tricarboxylic acid cycle can contribute to the ECAR. We thus performed a glycolytic rate assay (GRA), which eliminates the contribution of CO2 from the tricarboxylic acid cycle to ECAR. Proton efflux rate from glycolysis was increased in lung ECs exposed to hyperoxia (13C]lactate by a nuclear magnetic resonance (NMR) when cells were treated with glucose. As shown in 13C]lactate in MFLM-91U cells. In addition, we performed untargeted metabolomics to detect levels of intracellular metabolites by mass spectrometry (MS) in lung ECs exposed to hyperoxia (https://doi.org/10.1172/jci.insight.137594DS1). Specifically, we found that the levels of glucose 6-phosphate (G6P), G1P, fructose 1,6-bisphosphate , and dihydroxyacetone phosphate (DHAP) were significantly reduced, whereas levels of glycolytic products pyruvate and lactate were increased in primary mouse LMVECs exposed to hyperoxia   did not have any effects on hyperoxia-induced EdU incorporation  . Levels  (50 \u03bcM) . Levels poration . These rHyperoxic exposure has been shown to reduce EC migration . It is n+ (vWF+) cells was significantly increased in mice at P14 after exposure to hyperoxia as newborns  were exposed to hyperoxia for 3 days and allowed to recover in room air until P7 or P14. This model tests whether the effects of hyperoxia are persistent in vivo. Untargeted metabolomics were performed in the mouse lungs at P7 . Hyperoxnewborns . In additilation . These r+ cells between air and hyperoxia groups, hyperoxic exposure significantly increased the number of EdU+/vWF+ lung EC cells  or 6-AN  daily for 5 days between P9 and P13, or for 2 days between P12 and P13. The total number of EdUt manner . Lung ECt manner . Similart manner . In contse lungs . These dWe next measured lung levels of CD31, another EC marker, and found that hyperoxic exposure did not alter CD31 protein in mouse lungs, nor were lung CD31 protein levels changed by DHEA (20 mg/kg) or 6-AN (10 mg/kg) . Howeverpgd under the control of the human CDH5 promoter  for 48 hours with lipofectamine 3000 (Invitrogen)  were purchased from Seven Hills Bioreagents and cultured in UltraCulture medium (BioWhittaker) . There witrogen) .2 and 5% CO2) or air (21% O2 and 5% CO2) for 24 hours, followed by normoxia for 24 hours (2) for 72 hours in an A-chamber (BioSpherix) (Cells at 70%\u201380% confluence were exposed to hyperoxia (95% O24 hours . CultureSpherix) . The damLung alveolar formation starts at P4, and it peaks between P10 and P14 , 43. Thupgd or empty vector under the control of human CDH5 promoter at an optimized ratio of 1 \u03bcg plasmid DNA to 3 \u03bcL nanoparticles for 10 minutes at room temperature. Each C57BL/6J mouse received 3 \u03bcg of plasmid DNA via a retro-orbital injection at P9 , oligomycin , and 2-DG  into ports A, B, and C, respectively. The Seahorse XF Analyzer was calibrated, and the assay was performed using the Seahorse XF Glycolysis Stress Test Assay protocol as suggested by the manufacturer  and 2-DG (500 mM) into port A and port B, respectively. The Seahorse XF Analyzer was calibrated, and the assay was performed using the Seahorse XF GRA protocol as suggested by the manufacturer. Proton efflux rate was recorded, which was normalized to numbers of living cells in representative wells.Cells were incubated with the XF base medium without phenol red, which was supplemented with 5.0 mM HEPES (Thermo Fisher Scientific), 10 mM glucose, 2 mM glutamine (Caisson Labs), and 1 mM pyruvate (Invitrogen) for 1 hour in a non-COg for 10 minutes at 4\u00b0C. The top layer (aqueous phase) was collected for untargeted metabolomics analysis using a Thermo Fisher Scientific Ultimate 3000 LC coupled with a Q-Exactive Plus mass spectrometer. A total of 5 \u03bcL of each sample was injected on a Zic-pHILIC Column . The mobile phases were (A) 20 mM ammonium carbonate in 0.1 % ammonium hydroxide and (B) acetonitrile 97% in water. The gradient conditions were as follows: 100% B at 0 minutes, 40% B at 20 minutes, 0% B at 30 minutes for 5 minutes, and then back to 100% B in 5 minutes, followed by 10 minutes of reequilibration. Full MS spectra were acquired in switching polarity at 70,000 resolution, covering a range of mz 66 to 1000. Compound discoverer 3.0  was used to generate a list of features (mass/charge ratio [mz] and retention time) found in a pool sample . This list was used as an inclusion list for MS/MS runs in positive and negative ion modes separately. Using CD, the features that were selected for MS/MS in the first MS/MS run were then removed from the inclusion list, and the MS/MS experiments were repeated with the new list. By repeating this process 4 times, we were able to obtain MS/MS data for most of the features detected by CD. All the data were then combined and analyzed in CD. Likely elemental compositions were computed based on the accurate mass and isotope pattern, and a mzCloud MS/MS spectra database, a local MzVault database, and chemspider libraries were searched to identify possible candidates , or 50 mg of lung tissues were homogenized with 2 mL of chilled methanol. These mixtures were combined with 4 mL of HPLC grade chloroform  on ice. The mixtures were vortexed for 1 minute, incubated in an ultrasound bath for 5 minutes, and then combined with 2 mL of HPLC grade water . The samples were vortexed and centrifuged at 180013C]glucose (Cambridge Isotope Laboratories) for 24 hours during the air recovery phase after hyperoxic exposure. Intracellular [13C]-labeled deoxynucleotides or nucleotides from [U-13C]glucose were detected using the MS as described previously . A total of 1 mL of \u201380\u00b0C cold 80% methanol was added, which was incubated at \u201380\u00b0C for 20 minutes. Cells were then scraped off, and supernatants were transferred into microfuge tubes. Samples were pelleted at 4\u00b0C for 5 minutes at 14,000 rpm, and the supernatants were transferred into an Eppendorf microfuge tube. Cell pellets were reextracted with 200 \u03bcL ice-cold 80% MeOH and spun down, and the supernatants were combined. Samples were dried at room temperature under a vacuum and resuspended in 100 \u03bcL of acetonitrile containing 10 \u03bcM of adenosine-13C10,15N5-monophosphate  as internal standard (IS). A 6-point standard curve was prepared in the same acetonitrile plus IS from pure chemical standards  as a 1- to 10-dilution series with 100 \u03bcM as the highest concentration. Samples were analyzed by liquid chromatography\u2013MS (LC-MS) on a Vanquish LC coupled to an ID-X MS (Thermo Fisher Scientific). Sample or standard (5 \u03bcL) was injected on a ZIC-pHILIC peek-coated column . Buffer A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water; Buffer B was 97% acetonitrile in water. The LC program was as follows: starting at 99% B, to 40% B in 17 minutes, then to 0% B in 10 minutes, maintained at 0% B for 5 minutes, then back to 99% B in 4 minutes, and reequilibrated at 99% B for 11 minutes. The flow rate was maintained at 0.15 mL/min. Data were acquired on the ID-X in switching polarities at 500,000 resolution, with an AGC target of 1 \u00d7 105, and a m/z range of 65 to 1000. The combined extracted ion chromatogram for all m/z corresponding to all isotopologues for each compound was plotted, and the corresponding peak area was integrated. The total combined concentration of all isotopologues of each compound was calculated using this area divided by the area of the IS and using the standard curves. The relative intensity of each isotopologue for each compound was obtained by combining the mass spectra of the 3 scans at the top of each peak. In the obtained mass spectrum, the intensity of each isotopologue\u2019s m/z was extracted and used to calculate the relative abundances.One million to 2 million cells were cultured in a 60 mm dish with DMEM medium containing 20 mM [U-eviously . Briefly13C]glucose (Cambridge Isotope Laboratories) for 24 hours during the air recovery phase after hyperoxic exposure. The extraction of intracellular metabolites and the NMR analysis of [13C]-labeled lactate from glucose were performed as previously described . 1H-NMR spectra of extracellular extracts were acquired using a 9.6 kHz spectral width and 32 K data points. The acquisition time was 1.7 seconds, and the relaxation delay was 10 seconds with 128 scans. Proton decoupled 13C spectra were acquired on a Bruker 600 spectrometer. Proton decoupling was performed using a Waltz-16 sequence. 13C NMR parameters include a relaxation delay of 30 seconds, an acquisition time of 1 second, and spectral width of 36 kHz. Further quantitative processing of the experimental results was performed with proprietary software implemented in the software package MestReNova.Cells were incubated with 20 mM [1,2-escribed . In brieGlucose uptake was performed by detecting the uptake of 2-NBDG by culture cells using the 2-NBDG Glucose Uptake Assay Kit (Abcam) according to the manufacturer\u2019s instructions. In brief, cells were incubated with glucose-free HBSS medium for 30 minutes after washing with PBS. After washing with PBS, cells were incubated with 2-NBDG (100 \u03bcM) for 30 minutes at 37\u00b0C. Cells were then resuspended in 150 \u03bcL of prechilled PBS containing 0.5% BSA, and flow cytometry analysis was performed within 30 minutes.L-lactate was measured in cells and lung tissues using the L-lactate Assay kit  according to the manufacturer\u2019s instructions. All samples were deproteinized with the Deproteinizing Sample Preparation Kit-TCA  before analysis. Protein concentration in samples before deproteinization was measured for normalization.6 cells were pelleted and lysed in 800 \u03bcL extraction buffer provided using a Dounce homogenizer. DNA was sheared with a needle in lysates, and the enzymes consuming NAHPD were removed by filtering the samples through a 10 kD spin column . A total of 200 \u03bcL of lysates was heated at 60\u00b0C for 30 minutes to decompose NADP+ while NADPH was intact, which was used for assessing NADPH concentration. Protein concentration in samples before deproteinization was measured for normalization.Levels of NADPH were determined in cells and lung tissues using the kit from Abcam  according to the manufacturer\u2019s protocol. Briefly, 50 mg of tissue or 2 \u00d7 10In vitro proliferation was determined by Click-iT EdU Flow Cytometry Assay Kit from Invitrogen according to the manufacturer\u2019s protocol. Briefly, cells were incubated with 10 \u03bcM of a nucleoside analog of thymidine  for 2 hours. After washing and fixation, incorporated EdU during DNA synthesis was labeled with Alexa Fluor 488 azide (Thermo Fisher Scientific) in the provided reaction buffer for 30 minutes. Cells were analyzed using the BD FACSAria (BD Bioscience).+ cells were counted in 3 randomly selected high-power fields (HPF) using a Zeiss Axiovert 200M Fluorescence Microscope.For mouse lung EdU detection, mice will be i.p. injected with EdU  for 3 days and then sacrificed at 24 hours after the last EdU injection. Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 was used to determine proliferation in lungs, which were costained with the EC marker vWF as described previously . The numCell migration was assessed in cells using the Scratch assay . Once reWestern blot was performed as we described previously . The mem\u2013\u0394\u0394Ct method.Total RNA was extracted by the TRIzol reagent and purified using the RNeasy miniprep kit (Qiagen). RNA samples were quantified by the NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific). Then, 400 ng of total RNA were used for reverse transcription using the Taqman Reverse Transcription Reagents (Thermo Fisher Scientific). A total of 1 \u03bcL of cDNA was used for real-time PCR reactions by the 7300 Real-Time PCR System (Applied Biosystems). All Taqman gene probes were purchased from Thermo Fisher Scientific . Gene ex+/vWF+ and PCNA+/vWF+ cells in mouse lungs were counted in 3 randomly selected HPF using a Zeiss Axiovert 200M Fluorescence Microscope. Fluorescent intensity of PGD+/CD31+ cells in human lungs was evaluated using an ImageJ software. These experiments were carried out in a blinded manner.Lung sections were deparaffinized and subjected to heat-mediated antigen retrieval in a citrate buffer solution (Vector Labs) and then stained overnight at 4\u00b0C with antibodies against PGD, PCNA, vWF, and CD31 . After i2O, and we fixed them with 4% neutral buffered paraformaldehyde. These fixed lungs were embedded in paraffin and sectioned into 4 \u03bcm sections using a rotary microtome . Lung midsagittal sections with H&E staining were utilized to determine airspace Lm. A perpendicular line was drawn from the center of the respiratory bronchiole to the distal acinus (as defined by the pleura or the nearest connective tissue septum). The number of septa intersected by each line was counted as RAC, and a minimum of 8 counts were performed for each animal.Lm and RAC were measured in mouse lungs stained with H&E as previously described . In briet test was used for detecting statistical significance of the differences between means of 2 groups after checking the normality of data. The statistical significance of the differences among groups was evaluated by using 2-way ANOVA for overall significance, followed by the Tukey-Kramer test. Statistical significance was defined as P < 0.05.Statistical analyses were performed using GraphPad Prism 8. The results were expressed as mean \u00b1 SEM. The 1-tailed All animal experiments were reviewed and approved by the IACUC of Brown University. Utilization of human lung samples was done in compliance with the IRB guidelines of Women and Infants Hospital.HY designed the study, conducted experiments, acquired and analyzed data, drafted the initial manuscript, and revised the manuscript. JG, ZF, ALP, JFC, XL, HZ, XJ, YYZ, and MEDP performed experiments and data analysis. PAD assisted with the experimental design and revised the manuscript. All authors approved the final manuscript as submitted."}
+{"text": "Regular physical activity had been shown to reduce morbidity and mortality from chronic diseases such as cardiovascular diseases, hypertension, type 2 diabetes mellitus, dyslipidaemia, obesity/metabolic syndrome, osteoarthritis, osteoporosis, bronchial asthma and chronic obstructive pulmonary disease. Research had shown that physically active doctors were more likely to offer exercise counselling to patients. However, few studies looked into this association with counselling practices to patients with specific chronic diseases. This study aims to determine the association between physical activity levels of primary care doctors (PCDs) in Malaysian private practice with physical activity counselling to patients with chronic diseases.A cross-sectional study involving PCDs in private practice in 3 states was done. Participants were recruited from members of the Malaysian Academy of Family Physicians and attendees of a conference. A self-administered questionnaire obtained information on sociodemography, initiation of exercise counselling to patients with chronic diseases as well as physical activity levels using the International Physical Activity Questionnaire (IPAQ).The response rate was 32.3% (272/842). 47.1% of the respondents were post-graduate holders. 50% of participants had a moderate level of physical activity and 24.3% a high level. Most respondents answered \u2018always\u2019 or \u2018often\u2019 for initiation of exercise counselling to patients with cardiovascular diseases (59.9%), hypertension (72.8%), type 2 diabetes mellitus (78.6%), obesity/metabolic syndrome (86.4%), dyslipidaemia (81.6%), osteoarthritis/osteoporosis (41.9%) and bronchial asthma/COPD (29.5%). PCDs being physically active and non-smokers were associated with a higher initiation of exercise counselling to patients with cardiovascular diseases. Doctors with post-graduate degrees were more likely to offer exercise counselling to hypertensive patients.The association between PCDs\u2019 physical activity levels and their physical activity counselling varies between different types of chronic diseases. Primary care doctors with higher physical activity levels were more likely to initiate physical activity counselling in patients with cardiovascular disease during chronic disease follow up visits.The online version contains supplementary material available at 10.1186/s12875-022-01657-3. Prevention is one of the building blocks of primary care and nothing paves the way for that like a healthy lifestyle. Apart from a balanced diet and controlled levels of stress, physical activity constitutes an extremely important part of a healthy lifestyle. Regular physical activity is vital for well-being and health for people of all ages as it has been shown to reduce morbidity and mortality from many chronic diseases .Chronic diseases, which are also known as non-communicable diseases (NCDs) caused 71% of all deaths globally in 2016 and 73% of all deaths in Malaysia in 2014 [Physical inactivity is one of the behavioural risk factors causally linked with cardiovascular diseases, chronic respiratory disease, cancer and diabetes and it can lead to changes in metabolic profiles which include elevated blood pressure, increased glucose and lipid levels as well as overweight/obesity . RegularPrimary care doctors (PCDs) have the advantage of being able to offer physical activity counselling to patients in their practice, being in the frontline and seeing patients in the earliest stages of chronic diseases. Research had shown that physicians\u2019 own exercise habits influence their physical activity counselling of patients. Previous studies and systematic reviews had reported that healthcare providers who were physically active were more likely to counsel their patients on physical activity more compared to doctors who do not , 8. ThesIn Malaysia, primary care management of patients with chronic diseases is offered both in government and private health clinics . PrivateIn contrast with other previous studies, a study conducted amongst PCDs in Malaysian government health clinics found no significant association between physical activity levels of PCDs and physical activity counselling to patients with chronic diseases. Only a third of PCDs offered physical activity counselling to 50% or more of their patients . It is uThe general objective of this study is to determine if there is an association between physical activity levels of PCDs in private clinics with physical activity counselling to patients with major chronic diseases i.e. cardiovascular diseases, hypertension, type 2 diabetes mellitus, dyslipidaemia, obesity/metabolic syndrome, osteoarthritis/osteoporosis and bronchial asthma/chronic obstructive pulmonary disease. The specific objectives are a) to determine physical activity levels amongst primary care doctors in private practice from the Federal Territory of Kuala Lumpur, Selangor and Penang; b) to determine the frequency of initiation of exercise counselling by the participants to patients on chronic disease follow up; c) to determine the association between physical activity levels and confounding factors with frequency of initiation of exercise counselling to patients on chronic disease follow up. It is hoped that the findings from this study will help in formulating methods to improve physical counselling in patients with chronic diseases.This was a cross-sectional study conducted amongst PCDs in private practice from three states in Malaysia i.e. the Federal Territory of Kuala Lumpur (KL), Selangor and Penang from June to December 2016. These states were chosen as they were mainly urban with a high number of PCDs in private practice. All PCDs in private practice were included whereas those excluded were doctors who were away on prolonged leave, pregnant or who had conditions or disabilities limiting exercise during the data collection period.PCDs from KL and Selangor were recruited from Academy of Family Physicians Malaysia (AFPM). Universal sampling was conducted as the authors were not able to access the AFPM\u2019s member list in view of their confidentiality policy. Distribution of questionnaires was conducted by the AFPM\u2019s administrative staff to the 728 PCDs registered members in KL and Selangor. Self-addressed, stamped envelopes were sent of which 192 were returned by post\u00a0(10 were excluded as they did not fulfil the inclusion criteria). To further increase response rates, a reminder was given to participants of workshops attended by AFPM for their members in KL and Selangor and another 20 questionnaires were returned.Private PCDs from Penang were recruited from those who attended the annual Penang General Practitioner\u2019s Conference organized by the Malaysian Medical Association (MMA) in Penang. At the conference, 114 questionnaires were distributed to participants who fulfilled the inclusion criteria. A total of 78 questionnaires were returned in person or placed in designated labelled boxes at the end of each conference day. The recruitment process is summarized in Fig.\u00a0The study tool consisted of a self-administered questionnaire Appendix  divided The dependent variables measured comprised of frequency of exercise counselling to patients with chronic medical diseases . These conditions were chosen as exercise has been shown to benefit these conditions . The exeThe second section measured an independent variable which was physical activity levels of PCDs using the English version of the International Physical Activity Questionnaire (IPAQ) which has been validated in Malaysia . The shoData from this study was analysed using Statistical Package for Social Sciences (SPSS) software, Version 23.0. Descriptive statistics was for physical activity levels of private PCDs and frequency of initiation of exercise counselling to patients with chronic diseases. For exercise counselling frequency, Likert scores from 1\u20133 were classified as \u2018rarely initiates\u2019 and 4\u20135 as \u2018often initiates\u2019.To determine associations between physical activity levels and other independent variables (confounding factors) with initiation of exercise counselling for each of the five chronic diseases, bivariate regression was conducted for categorical variables and continuous variables that were normally distributed.For continuous variables that were not normally distributed, the Kruskal\u2013Wallis test was done.p-value\u2009\u2264\u20090.250) were included in subsequent multivariate analysis [To identify significant independent variables, bivariate regression was conducted between demographic variables and initiation of exercise counselling for each chronic disease (\u2018Often initiates\u2019 vs \u2018Rarely initiates). For continuous variables that were not normally distributed, the Kruskal\u2013Wallis test was performed. For each of the chronic diseases, independent variables with significant values  were either overweight (36.4%) or obese (23.3%). Most of the participants (98.2%) were non-smokers.Table The most common comorbidities in respondents were obesity/metabolic syndrome and dyslipidaemia at 23.2% and 14.3% respectively. The median total number of years working as a doctor was 8 (IQR 8.75) and median number of years working in primary care setting was 5 (IQR 7.38). Less than half (47.1%) had a post-graduate qualification. The mean number of patients seen on an average day was 40\u2009\u00b1\u200922.08.Table Most participants (50%) had moderate levels of physical activity and 25.7% had low levels. Only 24.3% had high levels levels of physical activity.p\u2009<\u20090.001, 95% CI 1.824 to 5.616) and hypertension . There were no significant associations found between physically active PCDs and increased initiation of exercise counselling to patients with osteoarthritis/osteoporosis and BA/COPD.Bivariate regression Table  showed sp\u2009=\u20090.001, 95% CI 1.518 \u2013 5.012) and  respectively. PCDs with post-graduate qualifications were more likely to counsel hypertensive patients on exercise . Initiation of exercise counselling to patients with cardiovascular diseases and diabetes mellitus was less likely with an increasing number of patients seen per day  and  respectively.Multivariate regression Table  revealedThe key findings of this study were that a majority of PCDs in private practice had moderate to high levels of physical activity and those who were physically active were more likely to initiate exercise counselling in patients with cardiovascular diseases.In this study, 74.3% of PCDs in private practice were found to be physically active (moderate or high IPAQ scores). A larger proportion of our respondents appeared to be more physically than the general Malaysian adult population with the physically active at 66.5% (41.1% at a moderate intensity and 25.4% at high intensity) . These fMost of the respondents in our study often initiated exercise counselling to patients with cardiovascular diseases, hypertension, type 2 diabetes mellitus, obesity/metabolic syndrome and dyslipidaemia. Similar trends were observed in other studies where in Northern Ireland, GPs reported comparable rates of exercise counselling to their patients with chronic diseases . MorishiOn the contrary, only about 32.8% of primary care doctors in government clinics in the Klang Valley provided exercise counselling to 50% or more of their patients with the majority to patients suffering from high cholesterol, obesity, type 2 diabetes mellitus and those who smoked or had a sedentary lifestyle . The conPrevious literature had shown an association between physical activity of doctors and exercise counselling where physically active doctors were more likely to provide exercise counselling , 27\u201329. Apart from physical activity levels of PCDs, there are other factors that could influence physical activity counselling to patients with chronic diseases. In the multivariate analysis, participants in our study with post-graduate qualifications were also more likely to offer exercise counselling to hypertensive patients which could be due to the further training in exercise counselling that they may have received as post-graduates. A similar trend was seen in government clinics where family physicians with post-graduate degrees were more likely to offer exercise counselling compared doctors without post-graduate qualifications . SimilarThis study to the best our knowledge currently is the first to look at associations between physical activity of general practitioners in the private sector from the federal territory of Kuala Lumpur, Selangor and Penang with initiation of exercise counselling to patients with specific chronic diseases. The findings from this study will pave the way for more research into the health and wellbeing of primary care doctors in private practice and their patient counselling practices.A limitation of this study is that it used a self-reported questionnaire, therefore, it was subjected to some degree of reporting bias. Another limitation is the low response rate despite the researchers\u2019 attempts in providing reminders.Similar poor response rates had been seen in postal as well as online surveys done in other countries ranging from 31 to 68.4% , 33\u201340. Sampling primary care doctors who attended the GP conference in Penang was also subjected to bias as only doctors who were proactive in learning and continuous medical education (CME) sessions would have had the time as well as resources to spare to attend them. As this study was conducted amongst primary care doctors in private practice only in Kuala Lumpur, Selangor and Penang which are urban areas due to limitations in human resources and time, results from this study may not be generalizable to all primary care doctors practising in Malaysia. There may have been differences in physical activity counselling practices between PCDs from urban and rural locations which were otherwise uncaptured here. Our respondents were predominantly female too, possible skewing results as females tend more to counsel patients more than male physicians , 35 FurtIncreasing PCDs\u2019 level of physical activity may increase their physical activity counselling to patients with cardiovascular diseases. Therefore, strategies to encourage doctors to stay active would be useful plans for the future such as involving and engaging PCDs in physical activity campaigns. In larger private practices, assigning a designated area for exercise or organizing fitness sessions for employees during protected time may be recommended.Positive associations were seen in physical activity counselling given to patients with other types of chronic conditions such as hypertension, type 2 diabetes mellitus, dyslipidaemia and obesity/metabolic syndrome although it was not significant. There were no associations with counselling of patients with osteoarthritis and osteoporosis although these diseases would benefit from regular exercise. The reasons are unknown, hence, this warrants further research to explore these reasons.Most respondents in this study were physically active with 50% physically active at a moderate level and 24.3% at a high level.Higher physical activity levels and being a non-smoker increased the initiation of exercise counselling to patients with cardiovascular diseases on follow-up visits. Private PCDs with postgraduate qualifications were more likely to initiate exercise counselling in hypertensive patients.Additional file1:\u00a0Appendix 1.\u00a0Fitness Questionnaire.\u00a0Appendix 2.\u00a0INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE."}
+{"text": "Demographers are interested in the degree to which marriage is driven by prenuptial pregnancy within particular communities. To help answer this question, we present a novel method which links marriage certificates to birth certificates, where the birth-mother is the marriage-bride, considering only births which occur in the first seven months after a marriage.To identify prenuptial births we employed an unsupervised graph-based record linkage method to link birth and marriage certificates. We first extracted related groups of individuals: babies and their parents from birth certificates, and brides, grooms, and their parents from marriage certificates. To link births with marriages, we employed techniques to address challenges such as changing attribute values over time (such as names and addresses), the ambiguity of attribute values giving priority to rare names over common names, and different relationships encountered at different points in time . Based on the obtained links we then extracted prenuptial births.Using two Scottish data sets containing a total of 38,451 births and 8,667 marriage certificates from the period 1861 to 1901 and employing different linkage thresholds we identified between 853 and 945 first birth-marriage links in the smaller  data set, and between 2,165 and 2,232 links in the larger (urban) data set. In the rural data set, between 16.9% and 17.7% of these links were with birth less than 8 months after marriage , where the corresponding ground truth contained 17.6% prenuptial births. For the urban data set, we identified between 51.3% and 51.4% prenuptial births. Our results show clear differences between rural and urban prenuptial pregnancies in 19th Century Scotland.We have presented an unsupervised graph-based record linkage method that compares attribute values of individuals and their relationships to link records based on the ambiguity of attribute values, attribute value changes, and temporal constraints. This linking process helps us to identify prenuptial births to help us understand the degree to which marriages may have been driven by pregnancy."}
+{"text": "The development of multimodal imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) allows the contemporary obtaining of metabolic and morphological information. To fully exploit the complementarity of the two imaging modalities, the design of probes displaying radioactive and magnetic properties at the same time could be very beneficial. In this regard, transition metals offer appealing options, with manganese representing an ideal candidate. As nanosized imaging probes have demonstrated great value for designing advanced diagnostic/theranostic procedures, this work focuses on the potential of liposomal formulations loaded with a new synthesized paramagnetic Mn(II) chelates. Negatively charged liposomes were produced by thin-layer hydration method and extrusion. The obtained formulations were characterized in terms of size, surface charge, efficiency of encapsulation, stability over time, relaxivity, effective magnetic moment, and in vitro antiproliferative effect on human cells by means of the MTT assay. The negatively charged paramagnetic liposomes were monodisperse, with an average hydrodynamic diameter not exceeding 200 nm, and they displayed good stability and no cytotoxicity. As determined by optical emission spectroscopy, manganese complexes are loaded almost completely on liposomes maintaining their paramagnetic properties. Manganese (Mn) is an essential bio-element for humans, is a cofactor of many enzymes, and is involved in multiple and fundamental cellular activities, including macronutrient metabolism, bone formation, and free-radical defense systems . Notwithd-transition metal belonging to the seventh group of the periodic ta-ble; in its +2 and +3 oxidation states, it shows paramagnetic properties, making it a potential candidate for MRI applications.Manganese is a 2+ ions in the tissues. A higher amount of Mn2+ results in a stronger and more detectable contrast, leading to better image quality  (MP) , Mn(II)(S2CNR2)2 , and Mn(III)(S2CNEt2)3 (MDE3) /Mn(III) complexes characterized by different water solubility were considered: [Mn(II)(PTA)(Cl)3 (MDE3) . NotablyThe manganese complexes were prepared and characterized according to the procedure reported in the literature . Briefly2\u00b74H2O complex with two equivalents of the sodium salts of the dithiocarbamate ligand (Na[(CH3CH2)2-N-C(=S)S]\u00b73H2O or Na[(CH3CH2OCH2CH2)2-N-C(=S)S] \u00b73H2O), after careful purging of all the reaction containers and water solvent by nitrogen bubbling and using the Schlenk technique. The preparation of the MDE3 complex was performed under open atmosphere by reaction of MnCl2 with three equivalents of Na[(CH3CH2)2-N-C(=S)S]\u00b73H2O, in water solution.The synthesis of MDE2 and MDB was performed under inert conditions by reacting the Mn(II)ClAll complexes were characterized with elemental analysis, ESI(+)-MS, UV/Vis, and infrared (IR) analysis as previously reported . In FiguMP, MDE2, and MDB were analyzed as powders, and curves of magnetization (M) vs. magnetic field (H) were measured at T = 300 K .The results obtained for the freshly prepared derivatives (black lines) clearly indicated a paramagnetic behavior. The slope of the curves represents the mass susceptibility, defined as \u03c7 = M/H (expressed in emu/g Oe), which is positive and independent from H in paramagnetic materials. Further analyses were repeated over time on the same samples stored in the open air, until the magnetic response no longer changed significantly. The magnetic behavior of MP did not change over time a. On theappl = 10 kOe. The results obtained for the three freshly prepared manganese derivatives are reported in The magnetization M was measured as a function of temperature between 10 K and 300 K in an applied magnetic field HAt T = 300 K, the values of \u03c7 were obviously equal to those derived from the M vs. H measurements see .eff is the effective magnetic moment per molecule of paramagnetic material , and \u03b8 (which has the dimension of a temperature) gives information on the type and strength of magnetic interactions between the magnetic moments in the paramagnetic regime.The thermal dependence of the susceptibility is expected to follow the Curie-Weiss law for paramagnetic materials, as expressed in Equation (1) .(1)\u03c7=0.eff coincides with the magnetic moment of the Mn ion and, according to Equation (1), isSince each investigated compound contains a paramagnetic Mn ion, then \u03bceff from the above relation, the value of \u03b8 is obtained by fitting the \u03c7 vs. T curves in In order to estimate \u03bcIn the case of MP, \u03c7 followed the dependence 1/(T \u2212 \u03b8) foreseen by the Curie\u2013Weiss law in the whole investigated temperature range, unlike that observed for MDB and MDE2. In MDB, the susceptibility tended to a plateau value at a very low temperature. This behavior indicates the existence of magnetic interactions between molecular moments so strong as to determine the onset of a magnetic state that could no longer be described as purely paramagnetic. The same effect was even more marked in the case of MDE2, where the \u03c7 vs. T curve deviated substantially from the 1/T trend below ~150 K and exhibited a strong decrease below ~60 K. Therefore, the fitting of the \u03c7 vs. T curve to the Curie\u2013Weiss law was performed for the whole investigated temperature range in the case of MP, whereas the 150\u2013300 K and the 50\u2013300 K intervals were considered for MDE2 and MDB, respectively  using the predicted MWs. For MnCl2, \u03bceff = (5.9 \u00b1 0.2) BM is in excellent agreement with the value expected for the Mn(II) ion in the high-spin state . The values measured in MP and MDE2 were also consistent with the high-spin state of Mn(II), within experimental error, while a larger value was measured in MDB.The \u03bcThe parameter \u03b8 was negative for the three compounds, indicating that the magnetic interactions between the magnetic moments were antiferromagnetic. The absolute value of \u03b8 is a measure of the magnitude of the magnetic interactions; therefore, it turns out that they were weak in MP and definitely strong in MDE2.eff, obtained by the analysis of these curves are reported in eff for MDB was consistent with that for the high-spin state of Mn3+ . As for MDE2, a \u03bceff as low as ~3.4 BM was estimated. This is in agreement with the previous observations that Mn(II) compounds are oxidized over time to Mn(III) if not stored under controlled conditions in the absence of oxygen \u00b73H2O were obtained from Aldrich Chemical , Na[S2CN(CH2CH2OEt)2] was obtained from Alchemy , and cholesterol (CH) was obtained from Merck-Aldrich . N-Lauroylsarcosin sodium salt (NLS) was from Fluka Chemie AG . Sodium lauroyl lactylate (SLL) was from Zschimmer & Schwarz Italiana S.p.A.  and Stepan Europe S.A.S. . Lastly, soybean phosphatidylcholine Phospholipon 90G (PC) was from Lipoid AG . Solvents of analytical grade were from Merck Serono S.p.A. . All other materials and solvents of high-purity grade were from Sigma-Aldrich . The heterocyclic phosphine 1,3,5-triaza-7-phosphaadamantane (PTA) and the Mn complexes MP, MDE2, MDE3, and MDB were prepared according to the procedure reported in the literature [Mn(II)Clterature ,17.v/v) containing PC, CH, and NLS or SLL in a 4:2:1 molar ratio to give a final concentration of 25 mg/mL was prepared. Afterward, the organic mixture was evaporated under vacuum  by means of a Rotavapor R-200 , and the obtained film was hydrated with 5 mL of water, swirled, and sonicated for 5 min. The addition in the selected formulation of manganese-based compounds  was carried out on the basis of their chemical characteristics either in organic phase (lipophilic) or in water during the hydration step (hydrophilic). Anionic liposomes with NLS and SLL, namely, LN and LS, were prepared by direct hydration followed by extrusion. Precisely, an organic solution of methylene chloride and methanol  equipped with a 5 mW helium neon laser with a wavelength output of 633 nm on aqueous diluted liposome samples (1:20 by volume). Plasticware was cleaned with detergent washing and rinsed twice with milliQ water. Measurements were made at 25 \u00b0C at an angle of 90\u00b0, with a run time of ~180 s. Data were interpreted using the \u201cCONTIN\u201d method .v/v), and the analysis was conducted at 25 \u00b0C. Values were obtained from three independent experiments performed in triplicate.The \u03b6 potential measurements were carried out by means of a Zetasizer Ultra . All the samples were diluted in disposable capillary cells  with deionized water  using GMS 1.4 software  as an image processing system.Liposome morphology was investigated by means of cryo-transmission electron microscopy (cryo-TEM). Liposome samples were vitrified and transferred to a Zeiss EM922Omega transmission electron microscope for imaging using a cryoholder , as previously described ,29. SampLIPID corresponds to the concentration of manganese in the lipid phase measured by ICP-OES, and MnTOTAL is the concentration of manganese used in the formulation.Optical emission spectrometry (ICP-OES) is an analytical technique used for quantitative and qualitative determination of metal ions in solution. Therefore, each sample was subjected to the ICP-OES to determine the MN concentration actually present. The encapsulation efficiency (EE) of manganese in LN and LS was determined by ultracentrifugation; specifically, 500 \u03bcL samples were loaded in a centrifugal filter  and subjected to ultracentrifugation  at 8000 rpm for 20 min. Then, the lipid phase was analyzed. All measurements were made with an ICP-OES device  equipped with an axial torch, segmented array charge-coupled device detector, and low-flow GemCone nebulizer with cyclonic spray chamber for sample introduction and choosing, among the several wavelengths, the readings at 259.372 nm. Each sample was prepared twice and subjected to analysis; the analyzed volume was 20 \u00b5L. For each condition, the average of the absorbance values obtained was calculated, and the manganese concentration was obtained by comparison with a calibration curve obtained after measuring known concentrations of the metal ion . The EE \u22127 emu sensitivity. To calculate the specific magnetization , the weight of the sample (few milligrams) was measured with a precision of 10\u22125 g.The magnetic measurements were carried out using a superconducting quantum interference device (SQUID) magnetometer  operating with a maximum applied field H = 50 kOe. The instrument allows measuring the sample\u2019s magnetic moment with 101 = 1/T1) and transverse (R2 = 1/T2) relaxation rates were measured at 25 \u00b0C at two magnetic field strengths, 0.47 T and 1.4 T (corresponding to 20 MHz and 60 MHz proton Larmor frequency), on a Stelar Spinmaster spectrometer working at adjustable field. T1 values were measured using the standard inversion recovery sequence  with a typical radio frequency 90\u00b0 pulse length of 3.5 \u03bcs. T2 values were measured using a standard CPMG sequence . Temperature was controlled with a Stelar VTC-91 heater airflow equipped with a copper\u2013constantan thermocouple (uncertainty of \u00b10.1 K). The relaxation rates of the solutions containing the paramagnetic complexes were subtracted from the corresponding diamagnetic contributions and then divided by the millimolar concentration of Mn ions to obtain the normalized millimolar relaxivities .The longitudinal  was added and incubated for 4 h. The conversion of MTT solution into a violet-colored formazane was obtained after addition, incubation (15 min), and shaking of 100 \u03bcL of DMSO. The solution absorbance, proportional to the number of living cells, was measured using a spectrophotometer at 590 nm and converted into percentage viability.Cell viability was evaluated using the MTT test ,32 on Hap < 0.05.Statistical analysis was performed using analysis of variance (ANOVA). The level of significance was taken at In this study, the possible use of manganese derivatives for diagnostic applications in multimodal imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) was investigated. As manganese(II/III) complexes, MP, MDE2, MDB, and MDE3 were considered due to their different water solubility. In particular, two different types of anionic liposomes carrying the different manganese complexes were prepared and analyzed in terms of morphology, size, and magnetic power, together with the loading capacity and in vitro activity. This study enabled the selection of liposomes based on SLL as the best in terms of size, encapsulation, retention, and physical stability over time. Moreover, LS did not show differences in loading hydrophilic or lipophilic manganese compounds, resulting in a suitable system to convey all the synthesized complexes. In addition, LS displayed good results in terms of safety toward cells and ability to cross the cell membrane. The encouraging results obtained concerning the magnetic properties after LS encapsulation allowed to select MBD as the manganese compound to be further investigated for a potential application in diagnostic imaging. Remarkably, the possibility to administer the manganese complex using liposomes can represent an innovative approach to hamper the toxicity issues related to manganese administration. The preliminary data shown in this study strongly support further preclinical studies aimed at understanding the potential applicability of these formulations for diagnostic purposes."}
+{"text": "This work studies the effect of interlayer adhesion on mechanical performance of fluorinated thermoplastics produced by fused deposition modeling (FDM). Here, we study the anisotropic mechanical response of 3D-printed binary blends of poly (vinylidene fluoride) (PVDF) and poly (methyl methacrylate) (PMMA) with the isotropic mechanical response of these blends fabricated via injection molding. Various PVDF/PMMA filament compositions were produced by twin-screw extrusion and, subsequently, injection-molded or 3D printed into dog-bone shapes. Specimen mechanical and thermal properties were evaluated by mode I tensile testing and differential scanning calorimetry, respectively. Results show that higher PMMA concentration not only improved the tensile strength and decreased ductility but reduced PVDF crystallization. As expected, injection-molded samples revealed better mechanical properties compared to 3D printed specimens. Interestingly, 3D printed blends with lower PMMA content demonstrated better diffusion (adhesion) across interfaces than those with a higher amount of PMMA. The present study provides new findings that may be used to tune mechanical response in 3D printed fluorinated thermoplastics, particularly for energy applications. Ideally, the mechanical response of a printed thermoplastic (multilayered specimen) would be similar to the isotropic mechanical response of the same thermoplastic fabricated via molding techniques (homogeneous specimen). By performing mechanical testing on these specimens, one can probe interface diffusion by observing behavior in the bulk [Additive manufacturing (AM) allows the production of complex geometries that are oftentimes not achievable by subtractive methods . In the the bulk ,5.The FDM process consists of a heated print nozzle through which a polymer filament passes undergoing melting. The extruded filament is then deposited onto a build platform in a chosen xy-plane, while building layer-upon-layer in the vertical z-dimension, allowing the production of complex geometries see . In thisgT) or melting temperature (mT) to achieve sufficient molecular mobility such that polymer chains can diffuse and entangle with one another across the interface. The polymer diffusion and entanglement formation determine much of the adhesion strength. In an optimum adhesion between surfaces, a failure will occur cohesively in the specimen rather than at the interface between two layers. There are many factors involved in the diffusion and entanglement mechanism, such as, temperature, contact time, molecular weight, degree of crystallinity, the strength of polar groups [Polymer surfaces brought into contact must be mutually soluble and above their glass-transition temperature  (PVDF) (Kynar 705) was generously supplied by Arkema  and arrived in pellet form. Poly (methyl methacrylate) (PMMA) with an average molecular weight of 141,000 g/mol was purchased from Sigma Aldrich  and arrived in powder form. \u201cPMMAXX\u201d is the nomenclature structure for samples used in this study where \u201cXX\u201d indicates weight-based PMMA concentration. For instance, a sample made with 25 wt% PMMA and 75 wt% PVDF is designated as \u201cPMMA25\u201d. All materials used in this study were obtained from commercial sources and dried at 50 \u00b0C under vacuum for 8 h to remove any moisture before use.Blends were prepared by melt blending PVDF and PMMA in a twin-screw compounder . Compounding was carried out at 190 \u00b0C and a screw speed of 100 rpm for 5 min. Different PVDF/PMMA blends were extruded at 100 rpm to form filament approximately 3 mm in diameter, containing 0%, 15%, 25%, 50%, 75%, 85%, and 100% of PMMA (by weight).HAAKE Type 3 specimen (557-2290) dog-bone tensile bars were produced via FDM and injection molding. All samples of each composition were fabricated under identical processing conditions to eliminate sample-to-sample errors. Examples of dog bones produced by FDM and injection molding are shown in PVDF/PMMA melt-extruded filaments were around 0.5\u20130.7 m in length and 2.9\u20133.1 mm in diameter. These filaments were used in an Ultimaker 2 to create the 3D printed dog-bones. Solidworks 2019  and Simplify3D  were used to model the dog-bones prior to the printing process. Printing of the specimens was done on a glass build plate heated to 90 \u00b0C through a 0.4 mm nozzle heated to 190\u2013210 \u00b0C. For blends from PMMA0 to PMMA25, the print nozzle was heated to 190 \u00b0C. For blends from PMMA50 to PMMA100, the print nozzle was heated to 210 \u00b0C. The layers were printed with 100% infill density, with an extrusion multiplier of 1, with high quality, with a layer height of 0.1 mm, at a print speed of 30 mm/s, and a x/y movement speed of 55 mm/s. PVDF/PMMA melt-extruded filaments were pelletized and introduced into a preheated cylinder (210 \u00b0C). A HAAKE Minijet Pro  was then used to form the dog-bone via injection molding. The cylinder was placed on top of a heated mold (90 \u00b0C) and fitted with a plunger. The plunger was pushed by a hydraulic press to extrude the molten blend into the closed mold. For blends from PMMA0 to PMMA25, the press was programmed to deliver an initial injection pressure of 420 bar for 20 s, and a secondary pressure of 200 bar for 15 s before releasing. For blends from PMMA50 to PMMA100, the press was programmed to deliver an initial pressure of 600 bar for 40 s, and a secondary pressure of 250 bar for 20 s before releasing. Upon completion, the mold was then removed from the machine and opened to eject the injected dog-bone.When injection molding PMMA (or blends with high wt% in PMMA), there exists a wide range of adjustable injection temperatures  since its decomposition temperature is approximately 270 \u00b0C . Further\u03c4 is the shear stress at the wall.Blends apparent viscosity was measured by monitoring the two pressure transducers located in the inside wall of the compounder chamber. Pressure data were taken at each transducer at 1 Hz for 1 min at a screw speed of 100 rpm using the HAAKE minilab software. Viscosity calculations were made using the pressure difference between the two transducers. Apparent viscosity can be calculated using Equation (1), where h and w are the height and width of the channel , \u0394L is the distance between the pressure transducers , and \u0394P is the pressure differential.The latter is given by Equation (2), where w represents screw speed.Similarly, the shear rate can be expressed as shown in Equation (3), where gT), melting temperature (mT), crystallization temperature (cT), and heat of melting (\u0394mH) were determined for the different processed blends. To assess the impact of PMMA on PVDF crystalline domains, the degree of crystallinity (cX) of each sample was calculated from the following Equation (4) [mH is the experimental melting enthalpy of the blend , Thermal analysis was carried out using a TA Instruments model Q20 differential scanning calorimeter (DSC) . DSC was performed on raw materials, extruded filament, injection molded samples, and 3D printed samples. PVDF/PMMA samples of approximately 5 mg were placed into an aluminum crucible and loaded into the DSC. Thereupon, these samples were heated at a rate of 20 \u00b0C/min, from 30 to 250 \u00b0C, and then cooled to 30 \u00b0C for two cycles under a nitrogen atmosphere. The glass-transition temperature (tion (4) , where \u039404.5 J/g ,29,30), DSC analysis was performed from the derivative of heat flow with respect to temperature. The derivative was used to establish the initial and final points of the exothermic/endothermic peaks. Since crystalline domains affect interface adhesion by preventing the diffusion of polymer chains, DSC was used to locate when crystallization enhancement occurs.E) was calculated from the initial linear portion of the stress\u2013strain curves. Maximum tensile stress (\u03c3) and elongation at break (\u03b5) were also reported. At least eight dog-bones specimens were tested for each composition and the average values are presented. Bluehill Universal software was used to measure and calculate stress\u2013strain data.Bulk tensile testing of dog-bones was carried out using an Instron 5569 Tensile Tester  with a 50 kN load cell. Dog-bones were placed in a pair of manual dual screw press clamps which were torqued to 40 N\u00b7m before testing. The displacement of the sample was measured using the crosshead of the machine frame. Tests were performed at room temperature using a crosshead speed of 50 mm/min, while axial strain was continuously recorded. The Young\u2019s modulus  and of thermoplastics fabricated via injection molding (homogeneous specimens) to study adhesive versus cohesive failure mechanism .\u22121 averaged from 32 scans with a spectral resolution of 2 cm\u22121. A background spectrum was collected before each sample scan.The attenuated total reflectance (ATR) sampling technique was used in conjunction with infrared spectroscopy to determine the presence and relative amount of PVDF \u03b2-phase within the blends. Thus, to identify the PVDF crystalline phases of PVDF/PMMA mixtures, absorption spectra were collected using a Thermo Scientific FTIR-ATR Nicolet iS20. The analysis was performed at room temperature in the range of 500 to 3000 cmw). The number of molecules per size corresponds to the molecular weight distribution. It is worth noting that, apart from the aforementioned effect that Mw has on diffusion, most polymer properties also depend on the molecular weight and its distribution.The basic idea behind this chromatographic method is that the polymer in solution passes through columns that absorb and filter it, while a detector measures the size of its molecular chains over time. The average size corresponds to the polymer molecular weight . GPC was conducted using a Waters Alliance 2695 separations module , an online multiangle laser light scattering (MALLS) detector fitted with a gallium arsenide laser (20 mW) operating at 658 nm, an interferometric refractometer operating at 65 \u00b0C, and 685 nm, and two PLgel mixed-E columns in series . The mobile phase was freshly distilled tetrahydrofuran (THF) delivered at a flow rate of 1.0 mL/min. Sample concentrations were 6 mg of polymer/mL of THF, and the injection volume was 100 \u03bcL. The detector signals were simultaneously recorded using ASTRA software, and absolute molecular weights were determined by MALLS using a For PVDF, the molecular weight and polydispersity were also determined via the same procedure. However, the mobile phase was 0.02 M LiBr/DMF (dimethylformamide) delivered at a flow rate of 0.5 mL/min. Furthermore, the polymer samples were pre-dissolved in 0.02 M LiBr/DMF by stirring for 3 h at 80 \u00b0C. The injection volume was set at 100 \u03bcL.w) of PMMA, which causes the melt blending process to consume more energy . In contrast, the relatively low PVDF surface energy reduces these interactions and plasticizes PMMA. More precisely, at low PMMA concentrations, the viscoelastic behavior suggests the blends have a high degree of partial miscibility, whereas at PMMA50 a more significant phase separation of the two polymers can be inferred. For higher PMMA concentrations, blends exhibit responses resembling pure PMMA.As shown in These viscosity trends suggest that interface diffusion worsens with the addition of PMMA, since it hinders the mobility of polymer chains. This can be easily understood by looking at the Newtonian polymer-sintering model proposed by Bellehumeur and colleagues . It can Differential scanning calorimetry (DSC) was performed to study the effect of the addition of PMMA on the thermal behavior of both processed thermoplastic and their respective mixtures. The DSC results of the investigated PVDF/PMMA blends are listed in gT) of PMMA, below which the overall chain mobility significantly decreases by forcing PVDF chains to diffuse through the rigid PMMA network to form crystals, thus altering melting behavior. In this way, crystallization indicates that there is still mobility in the system . These results suggest that PMMA50 is the limiting concentration beyond which the PVDF crystallization event is lost.Centration increases, the melt temperature, the melting enthalpy, and the degree of crystallinity decrease markedly for all processed blends. This behavior is caused by the partial miscibility of PVDF in PMMA, as PMMA is largely amorphous and does not contribute to the heat of fusion ,29, ThusAs mentioned in the previous section, the addition of PMMA raises the glass-transition temperature of the mixtures see . Thus, bThe thermal behavior of injection molded blends presents a similar profile with that of 3D printed and compounded (filament). However, the melt temperature of the PMMA15 concentration increases with respect to pure PVDF. This shift could be ascribed to the increase in \u03b1-phase crystals of PVDF. Similarly, the degree of crystallinity of the PMMA25 concentration increases with respect to PMMA15, which could be attributed to the slight increase in \u03b2-phase content. This was confirmed by the results shown in The above reasoning is extrapolated from the second heating-cooling cycle  of the DgT calculation of polymer blends does not apply in this case due to the approx. 160 \u00b0C difference between the PVDF and PMMA glass transition temperatures.The surface of the material experiences relatively high shear stress during production resulting in internal residual stress. Inside the nozzle, this shear stress aligns polymer chains that are in a low entropy state; they have very few degrees of freedom. Ideally, once the material exits the nozzle, forces acting on it are relieved. However, if the material cools too quickly, the polymer chains do not have enough time to reorder at the surface, resulting in strain-induced crystallization or other types of stress phenomena. This shear stress is more impactful for high PMMA concentrations due to their high glass-transition temperature, which hinders blends readjustment. Moreover, the high surface energy of PMMA increases the tackiness  of the system, facilitating interactions between the polymer and nozzle (or mold) surfaces. Therefore, both events demonstrate that higher PMMA concentrations add more shear stress to the material. Similarly, 3D printed components are more likely to experience higher residual stresses and process effects than the other two processes based on the surface area that is formed during production. It is then clear that in the 50% PMMA concentration composition does not promote crystallization. Rather, processing promotes strain-induced crystallization see . Lastly,Mechanical properties as a function of PMMA weight fraction are displayed in gT) of PMMA, which causes PVDF chains to not have enough space to move and form crystals. This loss of the crystallization impact on mechanical strength is responsible for the drop in PMMA50 tensile strength value. Note that the latter is not a bond formation issue because the drop in mechanical properties occurs for both processed samples. Thus, what changes as a function of the composition  is the fact that PMMA50 is no longer able to crystallize and, hence, it has lost part of its load-bearing ability.At PMMA50, a notable deterioration of mechanical properties can be observed. In particular, the tensile strength response of both IM and 3D printed specimens drops off due to loss of crystallization. As explained in gT . The relatively high glass-transition of PMMA creates diffusion limitations as the PMMA network becomes rigid below gT. These limitations hinder domain evolution at the printed filament boundary, effectively seizing polymer welds at high temperatures, thus limiting the degree of diffusion from layer to layer.From PMMA75 to PMMA100, the increase in PMMA content raises tensile strength simply because the overall PMMA tensile strength response is much higher than that of PVDF. In this way, the mechanism for improving this mechanical property is simply the fact that PMMA itself has a higher tensile strength than PVDF. Within this range, blends tensile strength responses are approaching the PMMA response. However, the difference between the tensile strength response of IM and 3D printed specimens is noticeably greater than it is for lower PMMA concentrations. These differences are greater because blends glass-transition temperatures have notably increased close to the value of virgin PMMA w and were tested below their gT (see next section).Concerning the elongation at break, a similar but decreasing trend can be observed with the addition of PMMA weight ratios. Starting from PMMA0, ductility decreases with increasing PMMA concentration. As seen in the tensile strength trend, a significant drop in elongation is identified at PMMA50. This drop in ductility can be explained by the fact that PMMA50 samples can only withstand a much lower load for a shorter time than the other bends due to (1) the loss in PVDF crystallization and (2) PVDF chains disrupting the PMMA regions, effectively reducing the load-bearing potential. Hence, their load-bearing potential drops. From PMMA75 to PMMA100, ductility follows the same decreasing trend that it had before PMMA50 until reaching PMMA100. High PMMA concentrations exhibit brittle behavior in response to applied loads mainly because they have high Mw and crystalline phases) of the material have not changed and remain fairly constant between processed specimens.Finally, Young\u2019s modulus trend matches tensile strength as it slowly increases proportionally to the amount of PMMA. In addition, the same drop in property can be observed at PMMA50. This intermediate composition does not have the crystallization impacts that help to increase load-bearing potential and does not have enough PMMA content to approach the modulus of the high PMMA concentration specimens. While the processing method strongly impacts specimen tensile strength and elongation, Young\u2019s modulus exhibits very small changes  between the two types of production. The latter can be explained because Young\u2019s modulus is a material-dependent property, whereas the former properties depend more on production. This material dependence can be visualized in that even if the geometry of the sample were to be changed, Young\u2019s modulus would not change. In contrast, the first two properties, which are more process-dependent, are very sensitive to voids and weld quality . As explained later in the GPC and FTIR analysis results, the internal properties , processed compositions underwent ATR-FTIR analysis. The latter measures the infrared spectrum of absorption of the sample over a range of wavelengths see . In this\u22121. As previously mentioned, PVDF can crystalize into five different crystalline phases (polymorphs): \u03b1, \u03b2, \u03b3, \u03b4 and \u03b5 [\u22121) and \u03b2-  phases are present in the spectrum of blends. More specifically, only concentrations up to 25% in PMMA content display these peaks. Beyond this limit concentration, \u03b1 and \u03b2 peaks diminish in intensity or vanish, which agrees with the crystallinity results discussed in the previous section. Moreover, pure PMMA displays at 1720 cm\u22121 the stretching frequency of its carbonyl group (C = O). However, for blends containing both PVDF and PMMA, this absorption band is shifted to higher wavelengths (1727\u20131729 cm\u22121) due to the interaction between the carbonyl groups of PMMA and the CH2 groups of PVDF, which indicates the formation of blends [\u22121 the bending frequency of CH3-O [ \u03b4 and \u03b5 ,15. The  \u03b4 and \u03b5 . Only ths 1727\u201317 cm\u22121 duef blends . It can of CH3-O .F(\u03b2) represents the amount of \u03b2-phase; \u03b1A and \u03b2A are the absorbance intensities for \u03b1- and \u03b2-phase at 762 and 840 cm\u22121, respectively; \u03b1K and \u03b2K are the absorption coefficients at the respective wavenumbers  [Investigation of the crystalline domains present in each mixture provides information about the diffusion environment. For instance, if PMMA interacts strongly with PVDF through its carbonyl group, then the \u03b2-phase should be more dominant than the \u03b1-phase. Consequently, the higher the \u03b2-phase fraction, the greater the intermolecular interaction between PVDF and PMMA, which will subsequently negatively affect diffusion. Alternatively, if it is mainly the \u03b1-phase that is present within the blends, there will be fewer PVDF-PMMA interactions and thus a higher possibility of diffusion across the polymer interfaces. Thus, the \u03b2-phase content of blends containing only \u03b1 and \u03b2 polymorphs can be calculated using the following Equation (5), where cm2/mol) ,34:(5)F works with relative amounts of the same spectrum. It is worth mentioning that ATR-FTIR spectroscopy was used instead of FTIR transmission spectroscopy because samples were very opaque, and light could not pass through properly. Reflectance is a superficial measurement, while transmittance is a bulk measurement.w and polydispersity of PMMA and PVDF. GPC curves are displayed in w) is higher in PMMA than PVDF since its narrow peak occurs early in the range of 10\u201314 min whereas the PVDF peak takes place later between 24 and 30 min. Moreover, PVDF exhibits a lower intensity peak around 40 min, which suggests that its low Mw chains act as a plasticizer and soften it. Therefore, PVDF chains require less energy  to experience motion than PMMA chains. In this way, PVDF chains can diffuse into PMMA and reduce large chain interactions with one another, improving blend plasticity.GPC was run to study the processing effects of injection molding, 3D printing, and twin-screw extrusion on the molecular weight MResults show that both processed thermoplastics did not experience degradation because they maintain a fairly constant molecular weight distribution throughout the eluded time for each production technique. This can also be inferred from the data presented in In this study, PVDF/PMMA dog-bone specimens were fabricated via FDM and injection molding. Specimen mechanical and thermal properties were evaluated by tensile testing and DSC, respectively. Tensile testing results proved PMMA to be the strongest material with the highest Young\u2019s modulus. In contrast, results revealed that PVDF has the highest elongation at break. When PMMA content ratios increased for each mixing ratio, the strength increased as well as Young\u2019s Modulus, whereas elongation at break decreased. However, PMMA50 was demonstrated to differ from this trend to be the weakest and most brittle composition due to the loss of crystallization. These results help to assess the impact of PMMA and the FDM process on PVDF/PMMA blends behavior and processability.DSC results revealed that the increase in PMMA content disrupted PVDF crystalline domains. The results suggested that PMMA50 is the limiting concentration beyond which the PVDF crystallization event is lost. Furthermore, PVDF crystalline phases were examined using the ATR-FTIR technique. Results revealed that for high concentrations of PVDF, an increase in PMMA content hindered nucleation of the \u03b2-phase PVDF polymorph. GPC results showed that both processed polymers did not experience degradation as specimens examined maintained a fairly constant molecular weight distribution throughout the eluted time. Finally, this work provides insight into the multiscale mechanical behavior of PVDF/PMMA structures processed through FDM from which interesting technical advances can be made in different fields by understanding these fundamental behaviors to the point of intentional manipulation."}
+{"text": "Interest in piezoelectric nanocomposites has been vastly growing in the energy harvesting field. They are applied in wearable electronics, mechanical actuators, and electromechanical membranes. In this research work, nanocomposite membranes of different blend ratios from PVDF and TPU have been synthesized. The PVDF is responsible for piezoelectric performance where it is one of the promising polymeric organic materials containing \u03b2-sheets, to convert applied mechanical stress into electric voltage. In addition, the TPU is widely used in the plastic industry due to its superior elasticity. Our work investigates the piezoresponse analysis for different blending ratios of PVDF/TPU. It has been found that TPU blending ratios of 15\u201317.5% give higher output voltage at different stresses conditions along with higher piezosensitivity. Then, TPU addition with its superior mechanical elasticity can partially compensate PVDF to enhance the piezoelectric response of the PVDF/TPU nanocomposite mats. This work can help reducing the amount of added PVDF in piezoelectric membranes with enhanced piezo sensitivity and mechanical elasticity. This has mainly followed the use of various clean and renewable energy sources due to their sustainability and environmental friendliness2. Moreover, energy harvesting technologies have recently been the main focus, where wasted energy from ambient environments is utilized. Such technologies can transform vibrations, heat, light, radiation, wind, and water into electrical energies for low-power devices1. Research has also extended further to include energy harvesting in biomedical applications3 and offered promising biomedical sensors and wearable electronics5, due to the ability to harvest kinetic energy in the form of vibrations from direct human activities such as walking, running, and finger tapping to heartbeat and respiration7. The kinetic energy is harvested based on three transduction mechanisms; piezoelectric, electromagnetic, or electrostatic. Because of their high energy density, simple design, and the ability to be scaled down to micro-and nanoscale devices, piezoelectric energy harvesters have gained the most attention10. Piezoelectric materials also possess the unique ability to convert mechanical energy into electricity directly, without an external input12. Therefore, numerous efforts have been put into developing high-performance piezoelectric nanogenerators using organic and inorganic materials15.Over the last few decades, extensive research regarding the use of alternative energy sources has been carried out16. Such materials have been seen to be applicable in a wide range of devices, with polymer-based materials being more preferred due to their intrinsic flexible nature, providing a great degree of bending and biodegradability18. Among all piezoelectric polymers, poly(vinylidene fluoride) (PVDF) films have shown the highest piezoelectric performances to date21. Due to the polar crystalline nature of PVDF, its ability to produce large voltages with low forces has made it favorable for piezoelectric applications23. The piezoelectric property of PVDF mainly depends on its \u03b2-phase, one of its four crystalline phases24. In addition to its lightweight, flexibility, resistance to solvents, and stability under high electric fields, it is considered as the optimum biomaterial for applications in energy harvesters, force sensors, and transducers.It has been found that organic piezoelectric materials have greater benefits than inorganic materials, including a higher level of processability26. Electrospinning has been the most promising as it can form nanofibers from solutions or melts with variable diameters. Additionally, it has been reported that the \u03b2-phase content in PVDF nanofibers produced by electrospinning is higher than that of PVDF cast films, thereby improving its piezoelectric properties27.PVDF nanofibers are the core candidate for such applications, especially wearable and implantable devices. The main techniques used to fabricate such fibers include electrospinning, melt spinning, and centrifugal spinning28. Such additives include carbon nanotubes (CNTs), graphene, and ZnO29. A previous study has successfully produced a nanogenerator from PVDF-ZnO nanocomposite, showing that adding ZnO particles increased its output voltage30. Furthermore, piezoelectric nanogenerators incorporating ZnO have successfully been implanted in live rats to harvest energy from heartbeats and breathing motion. That study has shown great potential for using PVDF-ZnO nanogenerators as a power source for implantable biomedical electronic devices, which suggests a strong potential for such devices in applications related to the normalization of the heartbeat and brain stimulation for the treatment of movement disorders31. Furthermore, PVDF and its copolymers have been utilized for pressure sensing applications, serving as healthcare monitoring devices for respiration signals. Composites of PVDF and graphene oxides were also developed for multiple sensory applications, exhibiting a high sensitivity for monitoring simultaneous artery pulse pressures and temperatures32. PVDF nanofibers have also been explored for the application of blood pressure sensors due to their excellent flexibility33, and has successfully been tested on an in vitro model where PVDF thin films were wrapped around the aorta, and periodic signals of output current and voltage were generated with the movement of the artery, showing a great sensitivity.Electrospinning also provides the ability to further enhance the piezoelectric properties of the fabricated PVDF nanofibers due to its ability to produce aligned fibers with hollow structures or various additives for improved performance34. Such characteristics may be required for applications in wound healing and filtration.Compared to multiple composites, the addition of thermoplastic polyurethane (TPU) has shown great potential for enhancing mechanical properties38. PVDF/TPU electrospun scaffolds were introduced for wound healing, where cell migration and fibroblast activities were seen to be improved due to the piezoelectricity of the composite material39. Another study has evaluated the changes in piezoelectric and mechanical properties regarding the addition of TPU with PVDF nanofibers40. The results showed more flexibility in dipole excitations inside PVDF due to the elastic content of TPU. (GO)/Bi2S3-PVDF/TPU composite nanofiber mat was developed for photothermal applications41, combining GO/Bi2S3 nanoparticles as a photothermal conversion material and electrospun PVDF/TPU membrane as a substrate. The results observed that the hybrid novel mat has about a 95% light absorption rate at a wavelength range of 400\u20132500\u00a0nm. In addition, the presence of TPU significantly improved the mechanical strength of the composite film. TPU and bismuth sodium titanate polycrystalline oxide  were blended with (PVDF) and casted with the aid of a blade coater to investigate their effect on the piezoelectric response of composite membranes42. The remarkable enhancement in face shear piezoelectric coefficient (d36) was revealed by adjusting the blending content of TPU due to micro-pore structure formation, which facilitates charge transfer under different stress types.Several studies have investigated PVDF/TPU composite mat characteristics and performance for different electrical and biomedical applicationsIn our work, we analyze the detailed mechanical and piezoelectric characteristics of different blending ratios of PVDF and TPU nanofiber mats synthesized by the electrospinning process. In detail, we check the optimum blending ratios to generate the maximum voltage at different applied forces. In addition, we show the impact of frequency of the applied force on the piezoresponse behavior of different blended nanofiber mats. This work is helpful for wearable electronics and energy harvesting units.\u22121 is supplied by . Known polymer concentrations have been dispersed in dimethylformamide .Polyvinylidene fluoride (PVDF)  is supplied by ARKEMA and thermoplastic polyurethane (TPU) with Polydispersity Index (PDI) of 1.83 and molecular weight of 107,020\u00a0g\u00a0molDifferent blending ratios of PVDF and TPU solution with a constant polymer concentration of 10% were prepared and processed through the electrospinning setup. Comparative study of the effect of TPU addition on the piezoelectric and mechanical properties of PVDF mat has been introduced through five different blending ratios of PVDF/TPU . The electrospinning process was performed by adding 10\u00a0mL of polymer solution into a plastic syringe tipped with a stainless steel needle. The positive voltages were provided from a high voltage power supply CZE1000R  to the metallic needle with gauge 18, for application of voltages around 25\u00a0kV with a constant feed rate of (1\u00a0mL/h) using a NE1000 syringe pump . Needle-to-collector distance adjusted to 10\u00a0cm. Random PVDF/TPU nanofibers composite was collected on a drum collector covered with aluminum foil and connected to the ground.\u22121 over a range of 4000\u2013400\u00a0cm\u22121 to study the chemical functional groups of blended mats.The morphology of PVDF/TPU nanofibers (NFs) was observed by scanning electron microscope  with an accelerating voltage of 15\u00a0kV. The nanofiber mats were placed on carbon tape fixed on aluminum stubs and sputter-coated with platinum. The diameter of NFs was analyzed using Image-J software . The average fiber diameter distribution was manually detected by measuring the length through fiber boundaries at different imaging scales . Fourier transform infrared spectrometer (FT-IR)  was adjusted in ATR mode. Samples were scanned 120 times at a resolution of 5\u00a0cmTesting the effect of TPU addition on the mechanical properties of the fabricated mats was performed by cutting the nanofiber membranes into equal rectangular pieces (1\u2009\u00d7\u20096\u00a0cm). The samples were fixed between holding frames with a gauge length of 4\u00a0cm. A universal testing machine  was used to perform the stress\u2013strain curve. The tensile test was conducted at a strain rate equal to 10\u00a0mm/min with zero initial loads using a load cell of 100\u00a0N.The synthesized PVDF/TPU nanofiber membranes were tested under a cyclic load using an excitation instrument constructed for this purpose Fig.\u00a0. The insPiezoelectric voltage signals from the PVDF/TPU nanofibers were analyzed through a simple impulse loading setup, as shown in Figure Figure\u00a0\u22121 for CH2 rocking, C\u2013C and CF2 stretching, 1175 and 1400\u00a0cm\u22121 for C-F, and C-H vibrations, respectively45. While the characteristic bands of TPU appeared at 1533, 1735, 2971, 3365\u00a0cm\u22121 corresponding to \u2013CONH\u2013 asymmetrical bond, C=O, C\u2013H, and N\u2013H stretching, respectively47. The resultant data in Fig.\u00a0The FT-IR spectra of nanofibrous composite membranes are shown in Fig.\u00a0\u03b1 and A\u03b2 are the intensities of absorbance bands at 764\u00a0cm\u22121 and 840\u00a0cm\u22121 respectively.The beta-phase content was calculated according to the following equation derived from Beer-Lambert law;45. Hence, the high \u03b2-phase content for TPU 15% can be attributed to the effect of TPU mechanical elasticity on facilitating the reorientation of electric dipoles inside the composite nanofiber under the exposure of applied mechanical excitation42. However, the more increase of TPU content beyond 15%; the composite loses the resulting polarizability and the corresponding beta-sheets content from the PVDF.As shown in Table 49.Figure\u00a042. The experiments showed that the intensity of face shear electro-mechanical coupling is remarkably affected by TPU addition. Significant enhancement of piezoelectric coefficient d36 is obtained when a small portion of TPU (\u2264\u20095%) is introduced into the composite. Within the range of 5\u201320%, a nearly linear increase of d36can be achieved; then, it decreases at higher TPU concentration (>\u200920%)42. The polarization inside the nanocomposite is mainly in the direction of mat\u2019s thickness due to the electric field direction inside the electrospinning process. However, TPU mostly affects a shear strain. Based on the resulted improvement of piezoresponse, we think that such shear strain helps in a better orientation of polarizability to make more aligned dipoles in the thickness\u2019 direction, and consequently enhanced generated output voltage at the sample applied normal force.Regarding the Force-Voltage analysis for different PVDF/TPU concentrations, Table To better highlight the effects of frequency (f) on the different PVDF/TPU composites, a comparison has been made between PVDF/TPU 30% and PVDF/TPU 10% with cyclic forces applied at a rate of 16\u00a0Hz and 8\u00a0Hz. It can be concluded that relatively lower TPU concentrations responded better to mechanical vibrational frequencies. In comparison, higher TPU concentration in the PVDF/TPU composite nanofibers resulted in unstable peaks and an undetectable electrical potential. Both Figs.\u00a0The piezoelectric response of nanofiber mats from different PVDF/TPU composites was analyzed under impulse loading impact from a fixed height of 1\u00a0cm. Within most samples, it was observed that the resulting voltage increased while increasing the exposed weight as shown in Fig.\u00a0In this work, we investigated the characteristics of piezoelectric elastic nanocomposites mats. The synthesized nanofiber mats were composed of PVDF with TPU. The piezoelectric performance of the nanocomposite is related to PVDF, and the elasticity feature is related to the blended TPU. Our synthesized mats have been used to generate electric voltage under the effect of different mechanical excitations, such as mechanical stresses, with both controlled forces and vibration frequencies, along with impulse loading via falling masses. The optimum piezoelectric response is found at a blending ratio of TPU between 15 and 17.5 wt%, based on output voltage and piezosensitivity. Although the ratio of PVDF is reduced, the mechanical elasticity of blended TPU causes an improvement in the piezoelectric response of the nanocomposite. This conclusion has been supported by different measurements of piezoelectric voltage at different amplitudes and frequencies of vibrational forces, along with impulsive fallen masses. Meanwhile, FTIR analysis showed that the beta-sheets of 85:15 nanocomposite are nearly equal to the pure PVDF nanofiber mats. This innovative elastic-piezo nanocomposite can be applied within energy harvesting membranes and wearable electronics."}
+{"text": "The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct \u201cS-R/x3\u201d to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics. Spike (S) proteins on the surface of SARS-CoV-2 initiate viral infection by binding to cell receptors and mediating the fusion of virus and cell membranes. Several conformations of S have been identified that are thought to exist at different steps of the virus life cycle, for example assembly, receptor-binding and entry. The function of a conformation termed \u201clocked\u201d has not been clearly understood, due to its transience. Here, we engineered a disulfide bond in SARS-CoV-2 S to stabilise the locked conformation for structural and biochemical studies. This allowed us to distinguish two distinct locked-1 and locked-2 conformations in S from the initial SARS-CoV-2 strain, only the locked-2 conformation still exists after introduction of the D614G mutation. Based on structural and biochemical characterizations, we propose that the locked conformations of S prevent the premature opening of the receptor binding domain during virus assembly and egress through intracellular compartments. Our engineered S provides a useful tool to facilitate structural research, serological research, and design of immunogens. The spike (S) protein of coronaviruses is responsible for interaction with cellular receptors and fusion with the target cell membrane . It is tFig 1A).The S protein of SARS-CoV-2 is a large, 1273 residue, type I fusion protein, that can be processed by host proteases into two parts, S1 and S2. In the prefusion, trimeric form, S1 forms multiple folded domains. From N-terminus to C-terminus, they are the N-terminal domain , receptor binding domain , Domain C (residues 320\u2013330 and 529\u2013592), and Domain D (residues 308\u2013319 and 593\u2013699) \u20136. NeutrSARS-CoV-2 spike protein has been captured in four distinct conformations by cryo-electron microscopy (cryo-EM): three prefusion conformations\u2013locked ,8\u201310, clin trans with the NTD of the neighbouring S protomer, hiding the receptor binding site. In the locked conformation, the three RBDs are in very close proximity at the 3-fold axis, and a disulfide-bond stabilized helix-turn-helix motif (FPPR motif) is formed below the RBD that can sterically hinder RBD opening  A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Daved H FremontAssociate EditorPLOS PathogensAndrew PekoszSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158X\u200bMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1:\u00a0The manuscript from Qu et al investigates the locked conformation of the SARS-CoV-2 spike, a conformation that is infrequently observed by cryo-EM studies of spike protein as compared to the closed or open conformations. The locked conformation has not been observed in high-resolution structures of spikes on virions, however, raising some concern as to whether the locked conformation is an in vitro artifact. To more easily study the locked conformation, the authors design a disulfide bond by substitution D427 and V987 with cysteine. Biochemical characterization, including non-reducing SDS-PAGE, confirms that the disulfide bond does form as intended and prevents ACE2 binding, as expected. Cryo-EM analysis of this variant shows that 76% of the particles are in the locked conformation and 23% are in the closed conformation. Data processing is able to discriminate two different locked conformations, named locked-1 and locked-2, that differ primarily by the position of residues in domain D. Introduction of the D614G substitution shifts the distribution of spikes toward the closed conformation, suggesting that D614G makes the spikes inherently less stable and more prone to open, which is consistent with data and conclusions published in 2020.The manuscript has several strengths. The design of a disulfide-stabilized locked spike, or mostly locked spike, is interesting, and may be of use to the field. The biochemical and structural characterizations are very thorough and are performed to a high standard. The writing, although technical in nature, is clear. The major weakness or limitation of the manuscript is the potential lack of physiological relevance. As noted by the authors and mentioned above, the locked state has not been observed in structures of spikes on the surface of virions. A potential explanation is that the locked state is some artifact of recombinantly expressed and purified spike protein. The authors primarily speculate about the functional role of the locked-1 and locked-2 states (lines 359 and 387), but do not experimentally support their claims. And even if the locked state is sampled by spikes on infectious virions in vivo, it is possible that the different locked-1 and locked-2 states observed in these studies is due to the artificial nature of the introduced disulfide bond. Overall, it is solid work, but may be too preliminary at this time.Reviewer #2:\u00a0Investigators have advanced prior studies of SARS-CoV-2 spike structures by \u201clocking\u201d spike ectodomains with an engineered (aa 427 to aa 987) disulfide bridge. Biochemical tests revealed the disulfide locked ectodomains did not display RBDs to ACE2 (RBD-down) but RBDs could be raised for ACE2 binding by DTT reduction. Cryo-EM showed that most disulfide locked trimers were in closed conformations. Further analysis revealed two distinct locked conformations. The D614G change reduced the proportion of locked conformations. These are important results that advance understanding of spike protein structures in different conformations.Prolonged incubation of the locked trimers resulted in conversion to a \u201cclosed\u201d conformation, and then exposure of the closed form to pH5 re-locked the trimers. These changes regulating locked to closed transitions involved specific subdomains, notably the NTD associated domains. The manuscript includes impressive detail on the structural differences between the locked and closed conformations, and the effects of a D614G substitution on these differences.Structures are used to infer models of spike dynamics during virus secretion. The suggestion is that virus spikes are assembled into locked conformations during virus assembly and egress. This is based on the assumption that viruses egress through acidic organelles, which lock the trimers. Once secreted, virus spikes at neutral pH slowly convert to closed conformations, which are prerequisite to RBD opening.The models are very intriguing, however, it is recommended that they be further considered. The results of Ghosh et al. indicate that organelles through which the viruses egress are at neutral pH \u2013 there are accessory proteins of the virus that neutralize endolysosome lumenal compartments. Furthermore, the natural spikes undergo furin cleavage in the TGN, which effects conformational changes that were not described in the models. In addition, the D614G change that is highlighted in the model is known to provide a stabilizing force, maintaining the S1-S2 heterodimers. It is difficult to appreciate this important S1-S2 stabilizing property in the context of the model involving locked and closed conformations. Finally, the notion that there are very slow transitions from the locked to closed, and then to open conformations of spike do not appear consistent with the very rapid cell membrane fusions that take place when spike-expressing cells are incubated with ACE2-expressing target cells. In this case, as soon as spikes appear on plasma membranes, they promote cell membrane fusions, suggesting that they are in open conformations.**********Part II \u2013 Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a \"Major Revision\" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend \"Reject\".Reviewer #1:\u00a0(No Response)Reviewer #2:\u00a0The models are very intriguing, however, it is recommended that they be further considered. The results of Ghosh et al. indicate that organelles through which the viruses egress are at neutral pH \u2013 there are accessory proteins of the virus that neutralize endolysosome lumenal compartments. Furthermore, the natural spikes undergo furin cleavage in the TGN, which effects conformational changes that were not described in the models. In addition, the D614G change that is highlighted in the model is known to provide a stabilizing force, maintaining the S1-S2 heterodimers. It is difficult to appreciate this important S1-S2 stabilizing property in the context of the model involving locked and closed conformations. Finally, the notion that there are very slow transitions from the locked to closed, and then to open conformations of spike do not appear consistent with the very rapid cell membrane fusions that take place when spike-expressing cells are incubated with ACE2-expressing target cells. In this case, as soon as spikes appear on plasma membranes, they promote cell membrane fusions, suggesting that they are in open conformations.**********Part III \u2013 Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1:\u00a0(No Response)Reviewer #2:\u00a0(No Response)**********what does this mean?). 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For an example see here: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at References:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.\u00a0If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice. 4 May 2022AttachmentD614G_ResponseToReviewers_220419.pdfSubmitted filename: Click here for additional data file. 9 May 2022Dear Briggs,We are pleased to inform you that your manuscript 'Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Daved H FremontAssociate EditorPLOS PathogensAndrew PekoszSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158X\u200bMichael MalimEditor-in-ChiefPLOS Pathogens <span style=\"font-family: \"Noto Sans\", sans-serif; font-size: 12px;\"> orcid.org/0000-0002-7699-2064 </span> ***********************************************************Reviewer Comments : 26 Jul 2022Dear Briggs,We are delighted to inform you that your manuscript, \"Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike,\" has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158X\u200bMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064"}
+{"text": "Background: Social behavior is mediated by steroid hormones, whereby various lines of evidence indicate that metformin might improve the symptoms of social withdrawal. This directly yields to the aim of the study to correlate the impact of metformin treatment on the potential alterations in steroid hormone homeostasis, which is ultimately impacting social behavior. Therefore, urinary samples of patients before and after treatment with metformin will be correlated to social behavior to elucidate potential changes in steroid hormone profiles and social behavior. Material and Methods: An observational study in healthy adults with a new indication for metformin. Steroid hormone analysis, including the most prominent androgen, estrogen, progesterone, aldosterone, corticosterone, cortisone and cortisol metabolites analyzed with gas chromatography\u2013mass spectrometry and a questionnaire on social behavior (Autism Spectrum Questionnaire (AQ)) will be administered prior to and after around a 12-week phase of metformin treatment. Discussion: It is likely that due to different pathophysiological mechanisms such as an effect on the respiratory chain in mitochondria or via AMP-activated protein kinase, a general alteration of steroid hormone levels can be detected prior to post treatment. The encompassing measurement of steroid hormones shall give hints concerning the involvement of specific cascades yielding potential pharmacological targets for future research. The oral biguanide metformin has been an effective first-line treatment of type 2 diabetes mellitus in adults for more than 60 years. It is to this day the most prescribed oral antiglycemic drug in the world and remains the foundation of type 2 diabetes mellitus treatment, according to the American Diabetes Association, due to its safety, efficacy and cost-efficiency ,2,3. MetSurprisingly, studies have shown metformin treatment reduces macrovascular events and mortality in diabetes mellitus type 2 patients exceeding the expected risk reduction through decreased blood glucose levels. More recent studies have further shown strong support for additional benefits in treating obesity, metabolic syndrome, polycystic ovary disease and depression while also having anti-inflammatory and neuroprotective effects, opening up the possibility for new off-label indications ,4,5.First, treatment with metformin mimics some of the benefits of caloric restriction, yielding increased insulin sensitivity and reduced low-density lipoprotein and cholesterol levels while inhibiting low-grade inflammation ,7,8,9,10Furthermore, AMP-activated protein kinase activity is increasing antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation ,9,10,11.Additionally, several lines of evidence indicate a beneficial effect of metformin on depression, neurodevelopmental disorders such as autism, neurodegenerative disorders such as Alzheimer\u2019s disease and genetic diseases with neurodevelopmental consequences such as fragile X syndrome ,29,30. ATo summarize, the effects of metformin on steroid hormones are, however, only partly elucidated (mainly restricted to the context of PCOS (polycystic ovary syndrome), yielding directly to the aim of this study ,43,44,45Several lines of evidence indicate that metformin seems to have an effect on steroid hormones ,48,49,50As a hypothesis with potential falsification, it shall be stated that a newly administered standard therapy with metformin has no influence on steroid hormone profiles in subjects having an indication for Metformin ,49,50,51As a hypothesis with potential falsification, it shall be stated that a newly administered standard therapy with metformin has no influence on social behavior in subjects having an indication for metformin ,51.There is no statistical correlation between steroid hormone levels and social behavioral changes .As we try to extend our research into the effects of metformin treatment not previously investigated , we intend to choose a simple study design.The study is designed as a clinical trial to investigate changes primary in steroid hormones and secondary in social behavior during metformin treatment. Therefore, a baseline data set will be obtained prior to intervention, and a follow-up at 12 weeks will be conducted. An accompanying social environment anamnesis will also be collected.The metformin indication itself is independent of study participation and is made following up-to-date diabetes guidelines. The study participation consists of urinary samples and a standardized Autism-Spectrum Quotient questionnaire obtained twice. All patient samples and data will be collected with prior informed consent.This study aims to analyze the effect of metformin treatment on steroid hormone profiles. Therefore, steroid hormones in patients with a new indication for metformin shall be measured with an encompassing gas chromatography\u2013mass spectrometry (GC\u2013MS) prior to treatment start and after 12 weeks of treatment.This study will investigate changes in social behavior under metformin treatment. Additionally, to the above-mentioned measurements, an autism questionnaire (Autism-Spectrum Quotient) ,53,54 shThis study will elucidate the potential correlation between steroid hormone levels and social behavioral changes.Changes in steroid hormone profiles of interest (see below) prior to metformin treatment start and after 12 weeks of treatment.Changes in social behavior measured by the Autism-Spectrum Quotient AQ (see below) prior to metformin treatment start and after 12 weeks of treatment.Correlation between steroid hormone profiles and social behavioral changes.Patients  older than 18 years with type 2 diabetes mellitus and an indication for metformin according to the American Diabetes Association, starting a new metformin treatment or are pausing treatment for example due to a further medical investigation for at least seven days. The main marker will be fasting plasma glucose levels of\u2265 7.0mmol/L and/or HbA1c \u2265 6.5% .Patients under 18 years of age.Clinically significant concomitant disease .Significant musculoskeletal disease.Active infection during sample collection.Immunosuppressive medical therapy.Hormonal/steroid treatment.Pregnancy.Psychiatric disease and known social-behavior-altering medication .Known or suspected malcompliance, drug or alcohol abuse.Inability to follow the procedures of the study, e.g., due to insufficient language skills, severe dementia.Life expectancy less than 6 months.Poor tolerability to metformin treatment with following treatment discontinuation within duration of follow-up.Pharmaceutical treatment with insulin or another pharmaceutical with a known strong effect on blood sugar levels.In the 1980s, a correlation between insulin, insulin-sensitizing drugs  and androgens was already being implied . In studTo research the primary endpoint of steroid hormone profile changes, we will collect urinary samples prior to metformin treatment start and after 12 weeks of treatment. Similarly, to research the secondary endpoint of social behavioral changes, we will conduct a questionnaire (Autism-Spectrum Quotient) along with social environmental anamnesis at both times.The additional medical risks of this study are to be assessed as very low . Risks arise only from the administration of metformin, which has to be assessed as a safe, very well-known and frequently used oral antiglycemic drug, the only severe side effect being lactic acidosis . In geneAs the detailed sample size calculation see  already Further, based on the number of patients per year in our clinical setting, it seems reasonable to win enough patients within 2 years. We estimate an average of one patient a week, yielding a total of 50 patients a year so that we will have enrolled a total of around 100 patients after 2 years. The recruitment will be conducted by the department of internal medicine of the Lindenhofgruppe and its associated General practitioners. Due to the common characteristics of patients with type 2 diabetes mellitus we expect to mainly recruit patients above 50 years of age and slightly more male than female patients. Furthermore, the obstetrics department of Inselspital Berne with its Patients with a potential indication of metformin due to PCOS will also be included.Analysis of urinary steroids will be conducted via gas chromatography\u2013mass spectrometry. Urine samples shall be taken prior to treatment start and after 12 weeks of treatment (gold standard: 15 mL from a 24 h urine collection). Urine sample preparation comprises pre-extraction, enzymatic hydrolysis, extraction from the hydrolysis mixture, derivatization and gel filtration. The recovery standard will be prepared by adding 2.5 \u00b5g of medroxyprogesterone to 1.5 mL of urine. The sample will be extracted on a Sep-Pak C18 column , dried, reconstituted in a 0.1 M acetate buffer, pH 4.6, and hydrolyzed with a powdered Helix pomatia enzyme  and 12.5 \u00b5L of \u03b2-glucuronidase/arylsulfatase liquid enzyme . The resulting free steroids will be extracted on a Sep-Pak C18 cartridge. A mixture of internal standards  will be added to this extract, and the sample will be derivatized to form the methyloxime-trimethylsilylethers. Analyses will be performed on a Hewlett Packard gas chromatograph 6890  with a mass selective detector 5973 by selective ion monitoring (SIM). One characteristic ion will be chosen for each compound measured. The derivatized samples will be analyzed during a temperature-programmed run (210\u2013265 \u00b0C) over a 35 min period. The calibration standard consists of a steroid mixture containing known quantities of all the steroid metabolites to be measured. Responses and retention times will be recorded regularly. In each case, the ion peak will be quantified against the internal stigmasterol standard. All procedures will be performed as described several times by us and others ,59,60.The following metabolites shall be measured as described above ,16:Androgen metabolites: Androsterone, Etiocholanolone, Androstenediol, 11-Oxoetiocholanolone, 11\u03b2-Hydroxyandrosterone, 11\u03b2-Hydroxyetiocholanolone, Dehydroepiandrosterone, 5-Androstene-3\u03b2,17\u03b2-diol, 16\u03b1-Hydroxydehydroepiandrosterone, 5-Androstene-3\u03b2,16\u03b1,17\u03b2-triol, 5-Pregnene-3\u03b2, 16\u03b1,17\u03b2-triol, Testosterone, 5\u03b1-DihydrotestosteroneEstrogen metabolites: Estriol, 17b-Estradiol.Progesterone metabolites: 17-Hydroxypregnanolon, Pregnanediol, Pregnanetriol, 11-Oxo-Pregnanetriol Aldosterone-Metabolites: Tetrahydroaldosterone.Corticosterone metabolites: TetrahydroDOC, Tetrahydrodehydrocorticosteron, Tetrahydrocorticosteron, 5a-Tetrahydrocorticosteron, 18-Hydroxy-tetrahydrocompound A, Cortisone.Cortisone metabolites: Tetrahydrocortisone, a-Cortolon, b-Cortolon, 20a-Dihydrocortison, 20b-Dihydrocortison.Cortisol metabolites: Cortisol, Tetrahydrocortisol, 5a-Tetrahydrocortisol, a-Cortol, b-Cortol, 20a-Dihydrocortisol, 6b-Hydroxycortisol, 18-Hydroxycortisol.In order to measure social behavioral changes related to typical behaviors of autism and expand the idea of autism being related to steroid hormone levels, we will be conducting an autism questionnaire prior to and after 12 weeks of treatment. The Autism Spectrum Quotient (AQ) is a questionnaire published in 2001 by Simon Baron-Cohen and his colleagues at the Autism Research Center in Cambridge, UK ,53,54. AData will be anonymized and stored in a pseudonymized manner. For females before menopause, the day of the menstrual cycle will be reported for further analyses. Personal data including ethnicity will be entered into a separate Excel file with restricted access only by the core staff of the study. Collection and management of the clinical trial data will be done using an electronic data capture system (Red Cap Cloud). Source data will be kept under lock and key. Password protection ensures that only authorized persons can enter the system to view, add or edit data according to their permissions. The complete study dataset is exported and transferred to the investigators as well as to the study statistician through a secured channel. Data will be archived at the Lindenhofspital. Independently from investigators, regulatory authorities can audit this trial. Direct access to source documents will be permitted for purposes of monitoring, audits and inspections. All involved parties must keep the participant\u2019s data strictly confidential. Any results of this study will be published in an anonymized manner. The study protocol and dataset shall be accessible to any regulatory authority after publication for at least 10 years.Per Morin-Papunen et al., 2003, the most prominent serum metabolite testosterone decreased by 2.7 \u00b1 0.3 to 2.0 \u00b1 0.2 nmol/liter in patients receiving metformin  . By takiMean and SEM (standard error of mean) of all metabolites will be calculated pre-versus post intervention for the respective sub-samples distinguished by age and sex. To analyze the distribution patterns of the measured values of each metabolite Kolmogorov\u2013Smirnov tests will be conducted. If normality distribution is present, the differences between pre-versus post intervention shall be analyzed with two-tailed dependent t-tests while correcting for multiple comparisons with Bonferroni correction. If the hypothesis of normal distribution must be rejected for metabolite concentrations pre-versus post intervention, Wilcoxon tests shall be conducted. If normal distribution is present, differences pre-versus post intervention will be further quantified with the calculation of effect sizes by Cohen with pooled standard deviation and 95% confidence intervals ,64. For Based on these preliminary analyses, structure equation modeling can be applied if indicated in order to combine findings from biochemical analysis and from questionnaires. All calculations will be performed with GraphPad Prism  and SPSS .The aim of this study is to analyze the effects of metformin treatment on steroid hormone profiles. To the best of our knowledge, there is no study directly elucidating the presumed effects on steroid hormones and social behavior in humans . The desWe are aware that the optimal study design would be a placebo-controlled crossover trial, which is, however, for practical reasons, impossible to conduct. Nevertheless, several lines of evidence suggested the involvement of steroid hormones in autism, especially increased androgens . Just reFor this purpose, we want to analyze the change in social behavior and steroid hormone profiles, as well as their potential correlation, under metformin treatment. Because only urinary metabolites of steroid hormones and not the blood serum levels themselves will be examined, there is a certain limitation concerning the reliability of measured outcomes; also, comparisons with other studies might be restricted. We chose this measurement because of its general availability and ease of use for both patient and physician to ensure easier recruitment and follow-up. As a general research question, we suspect that certain substances modulate social behavior via steroid hormones, which act as an intermediary in social behavior. Therefore, we will measure both urinary steroid hormone profiles as well as social behavior through an Autism Spectrum Quotient respectively questionnaire before treatment start and after.Although we expect our patient sample of adults not typically affected by social behavioral disorders or autism spectrum disorders to score in the lower spectrum of the questionnaire, we have chosen this tool because of its simplicity in use to ensure sufficient patient recruitment and follow-up. The questionnaire has been tested in both adults with autism spectrum diseases as well as healthy adults. In healthy adults, significant differences between genders and different backgrounds (mathematics vs. humanities) were recorded, while all scores remained in the lower spectrum . As discRegarding patient recruitment, we are well aware of potential effect alterations due to their type 2 diabetes mellitus, the difference in sex and baseline hormone levels , as well as an expected higher age. We chose this patient setting because we wanted to be able to recruit enough patients in a relatively short period while also ensuring safety for the participants, as they all would have received the metformin treatment without our study anyway. This patient collective also shares many characteristics with a majority of cancer patients who are also often severely impacted by social impairment due to their disease.Nevertheless, we hope to show the influence of metformin treatment on social behavior and/or steroid hormone profiles. Published in a recognized specialist journal, these findings could trigger further research within a more specific patient setting of social avoidant people in a randomized, double-blind placebo-controlled study.If we do succeed in showing a correlation between social behavior and steroid hormone profile changes under metformin treatment, this could result in a recommendation for further, more focused research examining the cascades of metformin\u2019s effects even further and the possibility to influence more biochemical cascades will arise. A better insight into the effects of one of the most common drugs worldwide may result in more specific and targeted treatment, maybe becoming a part of personalized medicine.The study is in the lowest class A . It is a therapeutic exploratory study per ICH E8 . This stAccording to the legal situation in Switzerland, the study is considered within the Humanforschungsgesetz (HFG)  and VeroThe trial will be conducted per protocol version 1.0. Patient recruitment will start in Winter 2021 and is expected to run consecutively until the end of December 2023. At the time of submission, zero patients have been included in the study. Data collection is expected to be completed in December 2023 and data analysis in Spring 2024.Clinicaltrials.gov database .Ethics approval to conduct this trial has been granted by the local Ethics Committee for the Region of Bern (Project-ID: 2020-02913). The trial will meet the criteria and principles of the Declaration of Helsinki and has been registered in the Informed consent to participate in the trial will be obtained by the study physician from all patients prior to entry into the study. Each patient will be informed that participation in the study is voluntary and that he/she may withdraw from the study at any time with no need for justification, and that withdrawal of consent will not affect his/her subsequent medical assistance and treatment. Furthermore, the patient will be informed on an obligatory basis that his/her medical records may be examined by authorized individuals other than their treating physician. All patients are covered by liability insurance for the total study duration."}
+{"text": "Acute flaccid paralysis (AFP) is a clinical syndrome characterized by the acute onset of weakness and paralysis with reduced muscle tone. This study explored the incidence and different aspects of AFP in Lebanese children between 2009 and 2019.AFP data were collected from the Lebanese Ministry of Public Health. Incidence rate according to year, age groups, clinical data, follow-up, diagnosis, and vaccination status was analyzed in the 11-years period.AFP incidence rates increased importantly from 0.63 per 100,000 in 2009 till 4.96 per 100,000 in 2019. Most of the patients were children under ten years of age, 40.6% of all cases were under five years old, and 37.9% were between 5 and 9 years old. Follow-up revealed that approximately two out of five patients experienced residual weakness. As for the final diagnosis, around 30% of cases were diagnosed as Guillain-Barre Syndrome (GBS). Most cases were children having received between 3 and 5 doses of polio vaccine.The rise in cases coincided with the Syrian refugee crisis in Lebanon and the progressively deteriorating economy in the country; yet, incidence rates were in the lower margin compared with other countries. Acute flaccid paralysis (AFP) is a rare clinical syndrome, having various infectious and non-infectious causes, with a broad array of potential etiologies that vary with age. AFP is characterized by the acute onset of weakness and paralysis with reduced muscle toneth centuryIn spite of its rarity and despite the Global Polio Eradication Initiative declared by WHO in the 20In the Middle-Eastern region, polio outbreaks have also erupted, often leading to AFP. In 2017, Syria was the victim of a vaccine-derived poliovirus type 2, resulting in 74 cases of AFP, the most recent of which showing onset of paralysis on 21 September 2017https://www.moph.gov.lb) has established an AFP surveillance program that reports cases of AFP since 2009. The registry categorizes reported cases according to many factors, including age group, region, and clinical data. In recent years, the reported AFP cases have reached relatively high numbers, and as far as we know, no previous studies have investigated the different aspects of AFP cases.On the local scale, the Lebanese MOPH's ESU  from the AFP surveillance system and publicly available on the MOPH official website. The database was screened between 2009\u20132019, and data were specifically selected to match the study's aim.Reported cases of AFP are classified as suspected, possible, and confirmed polio or non-polio cases according to laboratory tests and criteria listed in the MOPH's AFP Surveillance GuidelineChildren under 15 years of age presenting AFP for any other reason than severe trauma are suspected cases of poliomyelitis. Paralytic illness in a person of any age, in whom the physician suspects polio can also be considered a suspected case.Cases are classified according to laboratory results as polio-confirmed, polio-compatible, or polio-discarded cases.The laboratory isolates a wild poliovirus from the index AFP case or any of the contacts.There were no polio-confirmed cases registered in Lebanon between 2009 and 2019, and all non-polio AFP cases were classified according to the final diagnosis. AFP can be caused by several diseases, including Guillain-Barre Syndrome, transverse myelitis, and other pathologies .The yearly incidence rate was obtained by dividing the reported cases of AFP in Lebanon in that year by the total population for the specific age group, including Lebanese and non-Lebanese residentsThis descriptive epidemiological study includes data from public websites . The number increased moderately from 8 cases in 2009 to 50 cases in 2014 and abruptly attained a peak of 113 cases in 2015. This number slightly diminished in the following years . A compoConcerning the geographical distribution of AFP, the number of cases throughout these 11 years was evenly scattered among governorates excluding Mount-Lebanon , varying between 49 (Beirut) and 76 (Bekaa) . HoweverThe number of cases varies depending on the age group (p<0.001). Effectively, 239 cases out of 631 were observed in children between 5 and 9 years of age , and 256 cases were observed in children under five years of age . These numbers declined to 131 in the 10 to 14-years-old age group .From 2009 until 2014, more patients had no fever at the onset of the disease compared to those who did . From 20In every year observed, the number of patients who did not suffer from asymmetric paralysis exceeded the number of patients who did suffer from it (p<0.001). Nevertheless, the ratio of patients not suffering from asymmetric paralysis over the patients suffering from it was variable throughout the 11 years of the study, reaching a maximum of 10 in 2013 and considerably lower fluctuating values in the remaining years.Furthermore, a large proportion of patients experienced rapid progress within four days (p<0.001). This observation was most prominent in 2018 when the ratio of patients who progressed rapidly within four days over those who did not attain 11.7 per 100,000. This ratio was the lowest in 2011 when it reached 1.6 per 100,000.After follow-up, it was noted that 382 out of the total 631 cases (>50%) felt no residual weakness, while 137 cases still felt residual weakness . Twelve The cases of AFP recorded in Lebanon between 2009 and 2019 were diagnosed to be non-related to polio. 32.1% of these non-polio cases were attributed to Guillain-Barre, 1.6% to transverse myelitis, and the majority, which constitutes 62.0%, were attributed to other causes . No caseThe total number of cases recorded from 2009 to 2019 was moderately constant for patients who received 0, 1, 2, 6, 7, 8, and 9 OPV/IPV doses. Forty-four cases were observed at most in the case of 2 vaccination doses, and only 2 cases were noted throughout the 11 years of the study at nine vaccination doses. However, a peak was observed when three to five doses were given, reaching a maximum of 198 total cases at four doses . StatistAccording to the MOPH, Lebanon registered 631 cases of AFP between 2009 and 2019, averaging an incidence rate of 3.36 cases per 100,000 individuals under 15 years of age. Compared to selected regional countries , that nuAs depicted in the results section, data analysis revealed an overall positive growth rate. The incidence of AFP increased between 2009 and 2015 and was followed by a modest decrease till 2019. Most cases were children under ten years of age. Geographically, the most common governorate for AFP cases was Mount-Lebanon, probably due to its relatively large population and its crowdedness. The third of AFP cases were attributed to Guillain-Barre, while the majority of cases were from other causes. Poliomyelitis incidence was very low. Moreover, two out of fivehildren experienced residual weakness after follow-up. A higher incidence of AFP was observed among patients receiving four vaccination doses, and the curve shows a bell-like distribution. Furthermore, Lebanon seems to have a low average incidence rate compared to other countries.The incidence rate of AFP increased from 0.63 per 100,000 in 2009 to 4.96 per 100,000 in 2019, reaching a peak of 6.33 per 100,000 in 2015. The remarkable rise in the number of cases described in the results section coincides with the Syrian refugee crisis that led up to 1.5 million refugees from Syria seeking shelter in Lebanon. The population of Lebanon has remarkably increased by 30% because of the influx of Syrian refugees. That influx is highly skewed towards infant and children age groups, unlike the current demographic trends in aging in Lebanon. Studies have shown that Syrian refugees in Lebanon live in poor conditions and suffer mostly from communicable diseases and diseases affecting womenAs for future projections, it should come as no surprise that the incidence would get back to the pre-2011 situation, as the refugee population is stabilizing and blending into the Lebanese context, thus, becoming vulnerable to the microbiological realities just the same way as the Lebanese population. Prevention of outbreaks and implementation of vaccination programs throughout the country are needed, especially with the progressively deteriorating economy. In addition, the increase in cases can be partly explained by the enhanced efficiency of the surveillance programs throughout their decade of experience. Lastly, further screening of the vaccination program could reveal possible causes of vaccine-induced AFP due to defective vaccines.AFP Surveillance systems report each year the number of cases of polio and non-polio AFP and are used as a proxy for measuring the validity of the polio surveillance systemThe study reported that throughout the 11 years, there were almost as many cases of non-polio AFP in children under five years of age (40.6%) as there was in children between five and nine years old (37.9%), while the 10\u201314 years' group represented a minority in the study 20.8%) . The rem.8% . TheWith the advances in the eradication of poliomyelitis, GBS has become the most common cause of AFP in the worldThis study revealed that 83.2% of AFP cases received at least 3 doses of OPV/IPV vaccine, 13.8% received less than three doses , and the remaining cases were unspecified (3.0%). This vaccination rate of 83.2% is considered significantly high compared to the same rate in Congo (58.9%) between 2008 and 2014Moreover, the study showed that most AFP patients were individuals having received three, four, and five vaccine doses. The incidence according to vaccine dosage follows a bell-shaped curve almost every year, where most cases have received between three and five doses of IPV or OPV . InteresAccording to the MOPH, Lebanon registered 631 cases of AFP between 2009 and 2019, averaging an incidence rate of 3.36 cases per 100,000 individuals under 15 years of age. Compared to selected regional countries , that nuWe focused in this study on AFP cases occurring in children from 0 to 15 years. This study presented a lack of specification in the data, such as the absence of specification of the neurologic exam of the different presentations of AFP  and a lack of detailed description of the neurologic examination in the patients who improved spontaneously and those who did not, or worsened. Another shortfall in the study was the lack of a gender specification for the cases, which would have allowed a more significant comparison with previous similar studies. Under-reporting of AFP cases would be unusual in this study due to the sizeable involvement of international organizations with the refugee population and their focus on preventable infectious diseases such as Polio and Measles. Finally, due to the unexpected demographic dynamics and political situation of Lebanon, it would have been inaccurate to provide statistical projections of incidence rates in future years.The data provided by the MOPH was evaluated in terms of incidence rates of AFP in Lebanon, age distribution, causes of the disease, and vaccination doses, in order to get a better understanding of the disease and improve the plans of action towards its eradication. AFP incidence rate increased during the Syrian refugee crisis and is expected to stabilize in the upcoming years; still, Lebanon holds a lower-than-average incidence rate compared to regional and random countries. In the light of the ongoing refugee crisis and the deteriorating socio-economical norms, physician/patient awareness, prevention, and vaccination are crucial in flattening the curve of AFP and controlling the disease."}
+{"text": "KD: 1.3 \u00d7 10\u22129 M). Furthermore, E134Bf-H77scFv exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti-EGFR and anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients\u2019 survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf) and a mouse-dog chimeric anti-HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR/dHER2-positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity ( Therapeutic monoclonal antibodies (mAbs) for tumors have revolutionized over several decades due to the development of the hybridoma technique  and the 1 isotype) exhibits ADCC activity, and directs cytotoxic immune effector cells to EGFR-positive tumor cells  containing 1 \u03bcg/mL of E134Bf, H77Bf, E134Bf-H77scFv for 30 min at 4 \u00b0C. Then, cells were suspended in blocking buffer containing Alexa Fluor 488-conjugated anti-dog IgG  for 30 min at 4 \u00b0C. Fluorescence data were collected by the Cell Analyzer EC800, and analyzed by EC800 software ver. 1.3.6 .KD), GraphPad Prism version 8  was used.Serially diluted E134Bf (0.0006\u201310 \u03bcg/mL), H77Bf (0.0006\u201310 \u03bcg/mL), and E134Bf-H77scFv (0.0006\u201310 \u03bcg/mL) were suspended with CHO/dEGFR, CHO/dHER2, and D-17 cells. The cells were further treated with Alexa Fluor 488-conjugated anti-dog IgG (1:100). Fluorescence data were obtained by the Cell Analyzer EC800. To determine the apparent dissociation constant  were resuspended in DMEM supplemented with 10% FBS, and were used as effector cells. Target D-17 cells were labeled with 10 \u03bcg/mL of Calcein AM  ,40,41,42The target D-17 cells were incubated with 10 \u03bcg/mL calcein AM ,40,41,426 cells) were subcutaneously inoculated into the left flank of mice together with BD Matrigel Matrix Growth Factor Reduced . On day 8 after the inoculation, 100 \u03bcg of E134Bf, H77Bf, and E134Bf-H77scFv, and control dog IgG (n = 8) in PBS (100 \u03bcL) were intraperitoneally injected. Additional antibodies were intraperitoneally injected on days 15 and 22. Furthermore, on days 8, 15, and 22, canine MNCs (5 \u00d7 105 cells) were injected into the surrounding tumors. The tumor volume was measured on days 8, 10, 15, 17, 22, and 25, as described previously [BALB/c nude mice  were purchased from Charles River Laboratories, Inc. D-17 cells  and H77B (anti-dHER2), both of which possess the B type dog IgG backbone to confer the ADCC activity. We further produced the corresponding defucosylated forms, E134Bf  and H77B(KD) of E134Bf, H77Bf and E134Bf-H77scFv to D-17 cells using flow cytometry. As shown in KD values for the interaction of E134Bf, H77Bf, and E134Bf-H77scFv with D-17 cells were 6.0 \u00d7 10\u221210 M, 2.9 \u00d7 10\u221210 M, and 1.3 \u00d7 10\u22129 M, respectively.We first confirmed the reactivity of E134Bf-H77scFv to dEGFR and dHER2 expressed cells using flow cytometry. As shown in p < 0.01). There was no significant difference between E134Bf or H77Bf and the control dog IgG against D-17 in this experimental condition  in D-17 cells compared with that induced by the control dog IgG (13.8% cytotoxicity). These results indicated that E134Bf-H77scFv exhibited higher levels of ADCC and CDC activities against D-17 cells.We next investigated whether E134Bf-H77scFv was capable of mediating ADCC against D-17 cells. E134Bf-H77scFv showed ADCC (15.2% cytotoxicity) against D-17 cells more effectively than the control dog IgG , 15 (p < 0.05), and 17 (p < 0.01) than that of the E134Bf, H77Bf and control dog IgG (p < 0.01). D-17 tumors that were resected from mice on day 25 are shown in To evaluate the antitumor activity against D-17 xenograft tumor, E134Bf-H77scFv, E134Bf, H77Bf and control dog IgG were intraperitoneally injected into mice on days 8, 15, and 22 after the inoculation of D-17 cells. Furthermore, on days 8, 15, and 22, canine MNCs were injected surrounding the tumors. On days 8, 10, 15, 17, 22, and 25 after the inoculation, the tumor volume was determined. The E134Bf-H77scFv administration resulted in faster reduction of tumor volume on days 10 ( dog IgG A. Howeve dog IgG A. As sho377-RGDSFTHTPP-386) in domain III of EGFR [In this study, we developed a novel bispecific antibody E134Bf-H77scFv against dEGFR and dHER2 , and sho of EGFR . The EGFH and VL cDNAs should be considered, and the selection of objective bispecific antibody is required. In contrast, E134Bf-H77scFv can be produced by transfection with single combination of VH and VL cDNA, and purified by the conventional Fc binding beads.E134Bf-H77scFv is a novel modality for targeting dEGFR and dHER2. Because H77scFv are fused to the light chain of E134B but not to the heavy chain, E134Bf-H77scFv recognizes dual antigens without affecting the Fc-mediated ADCC activity. Furthermore, in case of bispecific antibody with two unique Fab domains, the combination of V2Mab-77, original antibodies of E134Bf-H77scFv, can be used in immunohistochemistry [2Mab-77, and E134Bf-H77scFv are expected to be used in clinics for both the diagnosis and treatment of canine tumors.Previously, different types of dual-targeting EGFR and HER2 bispecific modalities have been tested in preclinical studies. A bispecific affibody molecule to EGFR or HER2 was generated by linking a bivalent HER2-binding affibody to a bivalent EGFR-binding affibody with a linker sequence (Gly4-Ser)3. The bispecific affibody was shown to bind to both HER2-overexpressed SKBR-3 and EGFR-overexpressed A-431 cells . \u201cAffiMahemistry ,24. TherBispecific antibodies have been approved as therapeutic agents to cover unmet clinical needs ,51. A wiFor the development of E134Bf-H77scFv to treat canine tumors in the clinic, the effect of E134Bf-H77scFv on dEGFR homodimer and dEGFR/dHER2 heterodimer-mediated signaling should be investigated. Further, it should be determined whether E134Bf-H77scFv can induce the internalization of dEGFR and dHER2. Canine tumors represent an outbred population, and resemble human tumors in the initiation, disease progression, growth factors and oncogene mutations. A growing number of evidences has suggested that elevated expression of other HER family and their ligands confer the resistance to trastuzumab . For ins"}
+{"text": "Ferroptosis is a cell death pathway triggered by an imbalance between the production of oxidants and antioxidants, which plays an emerging role in tumorigenesis. It is mainly regulated at three different levels including iron metabolism, the antioxidant response, and lipid metabolism. Epigenetic dysregulation is a \u201challmark\u201d of human cancer, with nearly half of all human cancers harboring mutations in epigenetic regulators such as microRNA. While being the crucial player in controlling gene expression at the mRNA level, microRNAs have recently been shown to modulate cancer growth and development via the ferroptosis pathway. In this scenario, some miRNAs have a function in upregulating, while others play a role in inhibiting ferroptosis activity. The investigation of validated targets using the miRBase, miRTarBase, and miRecords platforms identified 13 genes that appeared enriched for iron metabolism, lipid peroxidation, and antioxidant defense; all are recognized contributors of tumoral suppression or progression phenotypes. This review summarizes and discuss the mechanism by which ferroptosis is initiated through an imbalance in the three pathways, the potential function of microRNAs in the control of this process, and a description of the treatments that have been shown to have an impact on the ferroptosis in cancer along with potential novel effects. MicroRNAs (miRNAs), defined as small noncoding RNAs of around 25 nucleotides that regulate gene expression at the post-transcriptional level, were initially described in 2005; currently, over 2700 mature miRNAs have been identified in humans with characteristic expression profiles . miRNAs Many diseases are associated with ferroptosis. Inhibiting or promoting the enzymes or genes involved in ferroptosis has an impact on the course of diseases. miRNAs can alter disease progression by modulating ferroptosis. Interestingly, the role of ferroptosis in cancer is not quite the same as in other diseases. Ferroptosis can inhibit the proliferation and cell cycle of cancer cells, which suggests that we should focus on ferroptosis-based therapies for cancer .Ferroptosis is caused by a redox imbalance between the production of oxidants and antioxidants, which is driven by the different expressions and activities of the multiple redox-active enzymes that produce or detoxify free radicals and lipid oxidation products. Accordingly, ferroptosis is regulated at multiple levels, including epigenetic, transcriptional, post-transcriptional, and post-translational levels . DiffereThe present review discusses the microRNA modulation related to ferroptosis with the aim of providing novel insight to increase knowledge about personalized cancer therapy.Ferroptosis is an iron-dependent form of programmed cell death. Defined by a novel term for the first time by Dixon et al. in 2012 , it has The term \u201cferroptosis\u201d was coined following the observations that RAS-mutated cancer cells were more sensitive to ferroptosis activation compared to cancer cells that lack RAS mutations ,10. ThisRecent studies reported that the well-known tumor suppressor gene p53 plays a role in ferroptosis . P53 wasFerroptosis is the consequence of an imbalance between the levels of reactive oxygen species (ROS) and the activity of the antioxidant defense, with a consequent accumulation of ROS in the plasmatic membrane, process widely known for causing many disorders in the cell . In this3+ form carried by a special transport protein called transferrin (TF). When it must enter the cell, Fe3+ is carried by transferrin near the plasmatic membrane, where the transferrin receptor 1 (TFR1) recognizes the complex and allows the Fe3+ to enter by endocytosis. Inside the endosome, Fe3+ is reduced to Fe2+ by the metal reductase Six-Transmembrane Epithelial Antigen of Prostate 3 (STEAP3) [2+ can then be released in the cytoplasm by the Divalent Metal Transporter (DMT1) and enter the labile iron pool (LIP) [2+ in the LIP reaches high levels, it can be stored mainly in ferritin or as heme.Ferroptosis is an iron-dependent type of cell death; therefore, the intracellular levels of iron play a major role in its induction. The crucial mediators are a group of factors that regulate iron at different levels inside and outside the cell. In the blood, iron circulates in the Fe(STEAP3) . Fe2+ caol (LIP) . When th2+ from the ferritin storage is the nuclear receptor co-activator 4 (NCOA4) that is able to bind the heavy chain of the ferritin complex and promote the ferritin degradation mediated by the lysosomes (\u201cferritinophagy\u201d) [Ferritin is an iron storage protein complex formed by two subunits consisting of a ferritin light chain (FTL) and a ferritin heavy chain 1 (FTH1) ,23. The ophagy\u201d) ,27. Any ophagy\u201d) .The regulation of intracellular iron levels is controlled by iron-responsive element-binding proteins IRP1 and IRP2 : when th2+ when induced by heme oxygenase-1 (HO-1) [2+ in the cellular LIP can be one of the first events triggering the ferroptosis machinery, since ferrous iron can easily be used in the Fenton reaction to produce free radicals such as hydroxyl radicals [The other iron storage protein, heme, can release the Fe1 (HO-1) ,32. The radicals . In thisradicals .Two years after the first definition of ferroptosis, in 2014, Yang et al. identified GPX4 as a protein playing a crucial role in this mechanism , althougThe deprivation of cysteine in the cell causes GSH depletion, which can inhibit the GPX4 function, thus leading to the accumulation of lipid hydroperoxides and to ferroptosis due to membrane damage. On the other hand, GPX4-independent pathways were described such as the FSP1 (ferroptosis inhibitor protein 1)-CoQ antioxidant complex that functions only in GPX4-deprived cells. In the membrane, FSP1 reduces ubiquinone to ubiquinol to limit the increase in ROS in the plasma membrane ,40.The lipid metabolism is the third pathway that comes into play to induce ferroptosis through lipid peroxidation. The final products of this process, such as malondialdehyde (MDA), cause membrane instability through damage of the lipid bilayer, which increases the cell permeability and can lead to cell death .The major players of this pathway are the Polyunsaturated Fatty Acids (PUFA), because they are more prone to go into peroxidation especially during ferroptosis development.PUFAs can be synthesized starting from the acyl-CoA synthetase long-chain family member 4 (ACSL4) that binds PUFA to coenzyme A (CoA) to produce acyl-CoA. Afterward, thanks to the action of several acyltransferases, acyl-CoA can be re-esterified in phospholipids ,42. LinoThe complexity of the mechanisms that regulate tumorigenesis and tumor progression leads to the difficult eradication of cancer cells. Apoptosis is one of the principal targets for cancer treatments; however, in the last few years, many observations indicate ferroptosis as a promising pathway to be investigated for cancer therapy. As previously discussed, iron homeostasis, the Xc-system, and lipid metabolism all play a central role in promoting ferroptosis . TherefoCancer cells require an increased amount of iron for survival in comparison to normal cells ; thus, tDihydroartemisinin is the semi-synthetic derivative of Artemisinin, first used as an antimalarial drug . Chen etGlutathione synthesis is essential to maintain a reduced state in the cellular environment, avoiding lipid peroxidation and ferroptosis as the final effect. Since SLC7A11, GPX4, and GSH are the focal points of this pathway, an increasing number of compounds are being studied to target these proteins. These are ferroptosis inducers (FINs), a set of molecules categorized into four classes according to their different mechanisms of action .Class I FINs aim to block GSH synthesis by directly inhibiting SLC7A11 and consequently cysteine uptake . This grClass II FINs target GPX4 by covalently binding selenocysteine in its catalytic site ,42. RSL3Class III FINs are represented by statins. They are hydroxy methyl glutaryl-CoA inhibitors and cause a depletion of GPX4 and CoQ10, but the hypothetical liaison between cholesterol and ferroptosis is still unclear . A novelClass IV FINs, such as FINO2, are compounds in a preliminary experimental stage and primarily act by increasing the oxidizing iron and then inactivating GPX4. Both class III and IV FINs have not yet been tested in vivo . The in It should also be considered that standard therapies (radiotherapy and chemotherapy) could induce ferroptosis . For insLipid peroxidation can be caused by multiple mechanisms, including GSH depletion and GPX4 inhibition . It is eWhile being the crucial player in controlling gene expression at the mRNA level, microRNAs (miRNAs) were recently shown to modulate cancer growth and development via the ferroptosis pathway ,68.2+, and MDA levels. Inhibiting GPX4 causes the antioxidant system to be disrupted, making it impossible for the lipid peroxides to be cleared in a timely manner from the cells, which would trigger ferroptosis [2+, and ROS; and subsequently destroy MMP  by blocking GPX4 [Whether by controlling GPX4, FSP1, or GSH, microRNAs fundamentally influence tumor ferroptosis via the antioxidant system. GPX4 is the most significant antioxidant component in the ferroptosis-regulation network. For instance, microRNAs have GPX4 as a primary target in hematological malignancies . Additioroptosis . Researcing GPX4 . A systeing GPX4 . The oveing GPX4 . A studying GPX4 .Another crucial regulatory component of the ferroptosis-signaling cascade is SLC7A11. Recently, it was discovered that several miRNAs target SLC7A11 to control the ferroptosis process in tumor cells through antioxidant pathways and by targeting 3\u2032UTR . It is hFerroptosis is directly linked to long-chain acyl-CoA synthetase 4 (ACSL4). In hepatocellular carcinoma (HCC), miR-23a-3b is considered as a prominent miRNA, and ACSL4 is found to be a target gene in Sorafenib-resistant cells in HCC. MiR-23a-3p targets the 3\u2032-UTR of ACSL4 and inhibits ferroptosis . FerroptA study performed by Liao et al. showed miR-130b-3p to be having an inhibitory function in Erastin- and RSL3-induced ferroptosis, as demonstrated by a decrease in lipid peroxidation and ferrous ion content. miR-130b-3p triggered the Nrf2/HO-1 pathway by reducing the expression of its target gene, DKK1, hence regulating ferroptosis. The ability of Erastin to fight tumors was inhibited by miR-130b-3p. An in vivo mice model was used to replicate in vitro findings; it was found that miR-130b-3p inhibition decreased the tumor volume, while miR-130b-3p ectopic expression increased the tumor volume in those mice models. In mice G-361 cells, Erastin\u2019s anticancer activity was consistently reduced by miR-130b-3p. These findings point to miR-130b-3p\u2019s capacity to suppress ferroptosis in melanoma cells, through the Nrf2/HO-1 pathway ,85.Ferroptosis-related gene SLC1A5 is a novel prognostic marker linked to ferroptosis cell death, although it was also studied as a negative regulator of ferroptosis in some cases. The overexpression of miR-137 prevents ferroptosis, suppresses iron build-up, and reduces lipid peroxidation by inhibiting the SLC1A5 glutamine transporter expression. However, the overexpression of SLC1A5 counteracts miR-137\u2019s protective action against ferroptosis. By focusing on the control of the glutamine transporter SLC1A5 as a novel therapeutic target for melanoma, miR-137 can inhibit ferroptosis in melanoma cells . AnotherLipoxygenases (LOXs) are a family of enzymes that produce oxylipin from polyunsaturated fatty acids. The LOXs consist of six isoforms including ALOXE3. In glioblastoma cells (GBM), ALOXE3 was identified as a tumor promoter. miR-18 was observed as a ferroptosis suppressor, which regulates ALOXE3 through the YAP signaling pathway. MiR-18 downregulates ALOXE3 and increases glioblastoma development and migration activity ,89.2+, and MDA [2+, and MDA in the CRC cells, thus stimulating CRC cell death and suppressing ferroptosis [The GLS2 gene was studied as both a positive and negative regulator of ferroptosis in different cases. In one study, miR-190a-5p was seen as a negative regulator of ferroptosis, which directly targets the GLS2 gene. In cardiomyocytes, the overexpression of miR-190a-5p was studied as an inhibiting factor for GLS2, which eventually downregulates ROS, Feroptosis .CircKIF4Ab was found to be an enhancer of the GPX4 being targeted by miR-1231 . ContrarMoreover, miR-387a-3p and miR-224-5p ,98 enhanOverall, ferroptosis can be considered a novel approach for the treatment of cancer cells, in particular for apoptotic-resistant cells. The understanding of the role of miRNA in the modulation of this pathway could be crucial in identifying potential therapeutic interventions and enhancing treatment efficacy .Functional enrichment analysis of miRNA through the Kyoto Encyclopedia of Genes and Genomes (KEEG) highlighted the role of the target genes in ferroptosis, which is an important pathway in cancer development, followed by iron metabolism, lipid peroxidation, and antioxidant defense. According to the KEGG pathway analysis results, a significant portion of the common pathways were associated with ferroptosis pathways. The antioxidant defense pathway (as shown in red in In addition to the miRNAs described above, the exploiting of ferroptosis-linked miRNAs as a therapeutic opportunity is worth considering. Although a significant number of miRNAs with a pro- or anti-ferroptosis activity were described , only a Ketamine, an intravenous anaesthetic that exhibits anti-inflammatory activity and analgesic and antidepressant effects suppresses the viability and proliferation of liver cancer cells both in vitro and in vivo. It was shown to downregulate GPX4 expression and upregulate miR-214-3p levels through the lncPVT1/miR-214-3p/GPX4 regulatory axis, thus promoting ferroptosis in liver cancer cells . PropofoThe mechanisms just described may offer new ways to hit tumors. However, some limitations must be considered such as the low specificity on the target. Trying to overcome this problem, different groups combined miRNAs with nanoparticles, which allows the molecules to be conveyed in the desired sites . Guo et The modulation of expression of miRNA is closely related to the degree of malignancy, drug resistance, and prognosis of tumors. Indeed, the expression of miRNAs can inhibit tumor cells through a variety of mechanisms, including inhibiting the cell cycle , inhibit"}
+{"text": "Ferroptosis is a new form of programmed cell death, which is characterized by the iron-dependent accumulation of lipid peroxidation and increase of ROS, resulting in oxidative stress and cell death. Iron, lipid, and multiple signaling pathways precisely control the occurrence and implementation of ferroptosis. The pathways mainly include Nrf2/HO-1 signaling pathway, p62/Keap1/Nrf2 signaling pathway. Activating p62/Keap1/Nrf2 signaling pathway inhibits ferroptosis. Nrf2/HO-1 signaling pathway promotes ferroptosis. Furthermore, some factors also participate in the occurrence of ferroptosis under hypoxia, such as HIF-1, NCOA4, DMT1. Meanwhile, ferroptosis is related with hypoxia-related diseases, such as MIRI, cancers, and AKI. Accordingly, ferroptosis appears to be a therapeutic target for hypoxia-related diseases. Ferroptosis is a newly discovered form of iron-dependent programmed cell death that is induced by the accumulation of iron-mediated lipid peroxidation  in serum and then combine with transferrin receptor (TfR) in the cell membrane. Subsequently, the TF-Fe/TfR complex is endocytosed into the cell. With the help of six-transmembrane epithelial antigen of prostate 3 (STEAP3) in the endosome, Fe3+ is reduced to Fe2+ and then Fe2+ is released into cytoplasm by divalent metal transporter 1 (DMT1)   family and is a cystine/glutamate antiporter system  and biliverdin  consist of HIF-1, HIF-2 and HIF-3 , ELAV-like protein 1 (ELAVL1) and carbonic anhydrase 9 (CA9) to effect ferroptosis. Ferritin, composed with FTL and FTH1, stores intracellular Fe2Furthermore, it is reported that hypoxia increases CA9 which blocks ferritin-mediated iron storage and increases lipid peroxidation by inhibiting to reduce oxidative stress and then ferroptosis , Parkinsonian disorders (PD), Amyotrophic lateral sclerosis (ALS), and so on. Studies suggest that hypoxia causes the reduction of ATP synthesis and the generation of ROS damage cells, and then leads to the dysfunction of mitochondrial and the disorder of oxidative phosphorylation, resulting in ferroptosis in neurodegenerative disorders  inactivation in cancer cells, which increases the uptake of cystine and the synthesis of GSH to develop the growth of cancer by inhibiting ferroptosis  have strong self-renewal, diffusion and metastasis, and resistance to various forms of anticancer therapy, which can easily lead to tumor recurrence Katoh . It is rHypoxia may promote cancer growth by inhibiting ferroptosis, which causes difficulties in cancer treatment. The effect of radiation on ferroptosis is mainly reflected in radiotherapy. Radiotherapy is one of the important treatments for malignant tumors, and the induction of ferroptosis is also one of the important factors. Radiotherapy suppresses SLC7A11 via activating ATM and increases lipid oxidative to cause the ferroptosis of tumor cells  is a new type of explosive disease characterized by pneumonia and acute respiratory distress syndrome (ARDS) which is accompanied with hypoxia and leads to multiple organ failure (Beckman et al. Intestinal I/R injury is another hypoxia-related disease. It is reported that hypoxia induced by Intestinal I/R injury decrease GPX4 activity and GSH levels, and then leads to ferroptosis (Deng et al. Ferroptosis is caused by iron-dependent accumulation of lipid peroxidation and the increase of ROS. The related pathways between ferroptosis under hypoxia mainly include Nrf2/HO-1 signaling pathway, p62/Keap1/Nrf2 signaling pathway. The Nrf2/HO-1 signaling pathway is currently a research hotspot in hypoxia-related diseases. However, the relationship between the p62/Keap1/Nrf2 signaling pathway and ferroptosis in hypoxia-related diseases still needs further research, and is a potential direction for future research. However, more researches on the signal pathways of ferroptosis under hypoxia are needed. Meanwhile, some factors also participate in the occurrence of ferroptosis under hypoxia, such as HIF-1, NCOA4, DMT1. Hypoxia-induced activation of HIF-1 has been shown to be closely related to ferroptosis. Because HIF-1 plays a role in ferroptosis in a context-dependent manner under hypoxia, the connection between HIF-1 and ferroptosis under hypoxia needs to be sorted out. Meanwhile, ferroptosis is related with hypoxia-related diseases, such as MIRI, cancers, and AKI. The research progress in ferroptosis and MIRI, IS, AKI, and cancers is rich, but research progress in ferroptosis and intestinal I/R injury still needs further study. In addition, hypoxic environment may inhibit the occurrence of ferroptosis and promote cancers growth, resulting in the unexpected effect of radiotherapy. It is needed that find a way to use ferroptosis to maximize the effect of radiation therapy. Inducing ferroptosis in ferroptosis-prone CSCs by controlling iron accumulation may be an excellent targeted therapy. Accordingly, ferroptosis appears to be a therapeutic target for hypoxia-related diseases."}
+{"text": "Ferroptosis is an iron-dependent form of regulated cell death with distinct characteristics, including altered iron homeostasis, reduced defense against oxidative stress, and abnormal lipid peroxidation. Recent studies have provided compelling evidence supporting the notion that ferroptosis plays a key pathogenic role in many diseases such as various cancer types, neurodegenerative disease, diseases involving tissue and/or organ injury, and inflammatory and infectious diseases. Although the precise regulatory networks that underlie ferroptosis are largely unknown, particularly with respect to the initiation and progression of various diseases, ferroptosis is recognized as a bona fide target for the further development of treatment and prevention strategies. Over the past decade, considerable progress has been made in developing pharmacological agonists and antagonists for the treatment of these ferroptosis-related conditions. Here, we provide a detailed overview of our current knowledge regarding ferroptosis, its pathological roles, and its regulation during disease progression. Focusing on the use of chemical tools that target ferroptosis in preclinical studies, we also summarize recent advances in targeting ferroptosis across the growing spectrum of ferroptosis-associated pathogenic conditions. Finally, we discuss new challenges and opportunities for targeting ferroptosis as a potential strategy for treating ferroptosis-related diseases. In the decade since ferroptosis was first reported, increasing evidence has emerged suggesting that ferroptosis may play a pivotal role in many biological processes such as tumor suppression and immunity, indicating that ferroptosis is important for maintaining health by regulating metabolism and redox homeostasis. Recently, a plethora of studies have shown that ferroptosis also plays a critical role in a variety of pathophysiological processes such as ischemic organ injury, stroke, cardiac myopathy, and neurodegenerative diseases; ferroptosis has also been implicated in many oncogenic pathways, suggesting it might serve as a target for novel cancer therapeutics.2Ferroptosis is a unique form of iron-dependent regulated cell death originally identified by screening RSL  compounds.7In mammalian cells, ferroptosis is regulated mainly by iron homeostasis, lipid metabolism, and glutathione-dependent redox balance. Thanks to rapid progress in the study of ferroptosis, numerous efforts have made to identify potent, druggable ferroptosis modulators for use in clinical applications, opening new avenues for developing novel treatment strategies to target many ferroptosis-related diseases, including cancer and heart injury. With respect to cancer, novel ferroptosis agonists have shown promise in several cancer types. On the other hand, antagonists of ferroptosis have been shown to help alleviate ferroptosis-related diseases such as ischemia/reperfusion (I/R)-induced damage, neurodegenerative diseases, and inflammatory diseases.Here, we provide a comprehensive update of recent efforts to target ferroptosis for treating a variety of relevant diseases, and we discuss the safety and efficacy of ferroptosis agonists and antagonists, as well as their pharmacokinetics profiles, their experimental stage in the drug discovery process, and opportunities for further clinical development. We also review the biological roles, molecular mechanisms, and clinical implications of ferroptosis, and we discuss insights into ferroptosis-focused translational research, as well as current knowledge gaps and opportunities. Finally, we discuss future research orientations that will lead to the clinical implementation of these novel ferroptosis modulators for treating a diverse range of diseases.8 this neuronal cell death was induced by the excitotoxin glutamate by inhibiting SLC7A11 (solute carrier family 7 member 11), a component of the cystine-glutamate antiporter system Xc\u2212 known to play a role in ferroptosis. Therefore, oxytosis and ferroptosis have been suggested to share several key characteristics, including their gene expression patterns, high activity of lipoxygenases, and high accumulation of reactive oxygen species (ROS).9 By 2003, a distinct form of erastin-induced cell death in RAS-expressing cancer cells was receiving considerable attention;10 this form of cell death was not inhibited by caspase inhibitors but could be suppressed by treating the cells with iron-chelating agents.11 Yang et al. subsequently found that RAS-selective lethal small molecule 3 (RSL3) could also trigger this iron-dependent form of cell death.12 Based on these characteristics, Dixon et al. named this type of cell death ferroptosis.1 Morphologically, cells undergoing ferroptosis generally have shrunken mitochondria with increased membrane density and reduced\u2014or absent\u2014mitochondrial cristae. Biochemically, ferroptosis is characterized by increased oxidative stress and depleted antioxidative defense. Although ferroptosis is generally well-accepted as a tightly regulated cellular metabolic process, its precise mechanisms such as how excessive phospholipid (PL) peroxidation drives ferroptosis and the tissue- and disease-specific epilipidome signatures remain unknown.Although the term \u201cferroptosis\u201d was first coined by Dixon et al. in 2012 Fig. , a simil13 Soon after, other groups identified ACSL4 (acyl-CoA synthetase long-chain family member 4) as a predominant mediator of ferroptosis.15 In addition, selenium was shown to protect against ferroptosis.16 In 2019, two groups independently reported that the FSP1 (ferroptosis suppressor protein 1)-CoQ10 (coenzyme Q10)-NAD(P)H pathway inhibits ferroptosis via a GPX4-independent mechanism.18 In addition, the GCH1 (GTP cyclohydrolase 1)-BH4 (tetrahydrobiopterin) pathway was reported as an additional GPX4-independent modulator of ferroptosis.19 Recently, Mao et al. identified the DHODH (dihydroorotate dehydrogenase)-CoQ10 axis, which is located at the mitochondrial inner membrane, as an important pathway that protects against ferroptosis.20 Even more recently, Mishima et al. reported that vitamin K is a potent FSP1-dependent inhibitor of ferroptosis by functionally screening vitamin compounds in Gpx4-deficient mouse embryonic fibroblasts.21The mechanisms that regulate ferroptosis have begun to emerge Fig. . For exaA growing body of evidence indicates that ferroptosis is regulated by a complex network involving iron homeostasis, lipid metabolism, and the oxidative-reductive system Fig. . For exa1 In contrast, iron overload has been shown to sensitize numerous cells types to ferroptosis.24 Therefore, maintaining iron homeostasis is critical in order to protect cells from ferroptosis. The intracellular labile iron pool (LIP) is regulated by the uptake, export, storage, and utilization of iron -mediated non-TF-bound iron (NTBI).23 Excess intracellular iron is sequestered largely by the principal iron-storage protein ferritin (FTH), which protects cells from ferroptosis. Consistent with this protective role, we found that mice lacking Fth in cardiomyocytes develop ferroptosis-induced cardiomyopathy.24 Notably, ferritinophagy\u2014an NCOA4 (nuclear receptor coactivator 4)-mediated autophagic degradation of ferritin in the lysosome\u2014has been shown to induce ferroptosis by releasing free iron from ferritin.27 Moreover, the iron exporter ferroportin (FPN) has also been shown to regulate cellular sensitivity to ferroptosis in vitro.28Because iron chelators such as deferoxamine (DFO) can block ferroptosis, ferroptosis was originally defined as iron-dependent.ron Fig. . Cellula15 ACSL4 catalyzes the conversion of free PUFAs to acyl-CoA derivatives (PUFA-CoAs), which can be further catalyzed by lysophosphatidylcholine acyltransferase 3 (LPCAT3) to produce PL-PUFAs. PL-PUFAs participate in the biosynthesis of cellular membranes and are highly susceptible to peroxidation due to their bis-allylic hydrogen atoms. Interestingly, loss of either ACSL4 or LPCAT3 increases cellular insusceptibility to ferroptosis by reducing the production of substrates for PL peroxidation.30 In addition, inhibiting lipoxygenases (LOXs), which are iron-containing enzymes, suppresses erastin-induced ferroptosis via iron- and oxygen-triggered free radical chain reactions, independent of their enzymatic activity.31The metabolism of lipids\u2014particularly PL-PUFAs\u2014critically regulates ferroptosis Fig. . Using a\u2013-GSH-GPX4 axis serves as a GPX4-dependent mechanism for scavenging PL peroxides via system Xc\u2013-mediated GSH synthesis.13 Moreover, inhibiting either component of system Xc\u2013  induces ferroptosis by disrupting cystine uptake, thereby limiting GSH synthesis.1 In this pathway, GPX4 serves as an essential regulator of ferroptosis.13 Second, in 2019 an FSP1-dependent metabolic pathway was shown to protect against ferroptosis via an GPX4-independent process.18 Specifically, two groups simultaneously reported that FSP1 traps lipid peroxyl radicals to suppress ferroptosis by reducing CoQ10 levels at the plasma membrane,18 a process that is independent of the system Xc\u2013-GSH-GPX4 pathway. Third, Mao et al. showed that mitochondrial inner membrane-located DHODH can protect against ferroptosis by reducing CoQ to form ubiquinol, an antioxidant that inhibits ferroptosis; thus, inhibiting DHODH in cancer cells potently activates ferroptosis in parallel with mitochondrial GPX4, independent of cytosolic GPX4 and FSP1.20 Finally, Liang et al. recently identified the PL-modifying enzymes MBOAT1 and MBOAT2 as novel sex hormone\u2012dependent ferroptosis inhibitors.32 Mechanistically, the authors showed that MBOAT1/2 suppress ferroptosis by remodeling cellular PLs to protect cells from ferroptosis independent of GPX4, providing novel ferroptosis-targeted therapeutic strategies to sensitize either estrogen receptor (ER) antagonists in ER-positive breast cancer and androgen receptor (AR) antagonists in AR-positive prostate cancer, respectively.With respect to oxidative and reductive reactions, several major pathways are involved in protecting against ferroptosis Fig. . First, Intracellular accumulation of iron and PL peroxides are two central biochemical events that occur during ferroptosis, and these processes have also been implicated in a wide range of diseases Tables  and 2. I33 Therefore, identifying novel cancer therapeutics that target pathways other than apoptosis is now an urgent unmet clinical need. Indeed, the notion of ferroptosis originated from lethal screening of compounds in RAS-mutated cancer cells.1 In support of this notion, ferroptosis has been functionally validated in cancer types with increased PL peroxidation, including hepatocellular carcinoma (HCC),34 pancreatic ductal adenocarcinoma,35 triple-negative breast cancer (TNBC),37 and renal cell carcinoma.39 Thus, these specific cancer types may be more sensitive to ferroptosis-inducing compounds.Apoptosis was long considered the principal form of cell death in various cancer types; however, apoptosis-based cancer therapies have limited clinical benefit.40 In addition, priming cancer cells with ferroptosis-inducing compounds can sensitize the cancer cells to subsequent immunotherapies.41 Taken together, these findings highlight the promise of targeting ferroptosis as a novel strategy for treating various forms of cancer.It is important to note that ferroptosis has also been closely linked to resistance to various cancer treatments. For example, several studies found that cancer cells with a high-mesenchymal state are more resistant to a variety of cancer treatments, but are particularly susceptible to ferroptosis-inducing compounds.42 Parkinson\u2019s disease (PD),43 and Huntington\u2019s disease (HD).44 In addition, ablating or inactivating GPX4 promotes neuronal damage and neurodegeneration.7 In experimental models of degenerative brain conditions, both ferroptosis inhibitors and iron chelators were shown to improve outcome and prognosis.48Iron deposition and lipid peroxidation are common pathophysiological features in various neurological diseases. Notably, the disease group known as neurodegeneration with brain iron accumulation (NBIA) refers to neurodegenerative disorders associated with altered iron metabolism in the lesion area and includes Alzheimer\u2019s disease (AD),42 Iron deposition in the brain, accompanied by a decrease in endogenous antioxidant capacity, has been associated with the pathogenesis of AD, and iron levels in the brain have been correlated with disease progression.49 Furthermore, accumulated iron interacts with the A\u03b2 peptide and tau protein via the formation of a peptide-hemin complex, possibly implicating ferroptosis.50With respect to AD, several studies have shown that dysregulated iron metabolism is linked to ROS production, mitochondrial dysfunction, and neurodegeneration.51 In the substantia nigra pars compacta of affected patients, iron was shown to accumulate in glia cells and dopaminergic neurons, with the level of iron correlated with disease severity.52 Moreover, reports suggest that the increased oxidative stress present in PD may be attributed to decreased GSH levels in the substantia nigra.53 In addition, the brain consumes high amounts of oxygen, making this organ more sensitive to lipid peroxidation.54PD is characterized by reduced dopaminergic neurons selectively in the substantia nigra and the presence of Lewy bodies.HTT) gene,55 has also been linked to ferroptosis in animal models.56 Excess iron remains a major cause of oxidative stress in neurons, which directly induces ferroptosis during the pathogenesis of HD.57 Interestingly, similar to PD, reduced levels of GSH have also been observed in HD.58 Together, these findings suggest that ferroptosis may be involved in the pathogenesis of HD.HD, a hereditary neurodegenerative disease due to an abnormally high number of CAG repeats in the huntingtin  refers to sudden-onset kidney failure and/or kidney damage characterized by a rapid loss of the kidney\u2019s excretory function caused by massive levels of cell death and inflammation.3 In addition, inhibiting glutaminolysis protected against I/R-induced ferroptotic heart damage in vitro, indicating that glutaminolysis plays an essential role in regulating ferroptosis during heart injury.25 Recently, Jacobs et al. suggested the possible presence of ferroptosis in the myocardium of a patient with COVID-19\u2012induced myocarditis.66 Using the antibody E06 to stain oxidized phosphatidylcholine, they found that a ferroptosis \u201csignature\u201d may be specific to injured cardiomyocytes in COVID-19\u2012induced myocarditis, as this signature was not present in either myocarditis of unknown etiology or in COVID-19 patients who presented without myocarditis.66 Recently, we summarized the role of ferroptosis in regulating cardiovascular disease, and we refer to the reader to this review.67In the heart, we first identified ferroptosis as the major pathogenic mechanism in both doxorubicin- and I/R-induced cardiomyopathy, and we showed that targeting ferroptosis significantly alleviated heart injury in mouse models.69 A subsequent study also showed that inhibiting ferroptosis prevented the death of primary oligodendrocytes.70 Notably, organotypic hippocampal slice cultures with ferroptosis-specific inhibitors were shown to prevent neuronal death and decrease hemoglobin-induced iron accumulation, suggesting a pathogenic role of ferroptosis in intracerebral hemorrhage.71 In addition, neurons obtained from an ischemic stroke mouse model were shown to have significantly decreased levels of GSH72 and increased lipid peroxidation,73 suggesting neuronal ferroptosis.74 In addition, carvacrol  has been shown to help protect hippocampal neurons from ferroptosis by upregulating Gpx4 expression in a gerbil model of cerebral ischemia.75 In spinal cord injury, recent studies suggest that ferroptosis contributes to secondary injury, and blocking ferroptosis may help repair traumatic spinal cord injury.76Previous studies using animal models of brain injury showed features resembling ferroptosis, including increased lipid peroxidation, increased intracellular iron levels, and decreased GSH levels.Slc7a11 expression77 or increasing non-TF-bound iron (NTBI).23 Ferroptosis was recently linked to various chemical-induced forms of liver injury such as alcoholic liver disease,78 methionine/choline-deficient\u2012induced non-alcoholic steatohepatitis (NASH),79 and arsenic-induced NASH.80 In addition, both ferroptosis and a concomitant accumulation of lipid ROS were also observed in CCl4-induced liver fibrosis,23 acetaminophen-induced liver damage,81 and I/R-induced liver injury.82The liver serves as a central metabolic organ and is highly susceptible to various toxic metabolites. We previously showed that high dietary iron directly leads to ferroptosis-induced liver injury, and this damage was aggravated by either knocking out 83 In addition, a ferroptosis signature was shown in tissue samples obtained from a patient with COVID-19\u2012induced MODS .66 Using an experimental model of MODS, Van Coillie et al. showed that excess iron can trigger ferroptosis in multiple organs; moreover, they showed that the novel ferroptosis inhibitor UAMC-3203 may be a viable therapeutic agent for the clinical treatment of MODS.83Critically ill patients with multiorgan dysfunction syndrome (MODS) were shown to present with a ferroptosis signature that includes increased plasma levels of MDA  and catalytic iron.84 Moreover, the functions of various immune cell types such as T cells, B cells, and macrophages can be affected by ferroptosis. For example, Gpx4-deficient CD4+ and CD8+ T cells undergo ferroptosis due to accumulated lipid peroxides.85 Thus, these ferroptotic T cells are unable to proliferate and have reduced function against the acute lymphoblastic choriomeningitis virus and Leishmania major parasites, suggesting that Gpx4 is required for maintaining T cell\u2012mediated immunity by protecting the cells against ferroptosis.85 With respect to B cells, various B cell populations have been shown to respond differently to ferroptosis induction.86 Similarly, in bone marrow cells inducible nitric oxide synthase (iNOS) is more abundant in M1 macrophages than in M2 macrophages, and M1 macrophages tend to be more resistant to iron deposits and ferroptosis.87 Interestingly, a recent study found that Gpx4-dependent ferroptosis in neutrophils drives the pathogenesis of systemic lupus erythematosus (SLE),88 suggesting that targeting ferroptosis-induced neutropenia may be a potential strategy for treating autoimmune diseases such as SLE.Ferroptosis has also been reported to directly modulate the host\u2019s immune response and inflammation during inflammatory and infectious diseases.Pseudomonas aeruginosa can infect both immunocompetent and immunocompromised hosts. Interestingly, this pathogen expresses lipoxygenase (which oxidizes AA-PE to 15-hydroperoxide AA-PE in host cells) and drives ferroptosis in human bronchial epithelial cells, as well as in clinically isolated cells from patients with persistent lower respiratory tract infection.89 Thus, targeting ferroptosis may be a viable strategy for managing P. aeruginosa infection.The aerobic gram-negative bacterium Mycobacterium tuberculosis is one of the major pathogens that causes tuberculosis, an infectious disease that affects the lungs, bone tissue, brain, and spine. Interestingly, M. tuberculosis has been shown to trigger ferroptosis in macrophages, accompanied by decreased GPX4 expression, increased iron content, and elevated levels of membrane lipid peroxidation.90 Similar results were also obtained in mice acutely infected with M. tuberculosis.90 Given these findings, ferroptosis may also be a promising therapeutic target in patients with tuberculosis and other infectious diseases.The bacterium 91 In addition, blocking ferroptosis was shown to significantly reduce atherosclerosis in ApoE\u2212/\u2212 mice fed a high-fat diet.92Notably, ferroptosis has also been closely linked to sterile inflammation, a process that occurs in the absence of pathogens and is commonly triggered by release of the intracellular contents from damaged and/or necrotic cells. Sterile inflammation is often triggered by acute conditions such as I/R-induced injury, trauma, oxalate crystal\u2012induced inflammation, and chronic inflammatory diseases such as atherosclerosis. For instance, ferroptosis increases the recruitment of neutrophils to damaged heart tissue under ischemic conditions, and ferroptosis-specific inhibitors can alleviate this inflammatory damage.Given that iron metabolism, oxidative-reductive pathways, and lipid metabolism coordinately control ferroptosis, altering these three pathways using genetic and pharmacological approaches has been shown to affect ferroptosis Table . Below, 3+, while Fe2+ serves as an electron donor to catalyze the Fenton reaction. During iron overload conditions, excess iron is involved in the generation of free radicals, lipid peroxidation, and DNA damage, which in turn promotes ferroptosis. At the cellular level, transferrin (TF) mediates the cellular uptake of Fe3+ via endocytosis of iron-loaded TF-bound transferrin receptor 1 (TFR1), followed by release from the endosome,94 in which Fe3+ is then converted to Fe2+ via the metalloreductase STEAP3.95 When the binding capacity of TF-2Fe3+ is saturated, ferrireductases reduce Fe3+ to Fe2+, which can be transported to the LIP via NTBI transporters such as DMT1 96 and SLC39A14.97 The LIP can also increase due to the degradation of either heme or hemoglobin, which are captured by hemopexin and haptoglobin, respectively. Upon binding to CD163 (a monocyte/macrophage-specific scavenger receptor)98 and LRP1 (LDL receptor related protein 1),99 heme-hemopexin and hemoglobin-haptoglobin complexes are internalized via endosomes, after which the rate-limiting enzyme heme oxygenase-1 (HO-1) catabolizes heme to produce biliverdin, releasing iron into the LIP. Iron is reserved primarily in ferritin, a complex consisting of 24 light chain (FTL) and heavy chain (FTH1) subunits.100 FTH also has ferroxidase activity, converting Fe2+ to Fe3+ to prevent iron-induced toxicity. Excess iron is exported by FPN, the body\u2019s sole iron exporter,101 which is regulated primarily by the hepatic peptide hepcidin,102 the master regulator of iron homeostasis. Thus, together with our recent finding that the newly identified E3 ubiquitin ligase RNF217 mediates the degradation of FPN,103 these results indicate that systemic iron homeostasis is tightly controlled by the hepcidin-FPN axis, and targeting dysregulated iron metabolism may directly affect ferroptosis  and can transfer iron in DFP-Fe to transferrin.114 DFP has been shown to improve motor function by decreasing iron content in the brain in patients with Friedreich\u2019s ataxia.115The term \u201cferroptosis\u201d originated from the rescue effect of the iron chelator DFO. In the clinic, DFO, deferiprone (DFP), and deferasirox (DFX) are commonly used iron chelators.3+ and a relatively long biological half-life.116 Studies have shown that DFX treatment can prevent the accumulation of hemosiderin  and can ameliorate ferroptosis-related kidney and neuronal damage.118Similar to DFP, DFX is a highly selective iron chelator showing a high affinity for Fe119 Compared to DXZ, the novel orally administered iron chelator CN128 is more potent and has fewer side effects and is currently being studied in a phase II clinical trial for treating \u03b2-thalassemia following regular blood transfusion.120 Finally, ciclopirox olamine, which is currently approved for treating cutaneous fungal infections, has also been used as an iron chelator in various model systems due to its inhibitory activity on iron-dependent ribonucleotide reductase.122Currently, dexrazoxane (DXZ) is the only iron chelator approved by the FDA for protecting against doxorubicin (DOX)-induced cardiotoxicity by chelating DOX-induced mitochondrial iron.25 Moreover, Wu et al. showed that knocking down TFR1 inhibits ferroptosis under cystine starvation conditions, whereas upregulating TFR1 significantly activates ferroptosis via the NF2-YAP signaling pathway.123 Using the TFR1-specific antibody 3F3-FMA, Feng and colleagues showed that TFR1 can serve as a marker for cells undergoing ferroptosis.124 Given that various cancer cells express relatively high levels of TFR1, antibodies that either neutralize of block TFR1 have been investigated as potential cancer therapies. For example, Horonchik and Wessling-Resnick found that the small-molecule TFR1 inhibitor ferristatin  can inhibit iron uptake by mediating the degradation of TFR1(ref. 125). Based on these results, it is reasonable to speculate that TFR1 antibodies may be suitable for treating ferroptosis-induced diseases, warranting further study.The TF receptor TFR1 is a glycoprotein that interacts with iron-bound TF in order to mediate cellular iron uptake. Gao et al. reported that TFR1-mediated cellular uptake of TF-bound iron is required for ferroptosis, showing that inhibiting TFR1 using RNA interference (RNAi) can efficiently block ferroptosis.2+ into the duodenum and out of the endosome during the TF cycle, but it can also transport NTBI.96 DMT1 inhibitors such as ebselen,126 pyrrolidine dithiocarbamate (PDTC),126 and benzylisothiourea127 have been shown to reduce iron-induced damage by potently reducing the DMT1-mediated cellular uptake of NTBI, indicating that DMT1 may be a promising target for regulating ferroptosis to manage ferroptosis-related diseases.DMT1  is known for its ability to transport Fe128 and FPN inhibitors such as VIT-2763 (ref. 128). Similarly, small molecules that directly upregulate hepcidin expression may affect ferroptosis by promoting intracellular iron accumulation. In addition, endogenous inducers of hepcidin such as the cytokine oncostatin M (encoded by the OSM gene)129 may promote ferroptosis. Conversely, clinical trials involving hepcidin antagonists such as PRS-080,128 NOX-H94,130 and LY2787106131 may suppress ferroptosis by reducing intracellular iron content. Thus, further studies are needed in order to ascertain the functional role of these FPN/hepcidin regulators in modulating ferroptosis under pathological conditions.Two strategies have been suggested to modulate ferroptosis by targeting iron export, namely hepcidin agonists such as minihepcidins132 Ferritinophagy has been shown to initiate ferroptosis via iron overload and lipid peroxidation.27 JQ1, a thienotriazolodiazepine that inhibits bromodomain proteins such as BRD4, has been reported to potentiate ferroptosis via ferritinophagy in breast cancer cells.133 Additionally, DpdtC  mobilizes iron by inducing ferritinophagy and can increase ferritinophagy-induced ROS production in MGC-803 cells, a human gastric carcinoma cell line.134 MMRi62, a small molecule compound initially identified as an inducer of apoptosis, is shown to potently trigger ferroptotic cell death in pancreatic ductal adenocarcinoma cells by increasing lysosomal ferritinophagy.135 On the other hand, Fang et al. recently found that a novel compound called 9a potently suppresses ferritinophagy-induced ferroptosis by competitively binding to NCOA4 and perturbing the interaction between NCOA4 and FTH1.136Under iron-deficient conditions, NCOA4 binds to iron-loaded ferritin, thus promoting lysosomal ferritin degradation and releasing iron into the LIP, a process known as ferritinophagy.137 Macrophage-mediated iron recycling is processed primarily in the spleen, and recycling iron is returned to the storage pool for utilization in various processes such as heme biosynthesis and Fe/S cluster formations in the mitochondria. The enzyme HO-1, encoded by the HMOX1 gene, catalyzes heme to produce carbon monoxide, biliverdin, and free iron; biliverdin and free iron can be used to generate bilirubin and sequestered by ferritin, respectively. Upregulation of HMOX1 expression has been shown to play a cytoprotective role138 and to increase the resistance of HCC cells to ferroptosis mediated by the p62-KEAP1 (Kelch-like ECH-associated protein 1)-NRF2 (nuclear factor erythroid-2-related factor 2) pathway.34 On the other hand, high HMOX1 expression can also be toxic due to high levels of ferrous iron,139 which in turn accelerates the Fenton reaction, particularly in the context of insufficient levels of free radical scavengers. Together, these reports support the notion that HMOX1 expression has a dose-dependent differential role.140 Targeting HO-1 has been proposed as a viable strategy for treating many diseases and conditions, including cardiovascular disease3 and inflammation.141 Notably, we previously showed that inhibiting HO-1 prevents ferroptosis-induced cardiomyopathy in mice.3 To date, many HO-1 agonists and antagonists have been proposed for use in various disease models. For example, Vreman et al. found that metalloporphyrins\u2014synthetic heme analogs used to treat jaundice in newborn infants\u2014can inhibit HO-1.142 However, several metalloporphyrins can induce phototoxicity and/or off-target adverse effects; given this poor safety profile, azalanstat was subsequently developed as a safer HO-1 inhibitor;143 however, studies are needed to test its efficacy in ferroptosis-related diseases.Macrophages in the spleen and Kupffer cells  in the liver maintain iron homeostasis by recycling iron obtained from senescent erythrocytes and damaged cells.\u2013-GSH-GPX4 metabolic pathway plays a key role in regulating ferroptosis. Intracellular cysteine is taken up primarily in the form of cystine via system Xc\u2013144 or converted from methionine via the transsulfuration pathway,145 or is transported directly by alanine/serine/cysteine transporters (known as system ASC).147 In addition, the KEAP1/NRF2 antioxidative signaling pathway, the glutaminolysis pathway, the FSP1-CoQ10-NAD(P)H pathway, and the recently identified DHODH-mediated pathway have all been recognized as central mediators of ferroptosis by directly regulating reductive-oxidative pathways. Below, we summarize these ferroptosis modulators that target the reductive-oxidative pathways  was developed and shown to have high (nanomolar) potency and high metabolic stability.152 The tumor-suppressing efficacy of IKE was then demonstrated in SUDHL6 (diffuse large B cell lymphoma) cell xenografts in mice.153 Capitalizing on the fact that IKE is soluble under acidic aqueous conditions, nanoparticles were used as an IKE delivery system to further improve its therapeutic index, with reduced toxicity compared to free IKE.153 Previous studies found that erastin was able to increase the sensitivity of various cancer cell lines to chemotherapy drugs, including doxorubicin, actinomycin D, cisplatin, temozolomide, and cytarabine,157 providing new motivation for exploring the feasibility of combination therapies. Recently, we solved the high-resolution structure of erastin-bound human system Xc\u2013 and showed that IKE is a highly potent inducer of ferroptosis, providing a structural basis for designing more effective ferroptosis modulators.158Erastin is a first-generation ferroptosis inducer that directly suppresses system Xc\u2013(ref. 159). Moreover, a lot of studies have shown that SAS has anti-tumor activity in various xenograft tumor models, including glioblastoma,160 prostate cancer,161 small cell lung cancer,162 pancreatic cancer,163 and TNBC.164 Based on its excellent safety profile in animal studies, several phase I and phase II clinical studies have been initiated. However, various doses of SAS failed to produce a clinical response in malignant glioma, and side effects were reported, including anorexia, gastrointestinal toxicity, and hematological toxicity.165 In addition, long-term high-dose (8\u201312\u2009g/day) treatment with SAS can induce several adverse side effects and should be avoided; therefore, combination therapy using SAS together with conventional radiotherapy and/or chemotherapy has been proposed as a promising therapeutic strategy.173 To mitigate its toxicity, studies involving SAS focus primarily on sensitizing agents and applications using nanoparticles, a novel strategy designed to increase the effectiveness of low-dose SAS.174The anti-rheumatic drug sulfasalazine (SAS) is clinically approved for treating inflammatory arthritis and inflammatory bowel disease. SAS has been reported to suppress the growth of lymphoma cells both in vitro and in vivo through the activation of ferroptosis by suppressing system Xc\u2013.176 However, the relatively limited clinical benefits of SRF and the emergence of drug resistance are major hurdles hampering its further development. On the other hand, several studies found that combination therapies can increase SRF\u2019s anti-tumor activity,178 as well as GSH starvation\u2012based nanoscience for cancer therapy.180 Notably, however, Zheng et al. recently reported that SRF failed to increase ferroptosis in various cancer cell lines.181 Thus, the precise role of SRF in ferroptosis, and its clinical value in the context of cancer, remains controversial and poorly understood.Sorafenib (SRF), a small molecular kinase inhibitor, is approved for treating various solid tumors, has well-characterized clinical efficacy and tolerability, and remains the only drug approved to treat advanced HCC. SRF has been shown to trigger ferroptosis in cancer cells by suppressing system Xc\u2013. For example, lanperisone has been shown to effectively target RAS-mutated cancers in vivo without causing overt toxicity.182 APAP is widely used as an analgesic and antipyretic, but can cause dose-dependent hepatotoxicity. Interestingly, Yamada et al. recently showed that APAP-induced hepatotoxicity is attributed to GSH depletion\u2012induced ferroptosis and proposed this as the predominant mechanism,183 providing new insights into the potential use of APAP in triggering ferroptosis in order to inhibit tumor growth. Indeed, several studies have shown a synergistic therapeutic effect of combining erastin and APAP to treat melanoma and lung cancer xenografts.185Interestingly, a subset of FDA-approved drugs such as the muscle relaxant lanperisone and the analgesic acetaminophen  have been shown to activate ferroptosis by functionally suppressing system XcGSH has a central role in the protection of cells from oxidative damage and toxic reactive species. Therefore, targeting pathways involved in GSH synthesis has been studied extensively as a major strategy for treating ferroptosis-related diseases.186 Indeed, we provided the first report that AUR can be used in vivo as to induce ferroptosis in mice by suppressing the activity of thioredoxin reductase,22 providing compelling evidence supporting its further clinical testing\u2014either alone or combined with other therapies\u2014for treating ferroptosis-resistant disease conditions such as cancer. To expand the potential clinical applications of AUR, several studies have investigated repurposing AUR; for instance, AUR has been repurposed as an anticancer drug in TNBC cells by inhibiting GSH biosynthesis.187 Recently, we found that malic enzyme 1 (Me1) regulates ferroptosis in hepatic I/R-induced injury, and its inactivation further decreased the production of NADPH and failed to restore GSH synthesis,188 suggesting that targeting Me1 may be a viable strategy for treating ferroptosis-related diseases by altering GSH synthesis.Auranofin (AUR) is a gold (I)\u2012containing compound used to treat rheumatic arthritis. Recent studies have shown that AUR has therapeutic potential for other diseases and conditions such as cancer, metabolic disease, and infectious and inflammatory diseases.189 Similar to erastin, BSO has also been used to deplete GSH and induce ferroptosis in several cancer cell lines.41 Based on early clinical trials, BSO is considered safe but has limited therapeutic benefit for treating refractory malignancies.190 To improve its clinical efficacy, researchers attempted to identify BSO-sensitive cancer types and patients, revealing that low GSH levels can serve as a marker for BSO susceptibility.191Buthionine sulfoximine (BSO) activates ferroptosis by inhibiting glutamate-cysteine ligase (GCL).192 and is used as a chemotherapy drug for treating brain cancer and lymphoma. Based in its mode of action, BCNU is believed to induce ferroptosis by directly inhibiting GSH synthesis. Recently, a combination of BCNU and sorafenib was shown to significantly suppress the in vivo growth of liver cancer by promoting ferroptosis.193 In addition, both BCNU and AUR have been shown to cause cell death in oxidant-enriched tumorigenic endothelial (EOMA) cells,194 possibly by activating ferroptosis. These findings may therefore increase the clinical application of BCNU for the treatment of HCC and endothelial cell tumors.1,3-bis-(2-chloroethyl)-1-nitrosourea  is a selective glutathione disulfide reductase (GSR) inhibitorN-acetylcysteine (NAC)195 is clinically approved to treat APAP overdose. As an antioxidant, NAC has been shown to inhibit ferroptosis by targeting cysteine metabolism. In addition to protecting the liver from APAP-induced ferroptosis, NAC has also been clinically shown to improve neurodegeneration-related symptoms by increasing cysteine levels and facilitating the synthesis of \u03b3-glutamyl-cysteine and GSH.196 Due to its poor bioavailability, however, NAC must be administered in fairly high doses and requires a long treatment time in patients with severe APAP overdose, thereby increasing the risk of an anaphylactoid reaction , fluid overload, and high fluid osmolarity.197 To overcome these issues, N-acetylcysteine amide (NACA), a modified form of NAC with increased membrane permeability198 and bioavailability (67% compared to only 15% for NAC),199 has been developed. To date, NACA has been shown to have antioxidant activity in several preclinical models, but is yet to be approved for clinical use.20013 Thus, numerous studies have focused on identifying novel activators of ferroptosis by targeting GPX4 in order to develop new cancer therapeutics.The essential antioxidant enzyme GPX4 is not only responsible for maintaining redox homeostasis, but is also recognized as the \u201cferroptosis gatekeeper\u201d by transforming lipid ROS into lipid alcohols. Direct inactivation of GPX4 has been shown to drive ferroptosis independent of intracellular cysteine and GSH levels.1 Although sharing the more common features of ferroptosis, RSL3-mediated ferroptosis does not include decreased levels of cellular GSH. GPX4 was subsequently identified as the primary target of RSL3 using an unbiased, affinity-based chemoproteomics approach.13 Moreover, genetically knocking down and overexpressing GPX4 were shown to cause sensitization and resistance to RSL3, respectively, providing further evidence that GPX4 is the target of RSL3.13 Since these previous studies, RSL3 has been widely used as a ferroptosis agonist in vitro in a wide range of cancer cell types, including pancreatic cancer, adrenocortical carcinoma, fibrosarcoma,201 and breast cancer15 cells. In addition, RSL3 may also serve as a chemosensitizer for cisplatin, doxorubicin, and actinomycin D by activating ferroptosis in various cancer cells, including neuroblastoma,202 osteosarcoma,155 lung cancer,203 and rhabdomyosarcoma cell lines.154 In mouse xenograft models, RSL3 is well-tolerated, with no overt toxicity or body weight loss observed even at the relatively high dose of 400\u2009mg/kg.205 As an experimental tool, RSL3 has been used extensively to study the role of ferroptosis in various disease models, including AKI, I/R-induced injury, and neurodegenerative disease.208As a covalent inhibitor of GPX4, RSL3 was originally identified as a pro-ferroptosis compound through chemical screening.209 and subsequent studies showed that ML162 can directly bind GPX4 and inhibit its activity.13 However, both RSL3 and ML162 have relatively poor selectivity and pharmacokinetics, as they covalently bind to GPX4 via a reactive alkyl chloride moiety.210 To develop a new GPX4 inhibitor containing a different moiety, Eaton et al. used masked nitrile-oxide electrophiles to create ML210, a more selective covalent suppressor of GPX4 with improved pharmacokinetics and comparable activity.211 However, given that the in vivo efficacy of all GPX4 inhibitors developed to date remains limited, efforts have focused on developing more efficacious GPX4-selective inhibitors.Similar to RSL3, ML162 was identified as a GPX4 inhibitor,212 FIN56 potently induces ferroptosis in all TNBC cell lines tested to date, suggesting that FIN56 may be an effective additional pro-ferroptosis anticancer drug.213 In contrast, the endoperoxide-containing 1,2-dioxolane FINO2 was shown to potently and selectively induce ferroptosis in engineered cancer cells by directly oxidizing iron and indirectly inactivating GPX4.214 In addition, the naturally occurring C28 steroidal lactone withaferin A (WA), one of the best-studied withanolides, has a diverse range of pharmacological activities, including anti-inflammatory, anti-tumor, and antioxidant properties. In both high-risk neuroblastoma cell lines and neuroblastoma xenografts, WA has been shown to dose-dependently induce ferroptosis via the dual mechanisms of inactivating GPX4 and activating the NRF2 pathway.202 Thus, natural products may serve as an additional resource for the discovery of new GPX4 inhibitors.Based on the structural optimization of CIL56 , FIN56 was developed as a highly selective inducer of ferroptosis that promotes GPX4 degradation as well as the GPX4-independent activation of squalene synthase (SQS).215 Mutations in either CKB or GPX4 significantly increase the ferroptosis-mediated cell death of HCC by inhibiting this phosphorylation.215 Importantly, the authors showed a positive correlation between phosphorylated GPX4 and HCC aggressiveness in clinical samples,215 suggesting a potential new strategy for treating HCC by blocking CKB/GPX4-mediated ferroptosis.Recently, Wu et al. identified creatine kinase B (CKB)-enhanced GPX4 as a novel mechanism for regulating ferroptosis, accounting for the resistance of HCC cells to ferroptosis by stabilized GPX4 via CKB phosphorylated GPX4 at residue S104.216 all currently identified GPX4 inhibitors have limited prospects for further clinical development due to their poor pharmacokinetics and specificity. Therefore, more studies are needed in order to develop GPX4-specific inhibitors with improved pharmacological properties.Although GPX4 inhibitors potently inhibit cells growth in vitro,217 which serves as the active site for GPX4.16 Ionic selenite (SeO32-) is commonly used to deliver Se to cultured cells. For example, treating cultured neurons with SeO32- was shown to increase GPX4 transcription and inhibit ferroptosis induced by either hemin or homocysteic acid.74 In addition, systemically treating mice with Tat SelPep, a selenocysteine-containing peptide that can cross the BBB, was shown to improve functional recovery following hemorrhagic and ischemic stroke by blocking ferroptosis.74 Together, these studies suggest that Se supplementation may help protect against ferroptosis-related tissue damage and disease.The essential micronutrient selenium (Se) is required for the production of selenocysteine,218 suggesting that dopamine may be a promising candidate drug for alleviating ferroptosis-related tissue injury and disease, as well as certain neurodegenerative diseases.The neurotransmitter dopamine has many physiological roles, particularly in controlling various functions in the central nervous system, including movement, memory, motivation, mood, and attention. Insufficient levels of dopamine production are known to contribute to the progression of PD, and dopamine-based therapies such as levodopa and dopamine receptor agonists have been used clinically to treat PD and various cardiovascular conditions. Interestingly, non-oxidative dopamine was reported to potently inhibit erastin-induced ferroptosis in both cancerous and non-cancerous cells by stabilizing GPX4,219Using a combination of computational prediction and experimental validation, Li et al. discovered eight new potential GPX4 activators and found that compound 1d4 was the most potent allosteric activator of GPX4, significantly increasing GPX4 activity by 50% when applied at 20\u2009\u03bcM in a cell-free assay and 61\u2009\u03bcM in cell extracts; moreover, they found that compound 1d4 potently inhibited ferroptosis when applied to HT-1080 fibrosarcoma cells.Curculigo orchioides Gaertn , an ancient medicinal plant well known for its immunomodulatory and rejuvenating effects. Recently, Wang et al. reported that curculigoside inhibits ferroptosis in IEC-6 cells  by upregulating GPX4 expression.220 In mice with dextran sulfate sodium (DSS)\u2012induced ulcerative colitis, curculigoside was shown to protect against disease progression by reducing ferroptosis via GPX4 induction,220 suggesting that activating GPX4 is a viable therapeutic option for ulcerative colitis. Another plant-derived bioactive compound, puerarin, is an isoflavone glycoside isolated from Pueraria lobata  with various pharmacological effects,221 including antioxidant, anticancer, and anti-inflammation properties, in addition to alleviating pain, promoting bone formation, attenuating insulin resistance, and exerting cardiac and neuronal protective effects. However, its molecular target and mechanism remain unknown. Interestingly, the anti-ferroptosis activity of puerarin in heart disease has been linked to the induction of FTH1 and GPX4 in rats.222 Given that the majority of bioactive natural compounds have relatively low bioavailability, future studies should focus on developing rationally designed combination therapies and/or their incorporation in nanoparticles.The diterpenoid triepoxide curculigoside is one of the primary bioactive phenolic compounds isolated from 223 Thus, NRF2 signaling is important for regulating ferroptosis.The p62-KEAP1-NRF2 signaling pathway plays a regulatory role in response to oxidative stress, environmental insult, and toxic chemicals. In response to oxidative stress, p62 activates NRF2 by directly binding to its ubiquitin ligase adapter KEAP1; as a result, NRF2 translocates to the nucleus and regulates cellular redox homeostasis by modulating the expression of target genes, including several genes encoding enzymes involved in both GSH synthesis and iron homeostasis.224 In addition, sitagliptin, a selective inhibitor of dipeptidyl peptidase 4 (DPP4) used to treat type 2 diabetes, was shown recently to inhibit ROS production, inflammation, and excessive autophagy by promoting Nrf2 translocation to the nucleus in mouse models of acute lung injury induced by severe pancreatitis.225 In addition, a modest dose of WA was shown to induce ferroptosis and increase the LIP by directly targeting KEAP1, mediating the NRF2-mediated upregulation of HO-1.202The polar hydrophilic alkaloid trigonelline can be extracted from many plant species, including coffee beans and fenugreek seeds, and has been shown to significantly sensitize cancer cells to ferroptosis inducers by inhibiting NRF2.10-NAD(P)H, glutaminolysis, and DHODH-mediated pathways, have also been linked to ferroptosis and are discussed in detail below.Other reductive-oxidative pathways, including the transsulfuration, FSP1-CoQ226 CH004, a pharmacological inhibitor of CBS, has been identified as a novel stimulator of ferroptosis in liver cancer both in vitro and in vivo,227 providing experimental evidence to support the therapeutic potential of targeting the transsulfuration pathway in liver cancer. Similarly, the CTH inhibitor propargylglycine has been shown to sensitize NSC-34 cells to both erastin- and RSL3-induced ferroptosis.228 In mice, propargylglycine was also shown to increase susceptibility to APAP-induced liver damage and mortality,229 possibly by inducing ferroptosis.When cysteine availability is restricted, the transsulfuration pathway is activated in order to increase the synthesis of cysteine from methionine. The enzyme cystathionine beta-synthase (CBS) activates the first step in this pathway, followed by the formation of cystathionine into cysteine by the enzyme cystathionine gamma-lyase (CTH). Sustained activation of the reverse transsulfuration pathway has been implicated in the resistance of ovarian cancer cells to erastin-induced ferroptosis.FSP1 gene was initially identified as a pro-apoptotic gene,230 but has also been identified as an anti-ferroptosis gene via FSP1\u2019s oxidoreductase activity, which reduces CoQ10 to ubiquinol (CoQ10H2).18 Moreover, the FSP1 inhibitor iFSP1 was shown to selectively activate ferroptosis in GPX4-knockout FSP1-overexpressing cells and was identified by screening 10,000 drug-like compounds;17 iFSP1 was also shown to potently increase the sensitivity of cancer cells to RSL3-induced ferroptosis.17 Recently, Yoshioka et al. identified the novel compound called NPD4928 as an FSP1 inhibitor;231 the authors then showed that NPD4928 increased the cytotoxicity of various cell types in response to GPX4 antagonists, indicating that NPD4928 and GPX4 inhibitors may work synergistically to treat cancer by inducing ferroptosis.231 Most recently, Hendricks et al. developed another FSP1 inhibitor called FSEN1, which they found sensitized various cancer cells to ferroptosis.232 Together, these findings suggest that pharmacologically inhibiting the FSP1-CoQ10-NAD(P)H pathway may provide a plausible strategy for sensitizing cancer cells to ferroptosis-resistant therapeutic agents. On the other hand, Fang et al. recently identified the diphenylbutene derivative compound 3f as a ferroptosis inhibitor, and they showed that this compound can protect against ischemic stroke in rats by increasing FSP1 protein levels,233 suggesting that novel agents that upregulate FSP1 may be used to treat ferroptosis-related diseases.The 234 Given that this reaction promotes ferroptosis by causing an accumulation of lipid peroxidation,25 blocking the glutaminolysis pathway has been proposed as a potential therapeutic approach for treating ferroptosis-induced tissue damage.Cellular uptake of the amino acid glutamine (Gln) is mediated primarily by specific transporters such as SLC38A1 and SLC1A5. Following its cellular uptake, intracellular Gln is converted by glutaminase (GLS) to produce glutamate, which can be further catalyzed to \u03b1-ketoglutarate either by glutamate dehydrogenase (GLUD1)\u2012mediated deamination or by transaminase-mediated transamination.236 In addition, compound 968 was shown to potently prevent I/R-induced heart damage in an ex vivo model.25Inhibiting glutaminolysis using L-g-glutamyl-p-nitroanilide , compound 968 (an inhibitor of GLS), or amino-oxyacetic acid  has been shown to block cystine deprivation\u2012induced ferroptosis in mouse embryonic fibroblasts, melanoma cells, as well as head and neck cancer cells.GLS, SLC7A11, and SLC1A5.238 In addition, low-dose PTX was shown to trigger ferroptosis by decreasing SLC7A11 expression by upregulating the well-characterized transcription factor p53(ref. 238). Moreover, a growing body of evidence suggests that p53 regulates ferroptosis either by transcriptional or posttranslational mechanisms such as modulating the downstream expression of SLC7A11,239SAT1 (encoding spermidine/spermine N1-acetyltransferase 1),240GLS2 (encoding glutaminase 2),25 and CDKN1A/p21 (encoding cyclin dependent kinase inhibitor 1A),241 or by directly inhibiting DPP4 activity in colorectal cancer cells.242Recent studies showed that low-concentration paclitaxel  suppressed cancer cell proliferation by promoting the production of lactate and changing the pH of the tumor microenvironment by downregulating glutaminolysis-related genes such as 243 Originally, targeting DHODH was shown clinically to improve autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Although functional studies characterized DHODH as a potential target for treating cancer, several potent DHODH suppressors such as brequinar, leflunomide, and teriflunomide were clinically assessed but failed to receive FDA approval. Importantly, however, brequinar was recently shown to act synergistically with the FDA-approved drug SAS to suppress tumor growth by potently inducing ferroptosis,20 suggesting that DHODH may indeed serve as a viable target for treating cancer. Interestingly, DHODH inhibitors have also been developed as antiviral agents to act against cytomegalovirus, Ebola, influenza, and SARS-CoV-2(refs. 245). Whether ferroptosis is involved in the multifaceted activities of DHODH inhibitors remains an open question; nevertheless, targeting DHODH may have promise as a novel therapeutic approach for cancer and certain viral infections.The flavin-dependent mitochondrial enzyme DHODH catalyzes the de novo synthesis of pyrimidine.30 In addition to PE, other PLs can also be oxidized upon the induction of ferroptosis. Because lipid metabolic pathways are important for regulating lipid peroxidation, targeting these pathways may provide a novel strategy for treating ferroptosis-related diseases - and adrenic acid (AdA)-containing phosphatidylethanolamine (PE) species (C18:0/C20:4 and C18:0/C22:4)\u2014were previously identified as the most susceptible substrates for lipid peroxidation.ses Fig. .Fig. 5Ta29 ACSL4 and LPCAT3 catalyze the addition of CoA to the long-chain polyunsaturated bonds of AA and AdA, respectively, thereby promoting the esterification of PUFAs to membrane PLs.30 PEs containing acylated AA or AdA (AA/AdA-CoA) can be subsequently oxidized by LOXs to form PL hydroperoxides (PE-AA/AdA-OOH), eventually leading to the membrane deposition of lipid ROS and driving ferroptosis.246 A number of inhibitors that target lipid metabolism have been shown to suppress ferroptosis, including LOX inhibitors, radical-trapping antioxidants , ACSL4 inhibitors, and deuterated PUFAs/MUFAs (by decreasing PUFA-containing PLs).A haploid genetic screen in KBM7 cells (a chronic myeloid leukemia cell line) revealed that the enzymes ACSL4 and LPCAT3 serve as ferroptosis regulators.247 that can regulate ferroptosis by mediating PL oxidation.248 A total of 6 LOX isoforms  are expressed in humans, and a total of 7 LOX isoforms  are expressed in mice.250 ALOX5/12 inhibitors, including AA-861,31 cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC),252 the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA),253 and BWA4C,248 have been either shown or suggested to suppress ferroptosis. In an effort to re-evaluate the selectivity profile of CDC for LOXs, Pergola et al. identified CDC as an ALOX5-specific antagonist with an IC50 value in the low nanomolar range (9\u201325\u2009nM) measured in cell-free assays.251 Zileuton, another widely used ALOX5-selective inhibitor, was the first FDA-approved orally administered drug for treating asthma. Zileuton was also shown to exert a neuroprotective effect by blocking ALOX5-mediated glutamate toxicity and ferroptosis in HT22 cells .254 With respect to ALOX12, ML355 is a potent and selective inhibitor255 that has been shown to reduce ALOX12-induced thrombosis and protect human pancreatic islets from ALOX12-triggered inflammatory injury.257 Recently, Zhang et al. reported that the compound IMA-1, which disrupts the interaction between ALOX12 and ACC1 (acetyl-CoA carboxylase 1), can reduce NASH progression in mice and Cynomolgus macaques,258 suggesting that the ALOX12-ACC1 complex may be a clinically viable target for the management of NASH.LOXs are a class of non-heme iron-containing dioxygenases259 and ischemia-induced myocardial injury260 by driving the peroxidation of PL-PUFAs and inducing ferroptosis, providing compelling evidence supporting the viability of targeting ALOX15 to treat I/R-induced injury in these tissues. Interestingly, Walters et al. functionally confirmed that ALOX15 is involved in inducing oxidative stress in human spermatozoa, supporting the notion of increased ALOX15 expression in sperm cells261 and suggesting that ALOX15 may be a potential target for the treatment of oxidative stress\u2012induced male infertility.261 Indeed, the ALOX15-selective inhibitor PD146176 was shown to protect against oxidative damage by decreasing the production of membrane PL peroxidation and 4-hydroxynonenal (4-HNE) in spermatozoa.261 Moreover, the ALOX15-specific inhibitor ML351 was recently shown to reduce ferroptosis in cardiac I/R-induced injury.262ALOX15 was recently reported to play an essential role in exacerbating I/R-induced cerebral injury263 suggesting that the regulation of enzymes other than LOXs may also contribute to lipid peroxidation by mediating the oxygenation of membrane PLs.Taken together, various LOX inhibitors have been shown to effectively inhibit ferroptosis. In addition, the enzyme POR (cytochrome P450 oxidoreductase) was recently shown to promote PL peroxidation in a LOX-independent manner,1 By scavenging free radicals, Fer-1 reduces ferroptosis-induced tissue injury and prolongs survival in mouse models of disease.64 However, Fer-1 has not been pursued in drug development, given its metabolic instability and poor pharmacokinetics. Currently, Fer-1 is used as a research tool for studying various ferroptosis-related processes. Compared to Fer-1, second-generation (SRS11-92) and third-generation (SRS16-86) RTAs have significantly higher activity but still contain an ester moiety.64 To develop an optimized set of RTAs, Devisscher et al. developed a series of compounds in which the ester was replaced with a sulfonamide, providing improved water solubility and better metabolic and kinetic properties.264 For example, they found that compound UAMC-3203 was more stable and more potent than Fer-1 (with a significantly lower IC50 compared to Fer-1).264 After intravenous injection, the terminal plasma half-life of UAMC-3203 is ~3 and 5\u2009h in mice and rats, respectively,83 suggesting that this compound has relatively good pharmacokinetics.One of the principal features of ferroptosis, namely PL peroxidation initiated by the formation and propagation of lipid radicals, can be blocked by RTAs, a class of lipid chain\u2012breaking antioxidants. Among the various RTAs identified to date, the now-widely used ferrostatin-1 (Fer-1) was originally identified as a potent ferroptosis-specific inhibitor in erastin- and RSL3-treated HT-1080 cells.5 However, Lip-1 also potently inhibits the enzymatic activity of CYP2D6 (with an IC50 of 4.1\u2009\u03bcM), a member of the cytochrome P450 superfamily of drug-oxidizing enzymes, indicating that Lip-1 may not be suitable for use in clinical trials.5Liproxstatin-1 (Lip-1), a spiroquinoxalinamine derivative, was identified by high-throughput screening and shown to inhibit ferroptosis in vivo with better pharmacological properties than Fer-1.265 Using three different cellular models of ferroptosis, Zilka et al. found that tetrahydronapthyridinols (THNs) inhibit ferroptosis induced by RSL3, cystine deprivation, and GPX4 inactivation.265 Compared to both Fer-1 and Lip-1, lipophilic THNs have similar potency, while hydrophilic THNs are ineffective at inhibiting ferroptosis.265In addition to inhibiting ferroptosis, potent RTAs such as Fer-1 and Lip-1 can insert into membrane PLs via arylamines. Notably, the catalytic activity of both Fer-1 and Lip-1 requires a relatively high temperature.266 Two diarylamine derivatives\u2014phenothiazine and phenoxazine\u2014have been shown to suppress ferroptosis in mouse embryonic fibroblasts.267 Interestingly, each phenoxazine molecule can trap >2 peroxyl radicals at moderate temperatures, suggesting new strategies for the development of more potent RTA-based ferroptosis inhibitors. Indeed, Mishima et al. showed that promethazine is more potent at protecting kidney function compared to Fer-1(ref. 268), suggesting that promethazine may be a viable tool for studying the role of ferroptosis in various disease models. Notably, edaravone\u2014an RTA clinically approved for treating acute ischemic stroke and amyotrophic lateral sclerosis135\u2014has been shown to protect against ferroptosis under various pathological conditions.269 In addition, Zilka et al. found that copper(II)-diacetyl-bis(N4-methylthiosemicarbazone) (CuATSM), a candidate drug for treating ALS and PD, suppresses ferroptosis via its RTA activity.270 Recently, a group of reduced forms of vitamin K (VKH2), including menaquinone and phylloquinone, was shown to have potent anti-ferroptosis properties,21 providing new mechanistic insights into the anti-ferroptotic activity of alternative RTAs.Diarylamines are RTAs commonly used to reduce the autoxidation of petroleum-derived products. The efficacy of this class of RTAs depends largely on the kinetics and stability of the rapid hydrogen atom transfer to one-electron oxidation by peroxidic species.271 and Barayeu et al.272 independently reported that endogenous sulfane sulfur (S0) species/hydropersulfides (H2S or RSSH) are potent RTAs. By supplying sulfur for S0 biosynthesis, cysteine can inhibit ferroptosis via a GPX4-independent mechanism.272 Genetic manipulation of enzymes involved in S0 biosynthesis clearly implicate S0 as playing a role in regulating ferroptosis by scavenging ROS and suppressing lipid peroxides.272 Both endogenous and exogenous S0 have been shown to provide cellular protection against ferroptosis.272 Together, these results suggest that regulating ferroptosis by targeting S0 warrants further clinical study.Recently, Wu et al.30 Thiazolidinediones (TZDs) such as rosiglitazone, pioglitazone, and troglitazone were originally identified as agonists of peroxisome proliferator-activated receptor \u03b3 (PPAR\u03b3) and are approved to treat adult type 2 diabetes. Interestingly, TZDs showed effects on suppressing ferroptosis by selectively inhibiting ACSL4273 in breast cancer cell lines and reduce mortality due to acute renal failure in kidney-specific Gpx4 knockout mice.15ACSL4 is a unique and important isozyme involved in the metabolism of PL-PUFAs such as AA, and deleting or inhibiting ACSL4 blocks ferroptosis by preventing the incorporation of PL-PUFAs into cell membranes.276 Notably, exogenous lipid supplementation can modulate both apoptosis and ferroptosis.277 Magtanong et al. showed that exogenous MUFAs can also specifically reduce the accumulation of lipid ROS in the plasma membrane and can displace PUFAs from their cellular location, suppressing ferroptosis in an ACSL3-dependent manner.277Similar to ACSL4 inhibitors, deuterated PUFAs and MUFAs can also inhibit ACSL4 by decreasing PL-PUFAs. PUFAs deuterated at the bis-allylic position (D-PUFAs) have been shown to inhibit ferroptosis in cells and animal models of PD and Friedreich\u2019s ataxia.+ T cells by blocking the uptake of oxidized lipids.278 Moreover, inhibiting stearoyl-CoA desaturase-1 (SCD1) with CVT-11127 or inhibiting acetyl-CoA carboxylase (ACC) with CP-640186 can affect the production of MUFAs, reducing the growth of lung cancer cells.279 Thus, Batchuluun et al. proposed that other potent ACC inhibitors should be examined for their ability to affect ferroptosis.280Molecules that affect the composition of intracellular lipids may also regulate ferroptosis. For example, sulfosuccinimidyl oleate (SSO), which irreversibly inhibits the fatty acid receptor CD36, has been shown to inhibit ferroptosis in CD8CDKN2A expression remodels the lipidome of glioblastoma cells, and patient-derived CDKN2A-deficient glioblastoma cells have higher levels of lipid peroxidation, thereby sensitizing the cells to ferroptosis in response to GPX4 inhibition.281 These results suggest potential new therapeutic strategies in which targeting cellular lipidome remodeling can induce ferroptosis in glioblastoma cells, particularly in cells lacking CDKN2A expression.Recently, Minami et al. found that loss of 108 The small molecule FIN56 potently induces ferroptosis via two mechanisms, namely by reducing GPX4 levels and by decreasing CoQ10 activity by targeting the enzyme SQS, which acts downstream of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase in the mevalonate pathway.213The mevalonate pathway regulates ferroptosis by altering GPX4 production by affecting the maturation of selenocysteine tRNA.212 and selectively kill cancer cells in a high-mesenchymal state.40 However, the concentration of statins needed to increase ferroptosis in cancer cells is 100\u2013500 times the recommended dose for decreasing cholesterol, and high-dose statins often cause severe side effects such as myalgia, rhabdomyolysis, and hepatotoxicity.283 To mitigate these side effects, it may be possible to deliver the statins in nanoparticles, leading to a high concentration specifically in tumor cells.284Statins are well-known inhibitors of HMG-CoA reductase (HMGCR) and are commonly used as cholesterol-lowering medications to prevent and/or treat atherosclerosis. Statins have also been shown to increase FIN56-induced cellular ferroptosisAlthough numerous ferroptosis-targeting agents have been studied, their properties often make them poorly suited for use as therapeutic compounds; indeed, their low solubility, high metabolic clearance, low cellular permeability, and systemic toxicity limit their clinical development. Therefore, multi-target ferroptosis modulators and nanomaterial technologies have been investigated in an attempt to target ferroptosis more effectively in vivo.Artemisia annua) extracts, are commonly used as effective antimalarial drugs. The semi-synthetic derivative and major active metabolite of artemisinin, dihydroartemisinin (DHA), has been shown to increase ferroptosis in lung cancer cells by inactivating the PRIM2/SLC7A11 axis,285 as well as in head and neck cancers286 and glioblastoma cell lines287 by inhibiting GPX4. In addition, DHA plays a role in ferritinophagy, thereby inducing free iron\u2012induced ferroptosis.288Artemisinins, which are derived from sweet wormwood . Recently, baicalein was shown to prevent ferroptosis in erastin-treated PANC1 cells (a human pancreatic cancer cell line) by reducing ferrous iron, inhibiting GSH consumption, and inhibiting GPX4 degradation, thereby suppressing lipid peroxidation.297 These results suggest that baicalein may prevent ferroptosis-associated tissue damage.The flavonoid baicalein (in Chinese: Huangqin) has long been used as a popular antibacterial and antiviral agent.Compared to small-molecule compounds, ferroptosis-inducing nanoparticles have been suggested to have a better preclinical profile. Importantly, nanoparticles can be loaded with chemotherapeutic drugs or tumor-selective molecules, making them ideally suited for developing nanoparticle-based cancer therapies as well as combination therapies.298 the PEGylated single-atom Fe-containing nanocatalyst PSAF,299 the nanoprobes UCNP@GA-FeIII300 and UCNP@LP(Azo-CA4),301 and the nano-platforms HA@MOF302 and PB@FePt-Ha-g-PEG303 can effectively deliver Fe2+ to cancer cells in order to accelerate the Fenton reaction and produce lethal amounts of ROS, specifically triggering ferroptosis. Interestingly, Zhang et al. found that the FePt@MoS2 nanocomposite efficiently induces ferroptosis by releasing >30% of loaded Fe2+ into the tumor microenvironment within 72\u2009h.304 In addition, GOD-Fe3O4@DMSNs, in which the nanoparticle is loaded with a natural glucose oxidase, has been shown to exhaust glucose in tumor cells and generate excessive levels of H2O2, thereby accelerating the Fenton reaction catalyzed by Fe3O4(ref. 305). In the relatively acidic tumor microenvironment, several nanoparticles can be reduced in order to release two molecules such as iron and doxorubicin.307 For example, FePt-NP2 can deliver iron oxide in order to enhance the chemotherapeutic effects of cisplatin specifically in cancer cells.308 Moreover, Shen et al. designed FeGd-HN@Pt@LF/RGD2, a cisplatin-loaded Fe3O4/Gd2O3 hybrid nanoparticle conjugated to lactoferrin (LF) and an RGD (Arg-Gly-Asp) dimer in order to potently induce ferroptosis specifically in brain tumor cells.298 Thanks to its small size (6.6\u2009nm) and LF receptor\u2012mediated transcytosis, FeGd-HN@Pt@LF/RGD2 nanoparticles can cross the BBB and deliver cisplatin together with reactants  to brain tumor cells, significantly slowing the growth of orthotopic brain tumors.298Many nanoparticles have been designed to increase ferroptosis by triggering the Fenton reaction in targeted tumor cells Fig. . For ins309 SRF@FeIIITA is a rationally designed nanoparticle spontaneously formed by a Fe3+ and tannic acid (TA) network\u2012like corona onto a nanocore containing the kinase inhibitor sorafenib (SRF). In the acidic lysosomal environment, SRF@FeIIITA releases SRF, thereby inhibiting the production of GSH and inducing ferroptosis.310 Another SRF-based nanoparticle, FaPEG-MnMSN@SFB, has been validated to effectively activate ferroptosis and suppress tumor growth via the dual GSH-depleting effects of MnMSN (manganese-loaded mesoporous silica nanozyme) and SRF.179 Moreover, SRF@MPDA-SPIO nanoparticles were generated using SRF and ultrasmall superparamagnetic iron oxide (SPIO) nanoparticles, making the particles visible on MRI and potently inducing ferroptosis by supplying iron.311 In addition, the biomimetic magnetic nanoparticle Fe3O4-SAS@PLT was generated using SAS-loaded mesoporous magnetic nanoparticles (Fe3O4) and a platelet (PLT) membrane camouflage, thereby activating ferroptosis by blocking the system Xc\u2212 pathway.174 Notably, Fe3O4-SAS@PLT\u2012induced ferroptosis significantly increased PD-1 (programmed cell death 1)\u2012based cancer immunotherapy and long-term tumor elimination in mice transplanted with 4T1 metastatic breast cancer cells.174 Recently, Li et al. reported the development of the multifunctional nanosheet system Au/Cu-TCPP(Fe)@RSL3-PEG-iRGD, which triggers ferroptosis in tumor cells by simultaneously blocking the GSH/GPX4 and FSP1/CoQ10H2 pathways via its nanocatalytic activity, as well as synergizing with RSL3 to further inactivate GPX4 in tumor cells.312An alternative nanoparticle-based strategy for triggering ferroptosis is to deplete GSH and/or inhibit GPX4 Fig. . For exa313 In addition, MON-p53, an encapsulated metal-organic network (MON) containing a plasmid expressing the tumor suppressor p53, is designed to treat cancer by switching apoptosis to ferroptosis, and long-term treatment  was shown to have potent anticancer activity in tumor-bearing mice.314 Similarly, RSL3 micelles\u2014nanoparticles composed of an AA-conjugated amphiphilic copolymer encapsuling RSL3\u2014have been shown to increase the efficacy of RSL3 in vivo, providing a novel approach to overcome multidrug resistance and improve cancer treatment.315 Moreover, a nanodrug consisting of nanoparticle ferritin\u2012bound erastin and rapamycin (NFER) with significantly improved drug-like properties, was proposed to increase ferroptosis by decreasing GPX4 activity as well as reducing lipid peroxidation levels.316 Interestingly, Sal-AuNP, a gold nanoparticle loaded with salinomycin, has been shown to induce ferroptosis in breast cancer stem cells by causing oxidative stress, mitochondrial dysfunction, and lipid oxidation.317 Recently, a hypercarbon-centered gold(I) cluster prodrug was engineered and then shown to accelerate ferroptosis in EJ cells (a human bladder cancer cell line) by inhibiting thioredoxin reductase (TrxR) activity.318Interestingly, nanoparticles can also be designed to directly deliver molecules that trigger ferroptosis Fig. . For exa319 and SPFeN320 have been studied as photothermal ferrotherapies for use in cancer, combining photosensitizers with ferroptosis-inducing molecules that can exert synergistic anticancer effects. Another photosensitizer\u2014chlorin e6 (Ce6)\u2014has been used in several delivery systems, including Ce6-erastin nanoparticles,321 SRF@Hb-Ce6,322 Ce6@MOF,323 and HAS-Ce6-IrO2.324 Liu et al. reported excellent chemodynamic/photodynamic synergy using mCMSN, a biodegradable cancer cell membrane\u2012coated mesoporous copper/manganese silicate nanosphere that increases ferroptosis via laser irradiation\u2012triggered ROS production and the GSH-activated Fenton reaction.325 To achieve a synergistic ferroptosis and immunomodulatory effect in treating cancer, Zhang et al. engineered a biomimetic magnetosome (Pa-M/Ti-NC) consisting of an Fe3O4 magnetic nanocluster (NC) as the core, pre-engineered leukocyte membranes loaded with TGF-\u03b2 inhibitor (Ti), and a PD-1 antibody (Pa) anchored to the membrane surface;326 they found that this magnetosome potently promoted ferroptosis in cancer cells via cooperative activity between the Pa and Ti components, causing reprogramming of the immunogenic microenvironment. Interestingly, NMIL-100@Gox@C was designed to activate ferroptosis and induce cancer starvation;327 specifically, the glucose oxidase (Gox) enzyme catalyzes glucose to generate sufficient levels of H2O2 in order to induce ferroptosis, simultaneously causing starvation of the cancer cells via the Gox-mediated consumption of glucose.327 Yang et al. developed an HLCaP nanoreactor that encapsulates lipoxidase and hemin in biodegradable PLGA (poly(lactic-co-glycolic acid)), which releases its contents in the acidic tumor microenvironment, synergistically inducing ferroptosis using the PUFA substrates produced in the tumor debris using radiofrequency ablation;328 importantly, these so-called tumor debris\u2012fueled nanoreactors have been shown to prevent tumor recurrence and metastasis.328 Similarly, the mesoporous carbon nanoparticle FeCO-Dox@MCN is designed to exploit the combined effects of chemotherapy, photothermal therapy, and gas therapy in order to increase carbon monoxide\u2012induced ferroptosis.329To maximize the efficacy of cancer therapeutics, nanoparticle-based combination therapies have been widely studied in various preclinical models. For example, CSO-SS-Cy7-Hex/SPION/Srfn330 The second is DEF-HCC-PEG, a deferoxamine-binding nanoparticle that protects cells against both ferroptosis and senescence.331 Importantly, ferroptosis is a rapidly developing field, with new mechanisms underlying ferroptosis being identified; thus, additional studies are clearly helpful for accelerating the development of clinical applications using nanoparticles to target ferroptosis.By contrast, relatively few studies have focused on ferroptosis-suppressing nanoparticles Fig. . Here, wOver the past decade, a rapidly growing body of evidence has shown that ferroptosis plays a key role in a wide range of diseases and conditions, including cancer, neurodegenerative disease, tissue/organ injury, inflammation, and inflammatory diseases. Despite many obstacles along the way, it is now widely accepted that targeting ferroptosis holds great promise for promoting the development and clinical translation of ferroptosis-based therapeutic strategies.To date, nearly all in vivo ferroptosis-related studies were based on preclinical animal models, and several challenges limit their translation to clinical practice.332 In addition, poor pharmacokinetics remains a major bottleneck for the further development of both ferroptosis antagonists such as Fer-1 and Lip-1 and ferroptosis agonists such as RSL3 and ML210. To develop novel bioactive ferroptosis-targeted drug candidates, researchers have studied natural products derived from microorganisms and plants, particularly traditional herbs. Furthermore, high-throughput screening, artificial intelligence, and other new technologies may also be used to identify more effective drug candidates to target ferroptosis.As discussed in this review, numerous molecules\u2014including experimental agents, naturally derived compounds, nanoparticles, and clinically approved drugs\u2014have been shown to modulate ferroptosis, revealing promising new strategies for the development of ferroptosis-based therapies. However, with respect to small molecule compounds, only ~15% of proteins in the human proteome are estimated to be druggable.Another challenge to ferroptosis-based drug discovery is the rational design of combination strategies to treat various ferroptosis-related diseases. It is generally well accepted that ferroptosis plays a pathogenic role in various diseases. In addition, many existing therapies can trigger apoptosis or other types of cell death. For conditions such as cancer, tissue injury, and neurodegenerative disease, various forms of cell death can occur simultaneously. Therefore, developing ferroptosis modulators that can act synergistically with existing therapies may serve as a promising strategy for treating these diseases, particularly drug-resistant cancer. Combining ferroptosis agonists with other anticancer therapies may also increase ferroptosis, as well as other types of cell death, thereby eradicating cancer more effectively.In addition to conventional ferroptosis inhibitors and inducers, new technologies such as proteolysis-targeting chimeras (PROTACs), RNA-based therapies, gene editing, and peptide and protein drugs have been applied to develop ferroptosis-targeting drugs.333 providing a promising novel strategy for the development of clinically applicable GPX4-targeted drugs. Similar to PROTACs, photodegradation-targeting chimeras (PDTACs),334 lysosome-targeting chimeras (LYTACs),335 and autophagy-targeting chimeras (AUTACs)336 have also been developed for use in TPD-based therapies. For example, Liu et al. reported the targeted photodegradation of GPX4 using a PDTAC conjugated with the photosensitizer verteporfin upon red-light irradiation, and showed that this PDTAC can induce immunogenic ferroptosis and may improve the efficacy of cancer immunotherapy.334Targeted protein degradation (TPD) is a new and challenging therapeutic modality that can degrade \u201cundrugged\u201d targets and other difficult protein targets such as proteins with a broad and shallow active pocket and/or \u201csmooth\u201d surfaces. Three major classifications of protein degraders have been developed, including PROTACs, monomeric targeted protein degraders, and molecular glues (MGs). PROTACs are heterobifunctional small molecules consisting of two ligands connected via a linker. The ligands are designed to bind to an E3 ubiquitin ligase and a target of interest. This chemically induced close proximity between the protein and E3 ligase results in the target protein\u2019s ubiquitylation and subsequent degradation via the ubiquitin-proteasome system (UPS). Recently, TPD has gained considerable attention due to encouraging results obtained from clinical trials involving ARV-110 and ARV-471, two PROTACs that target and degrade the androgen receptor (AR) and estrogen receptor (ER), respectively. Recently, several groups have attempted to develop PROTAC-based ferroptosis modulators. For example, Luo et al. developed the PROTAC dGPX4 to target GPX4 and showed in vivo that it potently induces ferroptosis selectively in cancer cells with no apparent side effects,337 Fulvestrant, a selective ER degrader, has been approved for treating ER-positive breast cancer, and some monomeric degraders have been tested in clinical trials.338 MGs are relatively simple small molecules that increase the interaction between E3 ligase and the protein substrate in order to induce the UPS-induced degradation of target proteins.339 Thanks to their low molecular weight, MGs are believed to have more favorable pharmacokinetics than other compounds. Thalidomide was developed as a MG to bring various targets in proximity to the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN for degradation.340 Lenalidomide, a thalidomide derivative, has been approved to treat multiple myeloma by inducing the degradation of oncoproteins. Thus, monomeric degraders and MGs represent promising new strategies for targeted protein degradation. However, to the best of our knowledge no reports have yet been published regarding the use of monomeric targeted protein degraders or MG-based degraders to target ferroptosis-related proteins.Unlike PROTACs, monomeric targeted protein degraders are designed to either directly bind to their target protein or induce their target protein\u2019s degradation. Therefore, monomeric degraders have a comparatively small molecular weight and can easily cross the BBB.341 Given that the traditional process of drug discovery is time-consuming and labor-intensive, it is now widely accepted that AI\u2014particularly machine learning\u2014can significantly increase prediction accuracy and accelerate the drug discovery process. Deep learning, which combines machine learning and AI, can serve as a particularly cost-effective platform for discovering new drugs, as it directly enables rapid machine-based decision-making using an artificial neural network. Today, AI-based technologies have been applied to virtually every step in the drug discovery process, including target identification, drug design, drug screening, chemical synthesis, and prediction of the drug\u2019s properties and mode of action.342 In addition, AI can be used to quickly identify lead compounds extracted from plants and microorganisms.343 Recently, AI was successfully used in cancer research and precision medicine.344 With respect to ferroptosis, Wu et al. used a combination of bioinformatics, network pharmacology, and AI to show that ferroptosis and the TGF-\u03b2 signaling pathway may contribute to the protective effects of celastrol, a plant-derived triterpene, against type 2 diabetes.345 It is therefore reasonable to speculate that AI will significantly accelerate the discovery of new ferroptosis-targeted drugs.AI, which was first defined by John McCarthy back in 1956 as the \u201cscience and engineering of making intelligent machines\u201d, primarily involves computer science, mathematics, and neuroscience, but can also be used for drug discovery.346 as well as vaccines against infectious diseases.347 With respect to RNAi, four RNAi-based therapies have been approved for use in patients, including patisiran for hereditary transthyretin amyloidosis, givosiran for acute hepatic porphyria, lumasiran for primary hyperoxaluria type 1, and inclisiran for heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease. These four approved RNAi-based compounds are based on small-interfering RNA (siRNA) technology, which decrease the level of specific mRNAs in order to reduce the corresponding protein levels. Currently, >50 clinical trials are investigating siRNA-based compounds. Unfortunately, however, no clinical trials are investigating ferroptosis-targeting agents using RNA-based technologies. Nevertheless, although the inability to precisely deliver RNA-based tools remains a major barrier to developing RNA therapies, new RNA-based modalities\u2014particularly CRISPR/Cas9-based gene editing\u2014will likely gain considerable traction in the near future.In addition to protein targets, a new line in the field of drug discovery focuses on RNA-based therapeutics, particularly for the management of genetic diseases. RNA-based therapeutics now include the use of messenger RNA (mRNA), RNAi, single-stranded antisense oligonucleotides, aptamers, ribozymes, and CRISPR-Cas endonuclease\u2012mediated gene editing. For example, mRNA-based therapies have been successfully used to develop personalized cancer vaccines,Peptide-based drugs represent a unique pharmaceutical niche between small-molecule compounds and biologicals. Compared to chemical compounds, peptide-based drugs are more efficient, safer, and better tolerated. This category of drugs has several other advantages as well, including high selectivity and reduced tissue accumulation. Biological drugs such as monoclonal antibodies bind with high specificity to their target proteins. Recently, bispecific antibodies\u2014which simultaneously target multiple antigens and/or epitopes using two specific antibodies\u2014have attracted considerable attention and may represent the next generation of antibody-targeted T cell\u2012based cancer immunotherapies. Furthermore, antibody-drug conjugates have high specificity and are highly efficient at delivering toxic small molecules in order to specifically target cancer cells.Ferroptosis is now generally accepted as playing a key pathogenic role in a wide range of diseases and conditions. As summarized in this review, a large amount of compelling evidence supports the notion that targeting ferroptosis can provide promising new options for the treatment of many ferroptosis-related diseases. As our understanding of ferroptosis-regulating pathways\u2014including iron metabolism, lipid metabolism, and oxidative-reductive pathways\u2014increases to reveal new druggable targets, ferroptosis modulators are likely to provide new opportunities to develop treatments for many currently incurable diseases. Although further studies are clearly needed, the novel technologies summarized in this review will facilitate the development of safer, more effective ferroptosis-targeted drugs."}
+{"text": "Ferroptosis is a form of regulated cell death characterized by iron overload, overwhelming lipid peroxidation, and disruption of antioxidant systems. Emerging evidence suggests that ferroptosis is associated with pregnancy related diseases, such as spontaneous abortion, pre-eclampsia, gestational diabetes mellitus, intrahepatic cholestasis of pregnancy, and spontaneous preterm birth. According to these findings, inhibiting ferroptosis might be a potential option to treat pregnancy related diseases. This review summarizes the mechanisms and advances of ferroptosis, the pathogenic role of ferroptosis in pregnancy related diseases and the potential medicines for its treatment. Cells are the fundamental organizing unit of life. Cell death, is therefore of critical importance in diverse aspects of mammalian development and homeostasis. Ferroptosis is a form of regulated cell death, coined in 2012. Generally, it is mainly characterized by iron overload, overwhelming lipid peroxidation, and disruption of antioxidant systems, particularly depletion of glutathione peroxidase 4 (GPX4) . MountinMammalian lipid bilayers consist of up to 62% of unsaturated fatty acids of which 35% are polyunsaturated fatty acids (PUFAs) . However2+ and H2O2 (Fenton reaction), subtract hydrogen from lipid to form lipid radicals (L\u2022) , which then interacts with adjacent PUFAs to form lipid hydroperoxide (LOOH), and many electrophilic species such as malondialdehyde (MDA), and 4-hydroxynonenal (4HNE) (The non-enzymatic phospholipid (PL) autoxidation is iron-dependent lipid peroxidation. Hydroxyl radicals, produced by the interaction of Feals (L\u2022) . After tl (4HNE) .in vivo model of acute renal failure, 12/15- LOX deletion cannot eliminate the cell death of GPX4 knockout mouse , a dioxygenase containing non-heme iron, has six isoforms in humans: 15-LOX-1, 15-LOX-2, 12-LOX-1, 12-LOX-2, E3-LOX, and 5-LOX . Althougut mouse . Therefout mouse . Similarut mouse . POR, idut mouse .2+ acts as a cofactor of LOXs or prolyl hydroxylase , which can be recognized by transferrin receptor-1 (TfR1) in the cell membrane. And then, Fe3+ endocytosis in endosomes, where it is reduced to Fe2+ by six-transmembrane epithelial antigens of the prostate 3 (STEAP3). Finally, Fe2+ is transported to the cytosolic labile iron pool via divalent metal transporter 1 (DMT1)  through the Fenton reaction . What\u2019s roxylase , which aroxylase . Consequ1 (DMT1) . Fe2+ alroptosis . It can roptosis . But ferroptosis . Ferroporoptosis , resistiThe SLC7A11-GSH-GPX4 axis is a classical signaling pathway of ferroptosis . GlutathThe ferroptosis suppressor protein 1 (FSP1)\u2013NAD(P)H\u2013coenzyme Q10 (CoQ10) pathway, acting in parallel to the SLC7A11-GSH-GPX4 pathway, is a potent suppressor of lipid peroxidation and ferroptosis . FSP1, kFerroptosis is associated with a great number of diseases. Multiple genes and signaling pathways, associated with lipid and iron metabolism, and antioxidant systems, play a role in inhibiting ferroptosis, providing new potential therapeutic drugs for these diseases .PUFAs, substrates of lipid peroxidation, are responsible for ferroptosis. Selectively bis-allylic deuterated PUFA, suppressing ferroptosis induced by RSL3 and erastin , is a pr2+ overload in intracellular iron pools, like endoplasmic reticulum (ER), may trigger ferroptosis H\u2013CoQ10 pathway, acting in parallel to the GPX4 axis, is a powerful antioxidant system in membrane structures . Idebeno2+ overload was a key mediator of ferroptosis , a ferroptosis inhibitor, decreased the mortality rate of trophoblasts  and high glucose (HG). Furthermore, it was found that HL and HG can induce GDM in pregnant rats, leading to the damage of rats\u2019 placenta  is common during pregnancy and is increasing in prevalence globally . The incplacenta . The expplacenta . Consequvia the Fenton reaction. As a result, oxidative damage leads to the injury and ferroptosis of pancreatic \u03b2\u2013cell in GDM  is a complication, most occurs in the third trimester, in 0.3%\u201315% of pregnancies in various populations . It is cExcessive ferroptosis occurred in spontaneous abortion rat model with low levels of GSH, GPX4 and increased levels of TFR1, ACSL4 and MDA . Some evvia significantly increasing GSH levels (2D3 is related to PRDs, which may result from ferroptosis, such as spontaneous abortion, GDM and PE (Trophoblast ferroptosis may provide a useful therapeutic target for pregnancy-related diseases. Quercetin, as an antioxidant, can significantly promote trophoblast invasion during early pregnancy H levels . AdditioH levels . Iron chH levels . SimilarH levels . ThiazolH levels . A recenH levels , indicatH levels . Thus, SH levels . CoQ10 iH levels . FurtherH levels . TherefoM and PE . VitaminM and PE . CertainFerroptosis is a form of regulated cell death involving lipid metabolism, iron metabolism and antioxidant system, regulated by multiple genes and signaling pathways. PRDs are mainly associated with placenta dysfunction due to trophoblasts injury and death. Recently, an increasing number of experimental studies are exploring role of ferroptosis in PRDs in order to provide new potential therapeutic drugs and therapeutic targets for it. However, there are numerous problems that have not been elucidated on the association between ferroptosis and pregnancy related diseases. Firstly, the exact molecular mechanism of transplacental iron transport is not clear, though much work has been done on it. Secondly, ferroptosis is a form of cell death that is associated with lots of signaling pathways, like hypoxia signaling , AMP-act"}
+{"text": "Conversely, insufficient ferroptosis has been linked to tumorigenesis. Furthermore, a recent study revealed the effect of ferroptosis on hematopoietic stem cells under physiological conditions. The regulatory mechanisms of ferroptosis identified to date include mainly iron metabolism, such as iron transport and ferritinophagy, and redox systems, such as glutathione peroxidase 4 (GPX4)-glutathione (GSH), ferroptosis-suppressor-protein 1 (FSP1)-CoQ10, FSP1-vitamin K (VK), dihydroorotate dehydrogenase (DHODH)-CoQ, and GTP cyclohydrolase 1 (GCH1)-tetrahydrobiopterin (BH4). Recently, an increasing number of studies have demonstrated the important regulatory role played by epigenetic mechanisms, especially DNA, RNA, and protein methylation, in ferroptosis. In this review, we provide a critical analysis of the molecular mechanisms and regulatory networks of ferroptosis identified to date, with a focus on the regulatory role of DNA, RNA, and protein methylation. Furthermore, we discuss some debated findings and unanswered questions that should be the foci of future research in this field.Ferroptosis is a form of programmed cell death characterized by elevated intracellular ferrous ion levels and increased lipid peroxidation. Since its discovery and characterization in 2012, considerable progress has been made in understanding the regulatory mechanisms and pathophysiological functions of ferroptosis. Recent findings suggest that numerous organ injuries ( Figure . Five years later (in 2008), the importance of ferrous iron, the system xc- (xCT) controlled cystine/cysteine redox cycle, and the glutathione peroxidase 4 (GPX4)-glutathione (GSH) redox system in lipid peroxidation and non-apoptotic cell death was highlighted by different research groupset al. demonstrated that histone deubiquitinase MYSM1  deficiency reduced the translation of ferroptosis-protective mRNAs, resulting in increased ferroptosis of human hematopoietic stem cells (HSCs), and HSC population maintenance was fully restored by the ferroptosis inhibitors , ferrostatin-1 (Fer-1), and vitamin E)Ferroptosis is regulated cell death driven by iron overload-triggered lipid peroxidation and is involved in a wide range of diseases, including cancers, cardiovascular diseases, degenerative diseases, infectious diseases, autoimmune diseases, and ischemia/reperfusion injury10, GTP cyclohydrolase 1 (GCH1)-tetrahydrobiopterin (BH4), dihydroorotate dehydrogenase (DHODH)-CoQ, and FSP1-vitamin K (VK)Figure . Additionally, epigenetic mechanisms such as DNA methylation, RNA methylation, protein methylation, and noncoding RNAs have gained increased attention. For instance, DNA methylation of key ferroptosis-related genes, such as GPX4, FSP1 and NFE2-like BZIP transcription factor 2 (NRF2) genes, has been shown to affect the expression of these genes, which participate in the regulation of ferroptosis6A modification regulators with ferroptosis-related genes have been established to effectively reflect the progression and prognosis of tumors6A modification can also regulate the fate of ferroptosis-related mRNAs, thereby controlling their protein expressionIn recent years, many key molecular mechanisms of ferroptosis have been identified, including GPX4-GSH, ferroptosis-suppressor-protein 1 (FSP1)-CoQ6A modification, and protein methylation and the underlying mechanisms of these effects on ferroptosis.Many reviews have summarized and discussed in detail the regulatory mechanisms, biology, and roles of ferroptosis in diseaseet al. demonstrated that various subcellular membranes undergo lipid peroxidation during ferroptosis, and the resulting lipid peroxides accumulate initially in the endoplasmic reticulum (ER) membrane and later in the plasma membraneFigure . For example, arachidonate 12-lipoxygenase, 12S type (ALOX12) inactivation abrogated p53-mediated ferroptosis in cancer cells, and ALOX12 also nullified p53-dependent inhibition of tumor growth in xenograft model miceFerroptosis is a form of programmed cell death regulated by multiple cellular metabolic pathways, such as iron metabolism, redox homeostasis, and mitochondrial activity, and executed by phospholipid hydroperoxides (PLOOHs), a form of lipid-based reactive oxygen species (ROS)Figure . In mammals, iron is mostly present in the form of heme, and heme oxygenase-1 (HO-1) catalyzes the degradation of heme into biliverdin, iron, and carbon monoxideLipid peroxidation in biological membranes is mediated by the nonenzymatic, iron-dependent Fenton reactionFigure . Nuclear receptor coactivator 4 (NCOA4) functions as a cargo receptor that mediates the delivery of iron-filled ferritin-H and ferritin-L to lysosomes via autophagy for breakdown, resulting in the release of ironInterestingly, the protein level of ferritin is regulated via a biological process named ferritinophagye.g., bafilomycin A1 and chloroquine) or knockout of autophagy-related gene 3 (ATG3) and ATG13, two core contributors to autophagy, reduced cell sensitivity to ferroptosis- activity and aggravates ferroptosisFigure . Nevertheless, more evidence is necessary to confirm a causal relationship between ferroptosis and autophagy and to indicate whether the factors in these processes show synergistic relationships. Furthermore, in any causal relationship that might be established, its dependence on specific treatments will need to be determined.In addition to ferritinophagy, upregulated microtubule-associated protein 1 light chain 3 II (LC3 II) expression and increased autophagic flux are observed during ferroptosis2\u2022- and H2O2 producing-proteins in signal transduction2O2 into H2OFigure .Lipid peroxidation has been identified as a key driver of ferroptosis; thus, the redox systems play a critical role in this process. More than 80-90% of total ROS are reportedly generated by mitochondrial electron chain Complexes I and III, and NADPH after oxidation by NOXs (NADPH oxidases) is another important source of O et al. demonstrated that depletion of glutathione causes the inactivation of GPXs in response to ferroptosis inducers. Furthermore, GPX4 overexpression inhibits the lethality of 12 ferroptosis inducers, which indicates that GPX4 is a central regulator of ferroptosishigh) have been observed to accumulate intracellular cystine when glucose levels are low, draining the cellular NADPH pool and leading to the aberrant accumulation of intracellular disulfides and subsequently to disulfidptosis, a previously uncharacterized form of cell death that differs from apoptosis and ferroptosisGPX4-GSH was the first known redox system to be implicated in the regulation of ferroptosis et al. generated a cDNA expression library derived from MCF7 cells (a ferroptosis-resistant cell line) and, through screening, discovered that the pro-apoptotic gene AIFM2 (apoptosis inducing factor mitochondria-associated 2) is a novel anti-ferroptotic gene, which they renamed \u201cferroptosis-suppressor-protein 1\u201d (FSP1)10, which is an antioxidant that prevents lipid peroxidation and ferroptosis10 is a novel anti-ferroptotic pathway that parallels to the canonical xCT-GPX4-GSH pathway. In addition, an FSP1-dependent noncanonical VK cycle has recently been reported to function as a ferroptosis suppressor, and FSP1 efficiently reduced VK to its hydroquinone VKH2 form, which function as a potent radical-trapping antioxidant to inhibit lipid peroxidation10 at the plasma membrane, mitochondrial CoQ10 is also reduced into CoQH2 in the mitochondrial inner membrane via the action of DHODH to attenuate ferroptosisR)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) synthesis, is a gene that antagonizes ferroptotic cell death4 is a critical cofactor for nitric oxide synthases (NOS); under BH4-deficient conditions, superoxide is generated instead of NO, increasing the susceptibility of cells to ferroptosis4 levels led to CoQ10 synthesis, thus alleviating oxidative damage and preventing ferroptosis et al. found that ferroptosis was non-cell-autonomously regulated by E-cadherin-mediated intercellular interactions, which repressed ferroptosis by activating intracellular NF2 (neurofibromin 2)-YAP  signalingAlthough xCT-GPX4-GSH is considered to be the primary mechanism that prevents ferroptosis, inhibition of GPX4 or GPX4 knockout fails to trigger ferroptosis in certain cancer cell lines, suggesting alternative resistance mechanisms6A modification and protein methylationMore importantly, epigenetic modifications are essential regulatory mechanisms for biological processes in the body, and recent studies have shown that ferroptosis is regulated by various epigenetic modifications, including DNA methylation, the RNA mFigure A. DNA methyltransferase 3A (DNMT3A), DNMT3B and DNMT3C are critical for de novo DNA methylation, which is maintained by DNMT1 through DNA replicationDNA methylation is an epigenetic modification that controls gene expression, typically occurring at the fifth position of the cytosine base in mammals to generate 5-methyl-cytosine (5mC). This modification is usually observed on cytosine-phosphate-guanine (CpG) dinucleotides, and approximately 60-90% of CpGs in the genome are methylatedet al. revealed that DNA methylation and somatic copy number alterations (SCNAs) may contribute to the differential expression of most ferroptosis regulator genes (FRGs) in tumorse.g., scavenger receptor class A member 5 (SCARA5), erythroferrone (ERFE), lipocalin 2, transferrin receptor 2 (TFR2), solute carrier family 11 member 1 (SLC11A1), and cytochrome B reductase 1 (CYBRD1)) were dysregulated in different cancers, and this outcomes may be associated with aberrant DNA methylation10, concomitantly increased the number of unsaturated fatty acyl chains in membrane phospholipids and decreased the number of long-chain saturated ceramides, which protected cells from ferroptosiset al. demonstrated that FADS1 and elongation of very long-chain fatty acid protein 5 (ELOVL5) were required to maintain intracellular levels of AA and AdA, and the expression of FADS1 and ELOVL5 was found to be frequently inhibited by an increase in the DNA methylation marks on at their promoter/enhancer regions in intestinal-type gastric cancer cells (GCs), which resist ferroptosisBy analyzing the data in The Cancer Genome Atlas, Liu Nrf2 promoter hypermethylation is observed in both COPD patients and CSE-treated HBE cells, resulting in NRF2 downregulation, inhibited GPX4 expression and ferroptosis occurrenceet al. found increased DNA methylation levels on several CpG nucleotides upstream of miR-4715-3p in upper gastrointestinal adenocarcinoma (UGC) tissue samples, which binds to the 3' untranslated region (3' UTR) of Aurora kinase A (AURKA) to inhibit its expressionFsp1 gene has been shown to be hypermethylated in T- and B- acute lymphoblastic leukemia cell lines and patient samples, in which it suppressed FSP1 expression and promoted ferroptosisFsp1 promoter prevented NRF2 from binding to its promoter and inhibited transcriptionPink1 (PTEN induced kinase 1) promoter, inhibiting PINK1 expression and promoting ferroptosis and brain damageGPX4 has been reported to be overexpressed in cancer tissues compared to normal tissues and to be associated with low levels of DNA methylation and enrichment of H3K4me3 and H3K27ac marks at its promoter, suggesting that epigenetic regulation may be involved in its aberrant overexpressionFigure B. Generally, hypermethylation in the promoter or transcriptional start site or hypomethylation in the coding sequence of a gene suppresses the expression of that gene. However, it should be noted that the expression of a methylated gene can be increased when either the coding sequence is hypermethylated or the promoter or transcriptional start site is hypomethylatedStudies have indicated that DNA methylation is critical for ferroptosis, as these marks regulate the expression of ferroptosis-related genes 6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), N3-methylcytosine (m3C), N7-methylguanosine (m7G), and pseudouridine (\u03a8), have been identified6A is one of the most abundant modifications of cellular RNA6A marks are usually located in 3' UTRs, although there are also a few m6A sites located in regions surrounding start codons and 5' UTRs6A marks are deposited by a complex comprising methyltransferase-like 3 (METTL3), METTL14, Wilms' tumor 1-associated protein (WTAP), and KIAA1429, with METTL3 being the major subunit with the methyltransferase activity and acting as the \u201cwriter\u201d6A mark6A-methylated RNA is recognized by \u201creaders\u201d, such as YT521-B homology (YTH) domain family proteins  and heterogeneous nuclear ribonucleoprotein family proteins (HNRNPA2B1 and HNRNPC), which determine the fate of the marked RNA, such as degradation, translation or splicingFigure A. Furthermore, the role of the RNA m6A modification in many biological processes and diseases has been elucidated in the past decade; these processes include autophagy, cell proliferation, stem-cell renewal and differentiation, and the diseases include tumorigenesis, and cardiovascular diseases6A marks are involved in the regulation of ferroptosis and thus affects the progression of diseases such as tumor growth and acute kidney injury6A modification regulators used in combination with ferroptosis-related genes can be diagnostic or prognostic markers for tumors in humansTo date, more than 100 RNA modifications, including N6A modification regulators have been shown to be aberrantly expressed in various types of tumors, and a prediction model comprising these regulators and ferroptosis-associated genes has been shown to effectively reflect the progression and prognosis of these tumor patients6A methylation-dependent manner6A modification of SLC7A11 mRNAs, which were then recognized by YTHDF2 to mediate their degradation6A methyltransferase, installs m6A modification on the 5'UTR of SLC7A11 mRNAs to facilitate their degradation in a YTHDF2-dependent manner, and hypoxia induces the downregulation of METTL14 by regulating HIF-1\u03b1 levels, thus protecting hepatocellular carcinoma (HCC) cells from ferroptosis6A reader, and it was shown to compete with the BTG2/CCR4-NOT complex to bind PABPC1, thereby inhibiting SLC7A11 mRNA deadenylation and promoting its stability, resulting in increased SLC7A11 expression and the eventual inhibition of ferroptosis6A demethylase FTO has been shown to inhibit SLC7A11 expression and thus trigger ferroptosis in an m6A-independent manner, attenuating the development of papillary thyroid carcinoma6A modification has also been observed to participate in ferroptosis by affecting the mRNA splicing of SLC7A116A-methylated SLC7A11 mRNA and then recruit the splicing factor proline and glutamine-rich (SFPQ), which recognize the splice site and thus promotes SLC7A11 mRNA splicing and maturation, ultimately preventing the ferroptosis of glioblastoma cells6A modification of SLC7A11 mRNA is critical for its expression regulation and ferroptosis. However, some results describing the impact of m6A on SLC7A11 expression are debated, and further investigation is needed. For example, more studies need to be conducted to explore the differences in m6A methylation sites in SLC7A11 mRNAs under different conditions, and these marks deposited on different sequence may lead to different fates of mRNAs after being recognized by different readers.Multiple m6A methylation. For example, in patients with lung adenocarcinoma (LUAD), the m6A reader insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is highly expressed and binds to m6A-marked mRNAs encoding anti-ferroptosis regulators ) to increase the stability of these mRNAset al. demonstrated that SLC3A2 is not directly regulated by the m6A modification of its mRNA but is positively regulated by the transcription factor homeobox A13 (HOXA13), whose mRNAs are m6A-methylated on its 3'UTR, and this modification is recognized by YTHDC2, which causes the degradation of HOXA13 mRNAs in LUAD cells6A methylation-dependent and -independent manner. GPX4 is another critical ferroptosis regulator6A modification on GPX4 mRNAs, increasing their stability and resulting in upregulation of GPX4. Enhanced GPX4 expression accelerates the proliferation and inhibits the ferroptosis of breast cancer cells6A methylation of GPX4 mRNAs, inhibiting GPX4 expression and increasing the ferroptosis of alveolar epithelial cells6A marks on the regulation of GPX4 expression under different pathological conditions. Do different methylation sites or the activity of different readers lead to different fates of methylated mRNAs?In addition to SLC7A11 mRNAs, the mRNAs of its partner SLC3A2 is also regulated by the m6A marks. For example, in non-small cell lung carcinoma (NSCLC) cells, exosomal miR-4443 targets METTL3 expression and negatively regulates METTL3-mediated m6A modifications on FSP1 and ferroptosis6A demethylation at two m6A residues in the 3'UTR of NFE2L2/NRF2 mRNAs, which are recognized by IGF2BP2, contributes to reduced mRNA stability and decreased NFE2L2/NRF2 expression in hypopharyngeal squamous cell carcinoma cells6A marks on the 3'UTR of NRF2 mRNA, and these marks are read by YTHDF1, enhancing the stability of NRF2 mRNA and inhibiting ferroptosis in bladder cancer cells6A modification on the mRNAs of the autophagy-related gene BECN1 results in these m6A-marked mRNAs resistant to degradation after recognition by YTHDF1, contributing to dihydroartemisinin-induced hepatic stellate cell ferroptosis and conferring protection against liver fibrosis6A-modified CBS (cystathionine \u03b2-synthase) mRNAs, leading to decreased mRNA stability and reduced CBS expression. Decreased CBS results in reduced methylation of the ACSL4 protein and facilitates polyubiquitination and degradation of ACSL4 via the ubiquitination-proteasome pathway, ultimately protecting gastric cancer against ferroptosisWith research advances in this field, the mRNAs of multiple ferroptosis regulators have been shown to modified with m6A modification is involved in ferroptosis by regulating the mRNA stability and protein expression of multiple ferroptosis-related genes Figure B. However, there are still at least three scientific questions that must be answered. First, what are the mechanisms underlying the RNA m6A modifications involved in ferroptosis? Second, what are the mechanisms by which specific m6A modification sites are recognized by different readers, which determine different fates of mRNAs after ferroptosis inducers treatment? Third, does targeting m6A marks to regulate ferroptosis has potential as a treatment for related diseases?Therefore, these aforementioned studies suggest that the RNA m Figure A and 5C. Histone methylation is the most common type of protein methylation, such as methylation of H3K27, H3K4, H3K9, H3K36, H4K20, and H4R3e.g., programmed cell death, proliferation, and autophagy) and diseases Protein methylation occurs mainly on lysine and arginine residues, and protein lysine methyltransferases (PKMTs or PLMTs) and protein arginine methyltransferases (PRMTs) are responsible for the addition of methyl groups to these residues177 et al. showed that the level of H3K9me2 was significantly increased in humans with multiple sclerosis and in a murine experimental autoimmune encephalomyelitis model. G9a suppressed anti-ferroptotic genes , reduced intracellular GSH levels, triggering ferroptosis and resulting in neuronal damage, while targeting G9a by its inhibitor UNC0642 alleviated inflammation-induced neurodegenerationSnai1, which inhibited the protein expression of Snai1, the main transcription factor that initiates the epithelial-mesenchymal transition (EMT). Overexpression of SETDB1 inhibited the EMT and caused ferroptosis, preventing pulmonary fibrosis significantly inhibits ferroptosis and is expected to be a potential treatment for degenerative diseases characterized by cell loss . On the other hand, enhancing H3K9 methylation can treat the diseases with excessive cell proliferation such as tumors by promoting ferroptosis.Our recent studies showed that compared to those in control cells, the protein levels of H3K9me1, H3K9me2, and H3K9me3 were increased in HASMCs treated with ferroptosis inducers . Further screening of the effects of several histone methyltransferase inhibitors on ferroptosis revealed that BRD4770, an inhibitor of H3K9me1/2/3, significantly inhibited ferroptosis in HASMCs, and its effect was comparable to that of Fer-1, a classical ferroptosis inhibitor. More importantly, BRD4770 delayed the progression of \u03b2-aminopropionitrile monofumarate (BAPN)-induced aortic dissection in mice by inhibiting the inflammatory response, lipid peroxidation, and ferroptosiset al. demonstrated that EZH2 mitigated the ferroptosis of tongue squamous cell carcinoma cells by suppressing miR-125b-5p but upregulating SLC7A11 expressionH3K27 methylation is an important histone mark that suppresses gene expression and is primarily catalyzed by enhancer of zeste homolog 2 (EZH2)In contrast, SET7 attenuated the inhibitory effect of OTUB1  on cystine starvation/erastin-induced ferroptosis by directly interacting with non-histone OTUB1 to catalyze its methylation on lysine 122. Interestingly, this effect is not achieved by affecting the deubiquitinase (DUB) activity of OTUB1 but by impairing its noncanonical activity, binding to the E2 enzyme UBC13In addition to lysine methylation, arginine methylation also plays a critical role in ferroptosis. For example, protein arginine methyltransferase 1 (PRMT1) has been reported to function as a suppressor of ferroptosis by increasing the level of asymmetrically dimethylated histone H4 on Arg 3 (H4R3me2a) to downregulate the expression of ACSL1 and then further decrease the lipid peroxidationFigure B and 5D. However, there are still few studies in this research field, and in-depth studies are needed to elucidate the relationship between protein methylation and ferroptosis and to develop strategies for targeting protein methylation to modulate ferroptosis and treat disease. Moreover, to date, there are few reports on the correlation between non-histone methylation and ferroptosis. For example, although the relationship between P53 and ferroptosis has been well establishedThese studies have indicated that protein methylation, such as H3K9 and H3K27 methylation, is a critical epigenetic mechanism in the regulation of ferroptosis 6A modification sites in ferroptosis, and how do RNA m6A modification sites and reader proteins jointly regulate the fate of related mRNAs in this context? Fourth, are methylated non-histone proteins, in particular those non-histones that have been linked to ferroptosis, involved in the regulation of ferroptosis? Fifth, how do other epigenetic modifications form a regulatory network with DNA, RNA, and protein methylation to regulate ferroptosis? Sixth, further exploration is needed to determine the potential of targeting DNA, RNA, or protein methylation to regulate ferroptosis and thereby treat and prevent disease. Moreover, whether combined inhibitors/inducers of ferroptosis and inhibitors/agonists of biomolecular methylation can function synergistically to mitigate related diseases needs to be explored in depth. Answers to these questions will not only advance our understanding of ferroptosis but will also provide important new insights into the biological functions related to DNA, RNA, and protein methylation.In the present review, we summarize the basis of ferroptosis and the evidence acquired to date to show how DNA, RNA, and protein methylation regulate ferroptosis. It is clear that methylation modification of all three types of molecules is essential for the regulation of ferroptosis and that these modified molecules exert influence mainly by regulating the expression of ferroptosis-associated proteins. There are at least six key unanswered scientific questions for which the answers are likely to drive advances in understanding the epigenetic regulation of ferroptosis in the near future. First, how do DNA, RNA and protein methylation mutually regulate and synergistically influence ferroptosis under certain conditions, and what is the spatiotemporal regulatory relationship among them? Second, what are the mechanisms by which DNA methylation in different regions during ferroptosis regulates related gene expression? Specifically, how methylated DNA in coding regions affects the expression of these genes should be investigated further. Third, what determines the specificity of the RNA m"}
+{"text": "Gut microecology is an important component of human health and environment. It\u2019s composed of gut microbiota, gut epithelial cells, immune system, which forms gut mucosal barrier and act in energy metabolism. Gut microbiota plays an important role in the proper functioning of human organisms, it coevolves and symbiosis with humans by combating pathogenic bacteria, assisting in nutrient digestion, maintaining the integrity of the gut epithelia, and promoting immunological development. The steady state of the gut microbiota is closely related to human health and both external and genetic factors affect its composition and function. Thus, gut microbiota-mediated immunomodulatory effects play an important role in diabetes mellitus and tumor immunity. However, the causal relationship between an altered gut microbiota composition and diabetes mellitus or tumor immunity remains to be elucidated. Host immune responses are an extremely complex process and an in-depth exploration of the gut microbiota-host-disease relationship will be helpful to identify potential therapeutic targets for diabetes mellitus and cancer. We encourage the exploration of the etiology and factors influencing diabetes mellitus and tumor immunity from a gut microecological perspective with the aim of addressing these challenging questions. In this Research Topic, we aimed to explore gut microbiota profiles and the specific processes by which bacterial components, metabolites, and other mediators of gut microbiota interact with the host to affect the immunity of patients with diabetes mellitus or cancer.Ye et\u00a0al.). In addition, the differences in gut microbiota between type 1 diabetes mellitus and type 2 diabetes mellitus patients and the role of gut microbiota imbalance in the pathogenesis of diabetes patients, are discussed, especially with regard to the incidence of leaky gut syndrome, immune dysfunction, and metabolic disorders. They also summarized the progress made in developing microbial therapies to prevent and treat diabetes, especially the current status and application prospects of fecal microbiota transplantation in diabetes. Liu et\u00a0al. expanded our understanding of the relationship between gut microbiota, an immune checkpoint inhibitor (ICI) response, and immune-related adverse events (irAE) occurrence. They studied the gut microbiota of patients who experienced a series of irAE in different cancers, particularly lung cancer; 1) with and without irAE; 2) with different severity of irAE; 3) with differences in microbiota composition between patients with and without irAE associated with colitis. Moreover, they explored the causal relationship between microbiota composition and immune-related colitis. Subsequently, their assessment of colitis and dynamic microbiota analysis led to more fundamental questions such as whether differences in microbiota cause immune-related colitis and whether immune-related colitis disrupts the gut microbiota. Huang et\u00a0al. sequenced the ulcerated and intact skin of patients with diabetes and found that skin had significantly higher bacterial richness and diversity than diabetic foot wounds. The microecological balance of the skin was disrupted, and pathogenic bacteria replaced the original microbiota. It is suggested that commensal bacteria of intact skin may be important in maintaining microecological balance and preventing the development of diabetic skin ulcers.This Research Topic included correlation analysis of gut microbiota composition with diabetes mellitus and tumor immunity. This review confirmed the differences in gut microbiota between healthy individuals and diabetic patients , explaining why it can be a treasure trove of potential probiotics, and may shed light on the mechanisms of action underlying TCM successes in disease treatment. To further investigate the effect of TCM on different levels of gut microbial abundance. Lin et\u00a0al. described gut microbiota characteristics of patients who were first diagnosed with diffuse large B-cell lymphoma (DLBCL), aiming to explore the correlation between immune indicators affecting patients and to clarify the role of the microecosystem-immune axis in the occurrence and development of DLBCL. In the future, it may be possible to improve the immune function of DLBCL patients by regulating the gut microbiota, increasing therapeutic efficacy and improving patient survival rates. Moreover, their study also revealed a correlation between changes in microbiota structure and host immunity, and this research suggests a better understanding of the specific mechanisms underlying the development of differential and dominant microbiota in DLBCL in future studies to identify new disease biomarkers and develop new therapeutic strategies.This special issue also discussed gut microbiota-associated molecules that can affect host immune responses; Yan et\u00a0al. established and validated a diagnosis model based on microbial amplicon sequence variants markers for light chain(AL) amyloidosis in China, and initially explored the important role of gut microbiota in other types of amyloidosis. Their characterization of gut microbial communities in AL amyloidosis patients revealed that alterations in the abundance of certain gut microbial species may play a crucial role in regulating metabolic function and inflammatory responses. They further revealed the association between baseline gut microbiota and disease severity, response to treatment, and prognosis. Subsequently, they suggested that the effect of the gut microbiota on clonal plasma cell proliferation may be the focus of future studies. To further investigate this relationship between gut microbiome, immunity, and complications in diabetics, these authors established a diabetic cornea wound healing model in rodents . They demonstrated that alterations in microvascular complications such as diabetic keratopathy and immune responses may potentially correlate with alterations in gut microbiota composition in diabetic patients. They were pleasantly surprised to find that these metabolic effects could be transferred to healthy lean individuals using a fecal transplant technique to produce a similar state of insulin resistance. Thus, the gut microbiota may be an important target in the treatment of metabolic syndrome and type 2 diabetes mellitus. The meta-analysis by Yan et\u00a0al. showed differences in enterobacteriaceae in gestational diabetes mellitus (GDM) and non-GDM populations, and explored inflammation and possible immune mechanisms associated with the pathophysiology of this disease. The results showed specific changes in the composition of the gut microbiota in GDM patients. This suggests that we can provide bacterial targets for the prevention or treatment of GDM by rebuilding homeostasis of the gut microbiota.This Research Topic also discussed the mechanisms whereby specific gut microbiota can affect the development of diabetes mellitus and tumor immunity; Mao et\u00a0al. used gut microbiota as a target to investigate differences in gut microbiota diversity between diabetic kidney disease (DKD) patients and non-DKD patients. Furthermore, the combined application of metagenomic and metabolomic approaches verified that a large number of metabolites produced by gut microbiota and circulating metabolites are key signaling molecules and substrates involved in metabolic reactions in the progression of DKD. In a surprising result, they confirmed the existence of an enteric-renal axis. The exploration of the molecular mechanism or pathway involved in DKD may be beneficial to the development of individualized prevention and treatment options. It is well known that short-chain fatty acids (SCFAs), a type of saturated fatty acid, are produced by the fermentation of the gut microbiome. Tao et\u00a0al. discussed the role of gut microbiota imbalance in the pathogenesis of diabetic nephropathy under the influence of SCFAs. At the same time, it may also underlie immune response by gut microbiota imbalance in diabetic patients, which damages the structure and function of the kidney. They also discussed the role of SCFAs in regulating energy metabolism, the inhibition of inflammation, and oxidative stress, and the regulation immune response. Thus, they emphasize that SCFAs may be valuable in the diagnosis and treatment of DKD.The final contribution to this Research Topic centers on the molecular mechanisms whereby bacterial components, metabolites, and other mediators of gut microbiota, interact with the host immune system; The importance of the gut microbiota is gradually being recognized by the field of microbiology due to the huge diversity seen in both its quantity and composition, and its ability to regulate metabolic organs. The reviews and original research in the present collection expand our understanding of the role of the gut microbiota and its metabolites as a critical endocrine organ, with a particular focus on the relationship between the gut microbiota and host diseases. We anticipate that future clinical validation of these findings and more detailed stratified analyses will identify novel biomarkers and disease-causing mechanisms that may permit prophylactic and/or therapeutic intervention.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."}
+{"text": "The digestion of food and the absorption of nutrients and other bioactive substances are mainly completed in the stomach and small intestine. The remaining food residue will enter the large intestine and be further decomposed by the microbial communities residing there. This procedure will release more nutrients from food and produce new products that may be beneficial or harmful to the host, and have a significant impact on the host's gut and overall health. Therefore, the intestinal microbiota is now considered an integral part of the body that plays an important role in the development of many local and systemic diseases, such as inflammatory bowel disease, colorectal cancer, obesity, type 2 diabetes, and Alzheimer's disease .In recent years, many scientists have shifted their interest to the interaction between diet, gut microbiota, and host health, and proposed to prevent diseases by regulating the composition of gut microbiota. In many experimental and clinical studies, dietary approaches to prevent diseases, such as adding probiotics, prebiotics, or synbiotics to food, have been proven to be safe and effective. The mechanisms by which specific foods or food components regulate specific bacterial species in the gut, the genes responsible for bacterial activity, the bioactive products they produce, and the pathophysiological effects of these products in specific bodily organs are increasingly being recognized. However, there are still many knowledge gaps in this field. For example, if substances from food sources can be absorbed without the help of the gut microbiota, it is difficult to distinguish between their direct effects and those mediated by the so-called \u201cgut-X axis.\u201d It is also very difficult to elucidate the exact mechanism(s) by which gut bacteria mediate the impact of food components on host pathophysiological responses even with the help of so many \u201c-omics\u201d technologies.Kadyan et al. found that resistant starch (RS) extracted from four different dietary pulse varieties can suppress aging-related metabolic disorders by regulating the gut microbiome of mice and ameliorating high-fat diet-induced gut leakiness and inflammation. Interestingly, they find that the impact of RS is food source- and gender-specific, and called for deeper research on the complexity of RS from aspects such as botanical origin, microstructure, impact on the gut microbiome, and the resulting pathophysiological responses of different genders . Li et al. show that the fermentation of Chinese medicinal food Astragalus membranaceus (FA) has produced many new bioactive chemicals, significantly enhancing its preventive effect on dextran sulfate sodium (DSS)-induced colitis and dysbiosis in mice. The correlation between key gut microbes that produce short-chain fatty acids (SCFA), such as Akkermansia and Alistipes, and colitis parameters explains the role of FA in balancing the immune responses and repairing damaged intestinal mucosa . The authors also acknowledged that it is difficult to distinguish between the effects of FA and those of FA-affected gut microbiota, which is a common problem faced by research aimed at establishing the mediating role of gut microbiota in response to oral substances, as they cannot rule out the direct effect of FA absorption into the body and exerting a systemic effect before encountering the gut microbiota. Zhang et al. observed the preventive effect of supplementing two types of algal oil on antibiotic-induced opportunistic pathogen infection and inflammation. They indicate that Parabacteroides and Melissococus were the main culprits of gut leakiness and inflammation caused by ceftriaxone sodium (CS) while supplementing with algal oil increased the abundance of a cohort of beneficial bacteria, reduced the bad ones, and repressed CS-induced inflammatory damages. They further analyzed the correlation between changes in gut microbiota and the fatty acid composition of algal oil and revealed interesting results .To add insights to this field, in this e-book, the role of diet, food, or food components in shaping the gut microbiota and preventing lifestyle-related diseases was studied in various experimental models, with a focus on changes in gut barrier function and the inflammatory reactions induced during this process. Bakshi et al. reviewed the formulation and production of IF and discussed its impact on the establishment of infant gut microbiota and its short- and long-term health consequences.To prevent lifestyle-related chronic diseases in adulthood, it has been proposed to use infant formula (IF) containing probiotics and prebiotics to help newborns establish a healthy gut microbiota. Therefore, In summary, in a world where adding bioactive ingredients to food products to nurture a robust and balanced gut microbiota and promote consumer health is increasingly becoming a trend, we should invest more in this research field to generate more practical insights to elevate our understanding of this hot topic and help us find better-targeted, personalized approaches for improving health, delaying aging and reducing the burden of disease.P-GL: Writing\u2014original draft, Writing\u2014review & editing. Z-WH: Writing\u2014review & editing."}
+{"text": "Lactobacillus and Bifidobacterium), prebiotics , synbiotics, postbiotics , dairy products, spices , fruits, vegetables, medicinal herbs, and so on, could exert protective effects against mental disorders by enhancing beneficial gut microbiota while suppressing harmful ones. In this paper, the mental disorder-associated gut microbiota are summarized. In addition, the protective effects of dietary components on mental health through targeting the gut microbiota are discussed. This paper can be helpful to develop some dietary natural products into pharmaceuticals and functional foods to prevent and treat mental disorders.The number of individuals experiencing mental disorders  has significantly risen in recent years. Therefore, it is essential to seek prevention and treatment strategies for mental disorders. Several gut microbiota, especially Firmicutes and Bacteroidetes, are demonstrated to affect mental health through microbiota\u2013gut\u2013brain axis, and the gut microbiota dysbiosis can be related to mental disorders, such as anxiety, depression, and other mental disorders. On the other hand, dietary components, including probiotics (e.g.,  Bacteroides uniformis, Roseburia inulinivorans, Eubacterium rectale, and Faecalibacterium prausnitzii exerted a positive effect on the maintenance of mental health by producing short-chain fatty acids (SCFAs) and regulating amino acid, taurine, and cortisol metabolism pathways [Mental health is one of the United Nations\u2019 Sustainable Development Goals, and mental disorders mainly include anxiety, depression, bipolar disorder, autism spectrum disorder (ASD), schizophrenia, and eating disorders . In 2019pathways . Additiopathways ,19. Therpathways . Therefopathways ,23,24,25pathways ,26,27. Fpathways . Anotherpathways .In this narrative review, we conducted a comprehensive search of Web of Science Core Collection and PubMed databases, gathered relevant high-quality literature published within the past five years, and retrieved the keywords of anxiety, depression, bipolar disorder, autism spectrum disorder, schizophrenia, mental disorder, mental health, gut microbiota, probiotics, prebiotics, postbiotics, dairy product, spice, fruit, vegetable, medicinal herb, and natural product. In this review paper, the relationships between gut microbiota and mental disorders are first summarized, followed by a discussion of the impacts of natural dietary products on mental health by regulating the gut microbiome, emphasizing the underlying mechanisms. This review paper could be helpful for people to make informed choices regarding natural dietary products for the prevention and management of mental disorders, and it may also promote the development of natural dietary products by the industry as pharmaceuticals and functional foods to maintain mental health. The composition of gut microbiota is complex; some microorganisms may protect mental health, while others may be related to the onset and development of mental disorders. In the following part, the association of gut microbiota with certain mental disorders is summarized below, and more detailed information can be found in Prevotella was increased, while the Firmicutes/Bacteroidetes ratio and the abundance of Faecalibacterium spp. were significantly reduced in individuals with social exclusion [Firmicutes spp. and microbiota that produce SCFAs, but more Fusobacteria and Bacteroidetes [Prevotella_9 and Lachnospira, as well as immunoglobulin proteins, but had an increase in the abundances of Lactobacillales, Sellimonas, Streptococcus, and Enterococcus [Firmicutes [Anxiety is among the most common mental disorders . It is ixclusion . Moreovexclusion . Additioroidetes . In addirococcus . Moreoverococcus . Anotherrmicutes .Firmicutes, but higher Bacteroidetes and Fusobacteria. At the genus level, several gut microbiota genera were positively correlated with anxiety, such as Prevotella, Lactobacillales, Sellimonas, Streptococcus, and Enterococcus, while some gut microbiota genera were inversely correlated with anxiety, suggesting that targeting these gut microbiomes could be a promising approach for preventing anxiety. At present, most studies focus on the genus level of anxiety-related gut microbiota. In the future, more research should highlight the species level of gut microbiota, considering that different species in the same genera could exert different functions, or even the opposite functions, on anxiety.In brief, patients/mice with anxiety showed dramatically decreased microbial richness and diversity. At the phylum level, anxiety patients/mice usually had lower Dialister and Coprococcus spp. were decreased in patients with depression [Prevotella, Klebsiella, Streptococcus and Clostridium XI, but lower levels of Bacteroidetes [Helicobacter pylori (H. pylori) infection could induce a pathological state of gastrointestinal flora. For instance, a cross-sectional study including 5558 Chinese people found a significantly higher risk of depressive symptoms in women infected with H. pylori, but not in men [H. pylori and cancer [Klebsiella aerogenes) compared to healthy controls [Dialister sp., which could modulate the immune and metabolic activity of the host.Depression is considered a major public health problem . Depresspression . Furtherroidetes . Moreovet in men . This cod cancer . In addicontrols . Furthercontrols . Anothercontrols . Anothercontrols . AdditioPrevotella, Klebsiella, and Clostridium, showed a positive relationship with depression. Moreover, gut microbiota dysbiosis might be a crucial factor in the pathogenesis of depression via influencing the protein expression in tissues related to the gut\u2013brain axis. Although the specific gut microbiota of people with depression varied from study to study, all these discoveries showed that gut microbiota composition significantly changed in depressive individuals and indicated that gut microbiota might be a novel target for the prevention and management of depression. Similar to the anxiety mentioned above, most of the studies about gut microbiota related to depression have been mainly at the genus level in recent years. More research studies should be conducted at the species level to further investigate the connection between gut microbiota and depression, since the effects of different species of gut microbiota in the same genera on depression could be different or opposite. In a word, gut microbiota in individuals/animals with depression were found to differ in composition and abundance from those in healthy controls. At the family level, certain gut microbiota were positively correlated with depression, such as Paraprevotella, but some others were negatively associated with depression, such as Streptococcaceae and Gemella. At the genus level, several gut microbiota genera, such as Clostridiaceae and Collinsella [Flavonifractor was associated with bipolar disorder (odds ratio (OR), 2.9; 95% CI, 1.6\u20135.2) [Faecalibacterium significantly decreased in bipolar disorder patients [Bifidobacterium (phylum Actinobacteria) or Lactobacillus bacterial counts between the two groups of 39 bipolar disorder patients and 58 healthy individuals [Bipolar disorder is a chronic and incapacitating illness that often reoccurs, leading to cognitive and functional impairment . Severallinsella . Additio1.6\u20135.2) . Anotherpatients . Moreovepatients . Howeverividuals . The disClostridiaceae, Collinsella, and the phyla of Actinobacteria and Coriobacteria, but lower abundances of Faecalibacterium and Ruminococcaceae. Further large-scale population studies are required in the future to investigate the impact of various gut microbiota on bipolar disorder, and the participants should be more representative, such as more races from different countries with different gender as well as age. In general, bipolar disorder patients had higher abundances of Clostridium bolteae than their unaffected family members [Clostridium paraputri, Clostridium bolteae, and Clostridium perfringens were found in the feces of Egyptian ASD children. In addition, Clostridium diffiicile and Clostridium clostridiioforme were only found in ASD children, while Clostridium tertium was only found in normal children [Actinobacteria, Proteobacteria, as well as Bacilli [Fmrl KO mice with autistic-like behaviors had a reduced population of Akkermansia muciniphila, accompanied with increased levels of TNF-\u03b1 and Iba1 [Faecalibacterium and Agathobacter. These bacterial strains were positively linked to the levels of 3-hydroxybutyric acid and melatonin, but negatively correlated with the level of serotonin [ASD is a heterogeneous neurodevelopmental disorder . Researc members . Anotherchildren . Moreove Bacilli . Additioand Iba1 . Furthererotonin . Clostridium diffiicile and Clostridium clostridiioforme. Moreover, certain gut microbiota were increased in ASD patients/animals, such as Actinobacteria, Proteobacteria, and Bacilli, whereas some were decreased, such as Akkermansia muciniphila, Faecalibacterium and Agathobacter. In the future, more studies about the association between gut microbiota and ASD need to be conducted to find out more beneficial or harmful gut microbiota, which could be targeted for the management of ASD.Overall, several species of gut microbiota were only found in ASD patients, such as Schizophrenia affects 1% of the world\u2019s population, and patients with schizophrenia may exhibit positive symptoms like hallucinations and disorganized speech, negative symptoms like an absence of interest and motivation, and cognitive deficits like impaired executive functions and memory . Haemophilus [Haemophilus abundance and negative symptoms of schizophrenia [Ruminococcus and Roseburia, as well as an increased abundance of Veillonella [Lachnospiraceae [Lactobacillus fermentum and Enterococcus faecium [Proteobacteria, but lower abundances of Faecalibacterium and Lachnospiraceae [Succinivibrio was positively correlated with the severity of schizophrenia symptoms, while the abundance of Corynebacterium was negatively related to the negative symptoms [Mounting evidence indicated that schizophrenia was associated with gut microbiota dysbiosis. For instance, a case-control study showed that gut microbiome dysbiosis was found in schizophrenia patients . A crossmophilus . Howeverophrenia . The disllonella . Anotherpiraceae . Additio faecium . Anotherpiraceae . In addisymptoms . Additiosymptoms .Lactobacillus fermentum, Enterococcus faecium, and Alkaliphilus oremlandii, could be only found in patients with schizophrenia. Some gut microbiota were positively correlated to the severity of schizophrenia, such as Lachnospiraceae, Veillonella, Collinsella, Lactobacillus, Succinivibrio, and Corynebacterium, whereas some were negatively correlated with schizophrenia, such as Coprococcus, Ruminococcus, Roseburia, Adlercreutzia, Anaerostipes, and Faecalibacterium. Furthermore, the gut microbiota could affect neurochemistry and neurologic function through modulating the metabolism of enteric and central nervous system function-related molecules, such as GABA. A supplement of beneficial gut microbiota, which were absent in patients with schizophrenia, might be useful for the treatment of schizophrenia. Alternatively, the reduction in harmful gut microbiota, which were only found in patients with schizophrenia, could be another therapeutic strategy via the administration of medicine or functional foods. Collectively, some special gut bacteria, such as Eubacterium hallii and Bacteroides eggerthii were correlated to the reappearance of post-traumatic stress symptoms in frontline healthcare workers during the COVID-19 pandemic [Mitsuokella, Odoribacter, Catenibacterium, and Olsenella [Fusicatenibacter saccharivorans [Agathobacter, Anaerostipes, and Lachnospiraceae, as well as the plasma level of TNF-\u03b1 [In addition to the mental disorders mentioned above, many other mental disorders are linked to the gut microbiota, such as anorexia nervosa, PTSD, and attention-deficit/hyperactivity disorder (ADHD). For example, it was reported that patients with anorexia nervosa had a reduction in Bacteroidetes and gut microbiota dysbiosis, contributing to anorexia nervosa-specific pathologies . Anotherpandemic . Anotherlsenella . A studyrivorans . Additioof TNF-\u03b1 . Anotherof TNF-\u03b1 .In short, some gut microbiota were associated with some mental disorders, such as anxiety, depression, bipolar disorder, ASD, schizophrenia, anorexia nervosa, PTSD, and ADHD , which cMany dietary components have been shown to exert protective effects against mental disorders through regulating gut microbiota, such as probiotics, prebiotics, postbiotics, fruits, vegetables, and spices, which are discussed below and are shown in Lactobacillus, could prevent and manage several mental disorders by modulating the gut microbiota. For instance, a study showed that Lactobacillus murine (L. murine) and L. reuteri could increase GABA content in the hippocampus and alleviate depression-like behaviors in Dcf1 knockout mice [L. rhamnosus zz-1 alleviated depression-like behaviors in mice induced by chronic unpredictable mild stress (CUMS) improved hypothalamic\u2013pituitary\u2013adrenal (HPA) axis hyperactivity, and increased monoamine neurotransmitters, brain-derived neurotrophic factor (BDNF), and tyrosine kinase receptor B (TrkB) through regulation of gut microbiota, such as recovering the relative abundances of Lachnospiraceae NK4A136, Bacteroides as well as Muribaculum [Pediococcus acidilactici CCFM6432 could alleviate anxiety-like behaviors caused by stress through inhibiting the over-proliferation of Escherichia shigella and promoting Bifidobacterium growth in C57BL/6 mice [Akkermansia muciniphila reduced depressive-like behavior in mice through reversing gut microbial abnormalities [Enterococcus faecalis strain EC-12 reduced anxiety-like behavior and enhanced Butyricicoccus and Enterococcus in mice [L. rhamnosus HN001 significantly decreased the depression and anxiety scores of women in the postpartum period [L. rhamnosus Probio-M9 enhanced the psychological and physiological qualities of life in stressed adults through increasing the relative abundances of certain species of gut microbiota [Probiotics have become a hotspot in the research fields of foods, nutrition, biology, and medicine, and can be used to prevent and manage several diseases, such as constipation, obesity, and cardiovascular disease. As mentioned above, several mental disorders were associated with abnormalities in gut microbiota. An increasing number of studies have revealed that probiotics, particularly the genus out mice . Moreoveibaculum . AdditioL/6 mice . Moreovemalities . In addi in mice . A studym period . Anothercrobiota . Lactobacillus reuteri NK33 and Bifidobacterium adolescentis NK98 improved mental health and sleep through modulating gut microbiota [Ruminococcus gauvreauii and Coprococcus 3 [Actinobacteria, Cyanobacteria, and S24-7_unclassified were decreased in the gut of stress mice, while multi-strains of probiotics recovered these changes as well as reduced depressive-like behaviors in mice [The multi-strain probiotic formulation could also exert preventive and therapeutic effects on mental disorders. A study based on 156 adults with subclinical symptoms of mental disorders showed that the mixture of crobiota . Anothercoccus 3 . Moreove in mice . Lactobacillus, Bifidobacterium, Pediococcus acidilactici CCFM6432, and Akkermansia muciniphila, showed significant preventive and therapeutic effects on several mental disorders, such as anxiety and depression. They influenced neurotransmitter metabolism, improved HPA axis hyperactivity, and increased the expression of BDNF and TrkB through modulating gut microbiota. Furthermore, multi-strain probiotic formulations might have more efficient actions on mental health protection compared with a single probiotic strain. It is also important to consider the interactions between different bacteria and their metabolites when exploring multi-strain probiotics formulations. The beneficial effects of more probiotics on different mental disorders (not only anxiety and depression) should be investigated by high-quality clinical trials in the future. In short, probiotics, such as The studies indicated that prebiotics and postbiotics could also be potential for the prevention and management of mental disorders, such as anxiety, depression, ASD, and schizophrenia ,113,114.Bifidobacterium in the 4-week intervention of 64 late adolescent females [\u00ae galactooligosaccharide (B-GOS\u00ae) prebiotic dramatically increased Lachnospiraceae and enhanced anti-social behavior in 30 autistic children [Bifidobacterium in a study based on 20 depression patients in Japan [Lactobacillus and Bifidobacterium [It was reported that dietary fibers could improve the relationship between gut microbiota and the central nervous system in schizophrenia patients through regulating gut microbiota . A doubl females . Anotherchildren . Howeverin Japan . The disacterium . Additioacterium . A study showed that SCFAs could ameliorate high fructose-induced depressive-like behaviors in mice by improving hippocampal neurogenesis decline and blood\u2013brain barrier damage . It was \u00ae and alpha-lactalbumin) as well as postbiotics (such as SCFAs) exerted protective effects on mental health, especially in combinative administration. Moreover, prebiotics could be a potential agent to alleviate the side effects of antipsychotics. In the future, the effects of more prebiotics and postbiotics on different mental disorders should be studied. Because synbiotics could exert the synergistic or additive effects of probiotics and prebiotics, more attention should be paid to the effects of synbiotics on mental disorders. In addition, because postbiotics are considered to be safer and more stable than probiotics, their effects on mental disorders should also be highlighted. Furthermore, more large-scale clinical trials are necessary to prove the influences of prebiotics and postbiotics on mental disorders, including health benefits as well as side effects.In a word, prebiotics , but decreased harmful bacteria  in the gut, and ultimately alleviated constipation and potentially depressive symptoms [Lactobacillus spp. in juvenile rats, which was positively correlated with changes in serotonin (5-HT)1A and 5-HT2C mRNA expression [Lactiplantibacillus plantarum ST-III-fermented milk improved the autistic-like behaviors in male ASD mice through modulating specific gut microbes, such as increasing the relative abundance of family Lachnospiraceae and genus Kineothrix [Dipteryx alata Vog.) and goat whey modulated gut microbiota  improved memory and relieved anxiety in elderly rats [Lactobacillus by 235%, decreased Phascolarctobacterium by 25% in gut microbiota, and improved hippocampal function [Dairy products are important components of both traditional and modern diets, and have a variety of health benefits. A double-blinded RCT based on 82 depressive patients with constipation found that the intervention of a fermented dairy beverage containing symptoms . Anotherpression . It was neothrix . In addirly rats . Moreovefunction . Howeverfunction . This diTo sum up, dairy products and their bioactive components showed neuroprotective effects via modulation of gut microbiota . Since fCurcuma longa), shows various biological activities [Capsicum annuum L.), alleviated lipopolysaccharide-induced depressive-like behaviors and reduced levels of 5-HT and TNF-\u03b1 by enhancing the relative abundances of certain gut microbiota, such as Ruminococcus and Prevotella [Zanthoxylum bungeanum may have a significant impact in alleviating and mitigating symptoms of depression via restoring the chronic unpredictable stress-induced gut microbiota dysbiosis, such as increasing Bacteroidales_S24-7_group, Lactobacillaceae, and Prevotellaceae, and decreasing Lachnospiraceae [Spices have a long history of being used as both food flavorings and traditional medicine, which possess many bioactive functions, such as anti-inflammatory, antibacterial, antifungal, and anticancer activities ,120. Mortivities ,122. It tivities . In addievotella . Furtherpiraceae .Zanthoxylum bungeanum, curcumin, and capsaicin) showed protective effects against anxiety and depression via regulating gut microbiota  enhanced memory and attention via regulating gut microbiota. The study further found that Cereboost\u00ae could increase levels of certain SCFAs in the intestinal tract through increasing the abundances of Akkermansia muciniphila and Lactobacillus by an in vitro model [Ginkgo biloba leaves could reduce stress-induced depression through reversing gut dysbiosis [Lycium barbarum polysaccharide during pregnancy could reduce the emotional injury of offspring caused by prenatal chronic stress through modulating the intestinal microbiota, such as enhancing the diversity of gut microbiota [Many other natural products also played vital roles in the prevention and management of mental disorders through the regulation of gut microbiota, such as fruits, vegetables, and medicinal herbs. A study showed that a higher intake of fruits and vegetables was positively correlated with better mental health based on 5845 Australian adults . Anothererium_uc . Additioro model . Anotherysbiosis . Moreovecrobiota . Additiocrobiota .Camellia sinensis, such as green tea and black tea) and tea-like beverages  should be given increased attention, because they are popular beverages with many bioactivities and beneficial effects.In a word, several fruits, vegetables, and medicinal herbs could alleviate the severity of mental disorders and enhance mental health via modulating the gut microbiota . In the Lactobacillus and Bifidobacterium), prebiotics , synbiotics, postbiotics , dairy products, spices , fruits, vegetables, and medicinal herbs could prevent and manage the mental disorders by modulating intestinal microbiota, including increasing beneficial gut microbiota and reducing harmful gut microbiota. Therefore, the supplement of dietary components mentioned above could be potential prevention and treatment strategies for mental disorders, except pharmacotherapy and psychotherapy. In addition, when the results from animal experiments are extrapolated to human beings, the differences between animals and humans should be considered. In the future, a greater number of experimental studies and high-quality large-sample clinical trials are required to explore the effects of more dietary components on mental disorders through the microbiota\u2013gut\u2013brain axis, and synbiotics and postbiotics need highlighting. Meanwhile, further elucidation and investigation of the underlying mechanisms of action is imperative. This paper is helpful for the public to choose natural dietary products to maintain mental health, and for natural dietary products to be developed into pharmaceuticals and functional foods for the prevention and treatment of several mental disorders.The gut microbiota and its metabolites could play an important role in mental health through the microbiota\u2013gut\u2013brain axis. The composition and abundance of gut microbiota, especially Firmicutes and Bacteroidetes, were associated with several mental disorders, such as anxiety, depression, bipolar disorder, ASD, and schizophrenia. At present, most studies about gut microbiota with mental disorders focused on the genus level, and more studies on gut microbiota should be carried out at the species level in the future, because the different species in the same genera could have different effects (even the opposite function) on mental disorders. Furthermore, due to the potential significant differences in the composition of the gut microbiome among individuals, it is crucial to accurately identify the changes of featured microbes that occur in each individual with mental disorders, which is important for personalized treatment of mental disorders through targeting gut microbiota. The epidemiological, experimental, and clinical studies have revealed that many kinds of probiotics (particularly"}
+{"text": "The gut microbiota is a community of micro-organisms that inhabit a host\u2019s gastrointestinal tract. Over a trillion microbes contribute to shaping this environment through the production of metabolites that can impact host physiology and function. Environmental factors as well as host attributes such as sex and age are important determinants of gut microbial composition and the metabolites they produce. There are a growing number of microbially-produced metabolites that have been associated with positive host health; however, the mechanisms and signaling pathways these metabolites activate have yet to be fully elucidated. Understanding these mechanisms will allow the field to better understand this intricate symbiotic relationship between gut microbiota and humans.Dysbiosis, or the imbalance of gut microbial communities, can disrupt host health and can even lead to several disease states, including endocrine disorders. The endocrine system involves the release of hormones from glands that enter blood circulation to exert a wide range of effects on growth, development, metabolism, sexual function and mood. The indispensable nature of a functioning endocrine system is exemplified by deficiencies or excess production of hormones in circulation that lead to endocrine disorders. Circulating levels of bacterial metabolites can induce or influence the occurrence of endocrine disorders. The emergence of pharmacotherapies aimed at reshaping host-microbiota interactions has added a new dimension for managing endocrine diseases and highlight the importance of this reciprocal relationship.The Cross-Talk Between Gut Microbiota and Endogenous Metabolites in Endocrine Diseases, with an update on this rapidly growing field. The goal of this topic is to identify the origin of endogenous metabolites generated by gut flora and its effects on endocrine physiology. This includes uncovering novel associations between endogenous metabolites produced by gut microbiota and host endocrine system and determining the signaling pathways recruited by these metabolites.The present Research Topic builds upon the first edition of the Research Topic \u2013 Guo et\u00a0al., examines oral microbiota and its relationship with intestinal microbiota in patients with type 2 DM. Dysbiosis of oral flora and transmission to the intestine provide novel avenues for diagnosing and treating type 2 DM. A systematic review from Hong et\u00a0al., investigates the involvement of gut microbiota and incidence of diabetic microvascular complications (DC). DC complications include diabetic retinopathy and diabetic peripheral neuropathy, which contribute to the high mortality and morbidity rate in DM patients. Their study found depletion of short-chain fatty acid-producing bacterial richness was associated with DC, and these results can inform future studies into the progression of DM to more severe disease states. Finally, the study by Ma et\u00a0al. characterizes changes in the gut microbiota in a rat model of DM following treatment with Yu-Ye Tang (YYT). YYT is a widely used, traditional Chinese medicinal product used to treat diabetes and gastritis, but its specific mechanism of action is unclear. This study combines in vivo measurements of host endocrine function and uses methods to detail changes in the microbial community and metabolites during disease and treatment with YYT. This study also provides a useful framework to investigate the effects of herbal formulations on modulating gut microbiota and host metabolism in future studies.Diabetes mellitus (DM) is the most common endocrine disease, afflicting over 400 million people worldwide across socio-economic boundaries. DM is also an endocrine disease linked with dysbiosis of the gut microbiota . SeveralZhang et\u00a0al. adds clarity to this issue by using a novel approach to combine host genetic variants to explore the causal association between gut microbial populations and metabolites with chronic liver diseases.Endocrine disorders also have a huge impact on liver function. Although many studies, including ones included in this Research Topic, have drawn associations between dysbiosis and DM, the link between gut microbiota and chronic liver disease is more controversial. The study by Larraufie et\u00a0al. examines the gasotransmitter hydrogen sulfide, which is generated in the gut by sulfate-reducing bacteria, and its effects on enteroendocrine cell metabolism and function. Finally, the review by Masse and Lu covers the main bacterial metabolites produced in the gut and the known signaling pathways recruited in enteroendocrine cells. The utility of targeting these mechanisms as a treatment for metabolic diseases such as diabetes and obesity are also covered in this comprehensive review.Tackling the underlying mechanism of action of bacterial metabolites on host health, the final two submissions examine mechanisms involving the hormone releasing cells of the gut, the enteroendocrine cells. This Research Topic spans the breadth of current research at the intersection of endocrinology and gut microbiology. These studies advance our understanding of the impact changes in gut microbial populations have on host endocrine function and provides insight on potential therapeutic strategies targeting microbial populations to improve human health.VL: Writing \u2013 original draft, Writing \u2013 review & editing. GR: Writing \u2013 review & editing."}
+{"text": "The intestinal microbiota is a diverse and complex microecosystem that lives and thrives within the human body. The microbiota stabilizes by the age of three. This microecosystem plays a crucial role in human health, particularly in the early years of life. Dysbiosis has been linked to the development of various allergic diseases with potential long-term implications. Next-generation sequencing methods have established that allergic diseases are associated with dysbiosis. These methods can help to improve the knowledge of the relationship between dysbiosis and allergic diseases. The aim of this review paper is to synthesize the current understanding on the development of the intestinal microbiota in children, the long-term impact on health, and the relationship between dysbiosis and allergic diseases. Furthermore, we examine the connection between the microbiome and specific allergies such as atopic dermatitis, asthma, and food allergies, and which mechanisms could determine the induction of these diseases. Furthermore, we will review how factors such as mode of delivery, antibiotic use, breastfeeding, and the environment influence the development of the intestinal flora, as well as review various interventions for the prevention and treatment of gut microbiota-related allergies. The gut microbiota represents the population of microorganisms that inhabit the human gut. Over the last decade, several studies have been conducted to determine the relationship between the microbiota and allergies in children. The findings suggest that the gut microbiota has a significant role in the promotion of these allergies. For instance, Penders et al.  conducteFirmicutes and a higher count of Bacteroidaceae. Another study conducted by Azad et al. in 2013 found that infants with a lower diversity of gut microbiota are at a higher risk of developing allergies later in life [Similarly, Melli et al.  conducte in life ,5. Addit in life .Firmicutes, including Lactobacillus and Clostridium, being the most prevalent phylum in the intestinal microbiota among adult subjects. On the other hand, Actinobacteria, including Bifidobacterium, were more abundant in samples obtained from one-year-old participants, with their relative abundance decreasing after the weaning period. The intestinal microbiota developed to resemble an adult-like gut microbiota by the age of three. A study conducted by Odamaki et al.  in 2016 Many studies have reported that the establishment of the human gut microbiota begins in fetal life through various sources; one of them is the detection of bacterial DNA in the placenta . It is wn = 416) reported that monozygotic twins have a more similar gut microbiota composition than dizygotic twins, highlighting the influence of genetic factors on the intestinal microbiome. Additionally, this study identified several heritable bacterial species, with the most heritable belonging to the family of twins [The role of host genotype in shaping the composition of gut bacteria has only been acknowledged in recent times. To investigate the genetic factors involved, the traditional approach used is to compare data between monozygotic twins (MZ) and dizygotic (DZ) twins . An exteof twins . Two yeaLactobacillus, Prevotella, or Sneathia spp. [Another significant factor influencing microbiome development is the mode of delivery . Infantshia spp. , while ihia spp. , resultihia spp. . Bifidobacterium and Lactobacillus [Roseburia, Clostridium, and Anaerostipes [Enterobacter, Staphyloccoccus, and Enterococcus, while in a full-term infant the colonization is mainly by Bacteroides, Bifidobacterium, Parabacteroides, and Escherichia [Breastfeeding is also essential to shape the microbiome of infants. Breast milk contains various prebiotics, such as human milk oligosaccharides, which selectively promote the growth of beneficial bacteria such as bacillus . Howeverrostipes . Dietaryrostipes . Antibiorostipes . Gestatiherichia . Furtherherichia . Shigella spp. A retrospective study over a 10-year period, conducted on 376 patients with Shigella [Shigella spp. The study also found that atmospheric temperature, humidity, and rainfall were significant environmental factors influencing the incidence of Shigella spp. Similarly, in a retrospective study spanning a decade, from 1 January 2009 to 31 December 2018, researchers examined 377 patients diagnosed with Salmonella spp. disease [Salmonella spp. cases and elevated humidity and atmospheric temperature levels. These environmental factors could have initiated dysbiosis which led to the child intestine vulnerability to Salmonella species. However, there is a limited amount of published research to support the hypothesis.The combination of these factors can disrupt the balance of the intestinal microbiota and cause dysbiosis, in some cases causing cardiac pathology such as heart failure and correlated with the severity of the disease ,27. HearShigella , reveale disease . The stuThe microbiome is involved in various aspects of body health in children. For example, analyses have illustrated that the microbiome is taking part in the development and maturation of the immune system in children ,32,33. TFirmicutes and a lesser proportion of Bacteroidetes compared to underweight subjects. Another study by Liu et al. [Research has revealed a correlation between dysbiosis, elevated levels of SCFA, obesity, and metabolic alterations. However, the precise connection between SCFAs and obesity remains uncertain ,38. SCFAu et al.  determinu et al. . In concBifidobacteria and Lactobacilli, play an essential role in sustaining immune homeostasis [Dysbiosis, defined as an imbalance or maladaptation in the microbiota, is increasingly recognized as a significant factor in the development of allergies in children . The noreostasis ,55. Theieostasis . Dysbioseostasis . This caeostasis ,59. Lasteostasis . The deceostasis . Some preostasis . Followieostasis .Bifidobacterium and Lactobacillus are among the most important microorganisms in the early life of children and they appeared to be effective in treating allergic rhinitis in the previous article. More research is required to confirm these findings. Consumption of live microbes known as probiotics in adequate amounts is known to provide health benefits to the host . The proBifidobacterium and Lactobacillus species [Prebiotics, which are non-digestible components that exist within food such as fibers and carbohydrates, selectively promote the growth and the activity of the microecosystem that exist in the human gut. Prebiotics function as substrates, selectively stimulating the growth and activity of health-promoting bacteria such as  species . When co species . These S species . These c species . They ha species . Clostridium difficile infection [Fecal microbiota transplantation is a method which can normalize the gut microbiota of the affected individuals through replacement of that microbiota with another microecosystem from a healthy individual , and it nfection , inflammnfection , and evenfection . The prinfection . The tranfection . Additionfection . In thisPrevotella spp. [Dietary approaches have been suggested as an important strategy to manipulate the gut microbiota. Research has demonstrated that a diet rich in fiber can enhance the presence of many advantageous gut microbes such as lla spp. . In contlla spp. . Therefolla spp. .Bifidobacterium [Enterobacteriaceae [Bacteroides plus other bacteria such as Clostridium and Lactobacillus [Staphylococcus, Escherichia coli, and Clostridia [Breastfeeding is another of these factors that has been shown to have a significant impact on the development of the microbiota during the early years of infant life, because it provides the infant with essential nutrients ,91. Seveacterium  and Enteeriaceae . The stubacillus . Other sostridia ,96. SomeAtopic dermatitis (AD) is a chronic skin condition characterized by recurring eczematous lesions and severe itching. The onset typically occurs in childhood, leading to its original classification as a pediatric disorder. However, recent reports have illustrated that atopic dermatitis is increasingly prevalent among adults [Staphylococcus aureus has been observed as well [g adults ,98. The g adults ,100. Thug adults . In manyg adults ,103. In  as well . This is as well . HoweverAsthma is a chronic respiratory disease that affects over 300 million individuals worldwide, making it one of the most common respiratory conditions [nditions . Despitenditions ,108,109.nditions ,111,112.nditions . This renditions . Additionditions ,116. A snditions  comparedFood allergy (FA) is characterized as an immune system response that is detrimental to particular dietary antigens. In recent decades, there has been an observable increase in the incidence of FA, which has become a significant health problem, affecting a notable proportion of adult and child populations, with nearly 5% of adults and 8% of children affected . Cow milLachnospiraceae and Streptococcaceae families. In contrast, the control group, consisting of non-food allergic children, presented a higher occurrence of Leuconostocaceae. Furthermore, egg sensitization was correlated with greater gut microbiome diversity, and the Lachnospiraceae and Ruminococcaceae genera were pivotal in this regard. However, neither the differences in gut bacterial diversity nor the compositional variations in the gut microbiome could anticipate the persistence or resolution of egg allergy at 8 years of age.The research conducted by Fazlollahi et al. studied a specific food allergy in a pediatric cohort, which centered on 141 children between the ages of 3 and 16 months. The participants were sourced from five different centers located in the United States. The principal aim of the study was to scrutinize and contrast the gut microbiomes of subjects with egg allergies versus those of control subjects . The finClostridia and Firmicutes in those whose milk allergy improved later in childhood [In contrast, a different study focused on exploring the link between the composition of the gut microbiota in early life and the resolution of cow\u2019s milk allergy. Research showed that infant intestinal flora in subjects between 3 and 6 months of age exhibited an increase in hildhood . There are many potential mechanisms involved in the relationship between the microbiota and allergies; one of them is immune system modulation in which the gut microbiota can modulate the immune system by inducing regulatory T-cells (Tregs) and promoting the production of anti-inflammatory cytokines such as IL-10 ,126. TreEnterobacter cloacae and Escherichia coli, alongside decreased levels of Streptococcus faecalis and Staphylococcal species. Due to increased permeability in the intestines of preterm infants, it can be considered that this increase will lead to increased translocation of bacteria and explain bacteremia and endotoxemia in these premature infants. A study that observed the gut microbiota of preterm twins over time revealed a decline in the diversity of the gut microbiota and a rising prevalence of Escherichia spp. prior to the onset of NEC. Conversely, this pattern was not observed in healthy twins who did not develop NEC later [Proteobacteria population and a 32% decrease in the population of Firmicutes between the samples collected one week earlier and less than 72 h before the onset of NEC. In addition, not only preterm birth increases the risk factor, but studies have suggested that even antibiotic use and formula feeding have been associated with the development of NEC [Another potential mechanism is the function of the gut barrier; the gut microbiome contributes to the maintenance of the gut barrier function, which prevents the entry of harmful substances and pathogens into the bloodstream . DysbiosEC later . AnotherEC later . Within t of NEC ,140. TheIn conclusion, this literature review has highlighted the vital role that the gut microbiota plays in the development and manifestation of allergies in children. The complexity of the human gut microbiota, which is influenced by factors such as host genotype, mode of delivery, breastfeeding, diet, antibiotic use, and environmental factors, underscores the intricacies involved in establishing a healthy microbial ecosystem. The association between the gut microbiota and atopic symptoms or sensitization, immune system development, body growth, and various diseases such as atopic dermatitis, asthma, and necrotizing enterocolitis demonstrates the far-reaching implications of a balanced gut microbiome. Interventions such as probiotics, prebiotics, fecal microbiota transplantation, dietary approaches, and breastfeeding have shown promising results in preventing and treating gut-microbiota-related allergies. Notably, the adoption of a Mediterranean-style diet, probiotics, and prebiotics may help to mitigate the risk of developing allergies, while breastfeeding is essential in establishing a diverse gut microbiome and providing critical nutrients to infants. Furthermore, the relationship between the gut microbiota and food allergies highlights the potential to develop novel treatments that target the gut microbiota to improve intestinal barrier function and modulate the immune response to food allergens. Despite the advances in our understanding of the gut microbiota and its implications on allergies in children, further research is warranted to elucidate the complex interplay between the microbiome, immune system, and disease development. By deepening our knowledge in this area, we can pave the way for more targeted and effective interventions that could potentially transform the prevention and management of allergies in children."}
+{"text": "In recent years, experts have recognized the critical role of gut microbiota in maintaining overall health. The microorganisms, including bacteria, fungi, and viruses, living in the intestine are referred to collectively as the intestinal microbiota, which play a significant role in several physiological processes in the digestion and metabolism of numerous food constituents   might lead to targeted therapies and personalized dietary recommendations.As a result of the ongoing expansion of study, several essential subfields of research have recently come to the fore. One of those areas focuses on the effect of food homologous plants on the microbial diversity in the gut. Numerous scientific investigations have shown a correlation between normal and abnormal microbiota in the gut. It is hypothesized that an enormous variety of helpful bacteria in the gut might contribute to better immune function, increased nutritional absorption, and protection against hazardous pathogens. This is because beneficial bacteria can inhibit the growth of harmful pathogens  exhibited promising effects on type 2 diabetes. DBD treatment improved insulin sensitivity, reduced inflammation, and enhanced microbial diversity in the intestines, suggesting its potential as an antidiabetic and anti-inflammatory agent . Similarly, Astragalus polysaccharides (APS) showed anti-inflammatory effects with lung injury induced by lipopolysaccharides (LPS). APS improved lung pathology and reduced inflammation, possibly through its modulation of gut microbiota .The interaction between food-homologous plants and the microbiota of the digestive system has implications for brain functioning. The gut and the brain can interact through the gut-brain axis. Emerging research shows that abnormalities in the gut microbiota composition, often known as dysbiosis, may contribute to developing mental health issues such as depression and anxiety . Low-nicotine tobacco (LNT) supplementation in rabbits showed promising results on health parameters, altering gut microbiota diversity and metabolites. LNT positively impacted blood status and carcass weight, indicating its potential as a feed additive .The interaction between plant-derived compounds and gut microbiota was also evident in studies involving animal models. Taxifolin, a natural flavonoid, enhanced semen quality in Duroc boars by modulating gut microbes and blood metabolites. It increases sperm motility and beneficial blood components while reducing harmful bile acids and cholesterol levels . Black Lycium barbarum polysaccharide (BLBP) demonstrated protective effects against lipopolysaccharide (LPS)-induced intestine damage. BLBP attenuated inflammation and improved intestinal morphology, possibly through the NAD(P)H dehydrogenase (quinone) 1/NF-\u03baB pathway and modulation of gut microbiota . The potential of seaweed polysaccharides as therapeutic agents was also explored. Seaweed polysaccharides alleviated hexavalent chromium-induced gut microbial dysbiosis, demonstrating their ability to restore gut microbiota balance .Other studies delved into the immunomodulatory effects of Chinese medicine compounds. The compound small peptide of Chinese medicine (CSPCM) alleviated cyclophosphamide-induced immunosuppression in mice by modulating gut microbiota and regulating Th17/Treg balance. CSPCM increases immune organ indices, anti-inflammatory cytokines, and beneficial gut bacteria while decreasing immunosuppressive Treg cells and harmful bacteria (Overall, the articles in this Research Topic collectively emphasize the importance of gut microbiota in mediating the health benefits of traditional Chinese herbal medicines, plant-derived compounds, and seaweed polysaccharides. These studies shed light on novel therapeutic strategies for various health conditions, including diabetes, inflammatory lung injury, immunosuppression, and gut microbial dysbiosis. The findings underscore the significance of understanding the complex interactions between food-homologous plants and gut microbiota for developing innovative and effective therapeutic interventions.Although studies on food-homologous plants have been conducted, many obstacles still need to be overcome. Determining which particular phytochemicals are responsible for the effects that have been seen is a significant obstacle. It is challenging to isolate and analyze the impact of individual components since many identical plants possess a complex variety of bioactive substances. This makes it tough to examine the effects of individual components. Moreover, the fact that the gut microbiota is uniquely composed presents a hurdle when formulating applicable dietary guidelines.In conclusion, food homologous plants' interactions with the microbiota of the digestive tract is fascinating. The chemical compounds present in such plants can change the layout and activity of the gut microbiota, which might positively affect health. However, further study is required to understand the underlying processes and locate the particular phytochemicals that are accountable for the impacts that have been reported. This information might pave the way for personalized dietary plans to improve gut and body health.MK wrote the first draft of the manuscript. All other authors have made a substantial, direct, and intellectual contribution to the work and approved it for publication."}
+{"text": "Gut microbiota and Gut-associated metabolites in bone health\u201d highlights the importance of the latest research in this emerging and interesting field of Osteomicrobiology. This Research Topic contains 4 contributions with authors from China and India. The compilation of these recent reviews and research articles will solidify our knowledge regarding the role of GM and GAMs in the maintenance of bone homeostasis. This Research Topic primarily consists of the following specific topics.The term microbiota refers to all the microbes  that exist in a particular niche in/on the host. Microbes cover all the host mucosal surfaces with the majority of them residing within the gastrointestinal tract (gut microbiota). The human gut microbiota comprises of ~100 trillion microbes (500-1000 species) which encode for more than 3.3 million genes. These microorganisms have co-evolved with humans and improve human health by regulating several biological processes. Establishing and sustaining beneficial exchanges between the host and its accompanying microbiota are the key requirements for the maintenance of the healthy life of the host. Gut microbiota regulates host physiology and development, influences host metabolism, and modulates the host immune system . StudiesGuan et\u00a0al. in their review focused on several mechanisms underlying gut microbiota-mediated regulation of bone health. One such mechanism is gut microbiota facilitating nutrient absorption as well as help to maintain intestinal barrier integrity which further enhances bone mineral density (BMD). In addition, microbiota regulates the immune system which has a significant role in maintaining skeletal homeostasis. Tu et\u00a0al. also reviewed another novel mechanism referred to as the entero-endocrine-osseous axis to summarize the interaction of gut microbiota with the endocrine system to promote bone health. Gut microbiota influences several bone metabolism-regulating hormones to maintain skeletal homeostasis. For instance, the importance of estrogen in preserving bone health is well known. Estrogen reduces bone resorption by maintaining systemic and bone marrow T cells homeostasis in addition to directly modulating the formation of osteoblasts and osteoclasts. The gut microbiota controls the metabolism of estrogen and raises its circulating levels. Another hormone testosterone can both reduce the apoptosis of osteoblasts and enhance the proliferation of osteoblast precursors. Probiotics administration and fecal microbiota transplantation can control testosterone levels while concurrently changing bone structure. Microbiota also influences other key bone-regulating hormones such as insulin-like growth factor-1, parathyroid hormone, serotonin, and gastrointestinal hormones. Taken together, it is clear that gut microbiota plays a crucial role in maintaining bone metabolism and it does so by interacting with several physiological systems in the body.Gut microbiota influences the homeostasis of several tissues including bone. Numerous studies in the past decade have evidenced the significance of gut microbiota in the regulation of bone health. Guan et\u00a0al. reviewed the role of depletion of gut microbiota in bone pathologies such as osteoporosis and osteoarthritis. On the other hand, other studies pointed out the beneficial role of gut microbiota in maintaining bone health. Taken together the role of gut microbiota is very complex and conflicting in the perspective of bone health and thus further research is still warranted and required to dissect the complete nexus between gut microbiota and bone health under both physiological and pathological conditions.There are several methods to dissect the effect of gut microbiota on bone health. Depletion of gut microbiota with a selective mixture of antibiotics or use of germ-free mice are some of the most common strategies to evaluate the effect of gut microbiota on bone health in various disease conditions. Guo et\u00a0al. reported that perturbation of gut microbiota composition with antibiotic treatment resulted in decreased BMD. However, probiotic treatment with Lactobacillus casei fermented milk reversed the effect of antibiotics and promoted fracture healing in osteoporotic mice highlighting the important role of gut microbiota composition in bone health. The association of gut microbiota composition with several bone ailments is being actively investigated by several groups. Dagar et\u00a0al. reviewed the role of dysbiosis in RA. The primary bacterial family in the gut most closely connected with RA is Prevotellaceae. Prevotella copri overgrowth was seen in several investigations in RA patients\u2019 fecal samples. One major lacuna that Dagar et\u00a0al. highlight in their review is that the majority of current studies in the field of RA have been devoted to describing the role of gut bacteria. Research on gut fungal and virome components in RA is a relatively new and developing subject. However, alterations in fungal and viral components likely have a significant effect on the development of RA. Dagar et\u00a0al. filled this gap and documented the role of various viral and fungal components in the pathogenesis of RA. Altogether the composition of complete gut microbiota  is an important factor in the regulation of bone homeostasis.Recent studies have highlighted that gut microbial composition significantly impacts bone health. Tu et\u00a0al.). Taken together, interventions targeting the gut microbiota are effective strategies for bone loss, and gut microbiota modifying therapies, especially the gut metabolites can be examined for their effectiveness at the clinical level in the near future.As dysbiosis is associated with several bone pathologies, modulation of gut microbiota composition could be an effective strategy for the prevention, management and perhaps the treatment of these bone pathologies. Gut microbiome-modifying medications suggest that altering the gut microbiome may be a promising therapeutic or adjunct approach for the treatment of RA. Interventions such as administration of probiotics, prebiotics, and fecal microbiota transplantation are the preferred methods for restoring the dysregulated gut microbiota. Numerous studies have highlighted the role of these interventions as a therapeutic strategy for various inflammatory bone conditions. However recently the GAMs are receiving significant attention as a treatment modality for gut microbiota-associated pathologies including bone disorders. GAMs that are considered the most prevalent are short-chain fatty acids (SCFAs) and bile acids (BAs). In recent years, a number of studies have underscored the role of SCFAs in skeletal homeostasis. It has been shown that SCFAs block histone deacetylases (HDACs) and activate free fatty acid receptors (FFARs) to prevent the development of osteoclasts. In addition to their direct impact on bone cells, SCFAs promote the differentiation of regulatory T cells while inhibiting osteoclastogenic Th17 cells. The crucial involvement of BAs in bone remodeling has been demonstrated by the positive connection between serum levels of total BAs and BMDs in postmenopausal women. BAs enhance osteogenic differentiation while inhibiting osteoclastogenesis (Gut microbiota has the potential of regulating bone homeostasis and dysbiosis of gut microbial composition can lead to several bone pathologies. Modifying gut microbial composition can thus be a promising or adjunct strategy for the treatment and management of various skeletal disorders. However, large-scale clinical trials are required to clearly define their efficacy in humans.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."}
+{"text": "NFIX) gene encodes a ubiquitously expressed transcription factor whose mutations lead to two allelic disorders characterized by developmental, skeletal, and neural abnormalities, namely, Malan syndrome (MAL) and Marshall\u2013Smith syndrome (MSS). NFIX mutations associated with MAL mainly cluster in exon 2 and are cleared by nonsense\u2010mediated decay (NMD) leading to NFIX haploinsufficiency, whereas NFIX mutations associated with MSS are clustered in exons 6\u201310 and escape NMD and result in the production of dominant\u2010negative mutant NFIX proteins. Thus, different NFIX mutations have distinct consequences on NFIX expression. To elucidate the in vivo effects of MSS\u2010associated NFIX exon 7 mutations, we used CRISPR\u2010Cas9 to generate mouse models with exon 7 deletions that comprised: a frameshift deletion of two nucleotides (Nfix Del2); in\u2010frame deletion of 24 nucleotides (Nfix Del24); and deletion of 140 nucleotides (Nfix Del140). Nfix+/Del2, Nfix+/Del24, Nfix+/Del140, NfixDel24/Del24, and NfixDel140/Del140 mice were viable, normal, and fertile, with no skeletal abnormalities, but NfixDel2/Del2 mice had significantly reduced viability (p\u2009<\u20090.002) and died at 2\u20133\u2009weeks of age. Nfix Del2 was not cleared by NMD, and Del2/Del2Nfix mice, when compared to Nfix+/+ and Nfix+/Del2 mice, had: growth retardation; short stature with kyphosis; reduced skull length; marked porosity of the vertebrae with decreased vertebral and femoral bone mineral content; and reduced caudal vertebrae height and femur length. Plasma biochemistry analysis revealed NfixDel2/Del2 mice to have increased total alkaline phosphatase activity but decreased C\u2010terminal telopeptide and procollagen\u2010type\u20101\u2010N\u2010terminal propeptide concentrations compared to Nfix+/+ and Nfix+/Del2 mice. NfixDel2/Del2 mice were also found to have enlarged cerebral cortices and ventricular areas but smaller dentate gyrus compared to Nfix+/+ mice. Thus, NfixDel2/Del2 mice provide a model for studying the in vivo effects of NFIX mutants that escape NMD and result in developmental abnormalities of the skeletal and neural tissues that are associated with MSS. \u00a9 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.The nuclear factor I/X ( A mouse model for Marshall\u2013Smith syndrome. NFIX) gene (MIM #164005), located on chromosome 19p13.2, consists of 11 exons GCCAA\u20103\u2032 present in the promoter regions of viral and cellular genes, and a variable C\u2010terminal transactivation/repression domain, which can potentially provide a range of preferential interactions with other proteins to either activate or suppress transcription. NFI transcription factors play important roles in the regulation of stem cell differentiation, quiescence, and differentiation during the development of organs that include lung, kidney, liver, blood, heart, skeleton, and the nervous system.The nuclear factor I/X (#164005),2, 3ons Fig.\u00a0 that encNFIX gene can lead to two rare allelic disorders, Malan syndrome  and Marshall\u2010Smith syndrome . MAL is an overgrowth disorder, characterized by a slender habitus, long hands and advanced bone age, moderate to severe intellectual disability, unusual facial phenotype consisting of a long, triangular face with a prominent forehead, everted lower lip and prominent chin, and behavioral problems, which are usually dominated by anxieties and, less frequently, by aggression  and lead to NFIX haploinsufficiency. MSS is characterized by short stature with skeletal abnormalities that may include kyphoscoliosis, abnormal bone maturation, craniofacial defects, and osteopenia and be associated with delays in motor and neural development that lead to moderate to severe mental retardation, limited or absent speech, and postnatal failure to thrive. In addition, MSS patients may have distinctive facial features that include a high forehead, proptosis, blue sclera, anteverted nares, small and retracted mandible, gingival hypertrophy, and hypertrichosis .1, 9) Table\u00a0. The misn 2 Fig.\u00a0, which eis Table\u00a0. MSS patain Fig.\u00a0. The difin vivo consequences of Nfix exon 2 deletion, which encodes the conserved N\u2010terminal DNA binding and dimerization domain, have been studied in mouse models. In one study wherein Nfix exon 2 was replaced with an in\u2010frame lacZ reporter gene, Nfix+/lacZ mice were reported to have normal survival, but reduced body weight, while NfixlacZ/lacZ mice developed skeletal abnormalities due to defects in ossification that resulted in kyphosis and neurological abnormalities such as partial agenesis of the corpus callosum that was associated with hydrocephalus. In other studies wherein an Nfix null allele was initially generated via Cre\u2010recombinase\u2010mediated excision of Nfix exon 2, the heterozygous Nfix+/\u2212 mice also had normal survival but with neurological abnormalities, and the homozygous Nfix\u2212/\u2212 mice had neurological defects that included dysgenesis of the corpus callosum but did not have skeletal abnormalities. Moreover, Nfix\u2212/\u2212 mice are reported to have severe delay in intermediate progenitor cells during forebrain development and smaller muscle fibers with impairment of muscle regeneration despite the lack of skeletal defects  Table\u00a0. These NAll animal studies were approved by the Medical Research Council Harwell Institute Ethical Review Committee and were licensed under the Animal (Scientific Procedures) Act 1986, issued by the UK Government Home Office Department (PPL30/2433 and PPL30/3271). and genotyping was performed by PCR amplification using genomic DNA and confirmed by RT\u2010PCR using total RNA extracted, as described in Data\u00a0Mice were generated using the CRISPR/Cas9 system,22 andNfix cDNA expression constructs and luciferase reporter constructs were utilized for qRT\u2010PCR, Western blot, and immunofluorescence analyses, as detailed in Data\u00a0Murine embryonic fibroblast (MEF) cells and monkey kidney fibroblast (COS\u20107) cells that were used for RNA sequencing analysis or transiently transfected with wild\u2010type (WT) or mutant murine  and skeletons and tissues of WT and mutant mice were prepared and used for imaging and histological analyses, as detailed in Data\u00a0Blood samples were collected and used for plasma biochemical analysis,22 andp\u2009<\u20090.05 was considered significant for all analyses as described in Data\u00a0Data are expressed as mean and standard deviation (SD) or standard errors of mean (SEM). All analyses were performed using Prism (GraphPad), and a value of NFIX gene, the CRISPR\u2010Cas9 system was used to target exon 7 of the murine Nfix gene. Following injection of Cas9 mRNA and Nfix guide RNA into C57BL/6J embryos, founder mice were generated from which three mutant Nfix lines comprising deletions of two nucleotides (Del2), 24 nucleotides (Del24), and 140 nucleotides (Del140) were established. More specifically, Nfix Del2 consists of a frameshift two\u2010nucleotide deletion from position +49,580 to +49,581 relative to the translation start site (TSS), Nfix Del24 contains an in\u2010frame 24\u2010nucleotide deletion , and Nfix Del140 contains a 140\u2010nucleotide deletion  and comprised 53 nucleotides from exon 7 and 87 nucleotides of intron 7  were viable and intercrossed within each line to generate WT (Nfix+/+), heterozygous  and homozygous  mice. Genotypes were confirmed and validated by PCR amplification of exon 7, Sanger DNA sequencing, and, in the case of Nfix Del2 mice, using NlaIII restriction endonuclease digestion analysis . Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice were viable, normal, and fertile, but NfixDel2/Del2 mice were subviable by 21\u2009days post term (P21) due to early death around 2\u20133\u2009weeks of age  from the expected Mendelian ratio at P21 due to early death at 2\u20133\u2009weeks, indicative of reduced viability of the NfixDel2/Del2 mice. Moreover, the numbers of Nfix+/+, Nfix+/Del2, and NfixDel2/Del2 mouse embryos at day 18.5 (E18.5) did not deviate from the expected 1:2:1 Mendelian ratio , due to alternative splicing of exons 7 and 9 and the use of different transcription initiation sites (ENSMUSG00000001911.16). Thus, alternative splicing may produce WT Nfix transcripts that retain exon 7 (Nfix long isoform) or shorter conserved isoforms that lack exon 7 (Nfix \u0394Ex7)  and short WT \u0394Ex7 (194\u2009bp) isoforms , but the NfixDel2/Del2 and NfixDel24/Del24 MEFs had mutant Nfix long isoforms of 315 and 293\u2009bp, respectively, and the Nfix WT short isoform (\u0394Ex7 of 194\u2009bp) . Nfix+/Del2 and Nfix+/Del24 MEFs were confirmed to express a WT Nfix long isoform, a mutant Nfix long isoform, and the Nfix WT \u0394Ex7 short isoform . In contrast, NfixDel140/Del140 MEFs had only the Nfix WT \u0394Ex7 short isoform, thereby suggesting that the 140\u2010nucleotide deletion, which comprised 53 nucleotides of the 3\u2032 end of exon 7 along with 87 nucleotides from intron 7 that included the donor splice site, led to exon 7 skipping. Sanger DNA sequence analysis confirmed that the sequence of this Nfix short isoform from the NfixDel140/Del140 MEFs matched the consensus murine sequence of the Nfix WT \u0394Ex7 short isoforms . Therefore, the 140\u2010nucleotide deletion in the Nfix Del140 MEFs led to skipping of exon 7 and alternative splicing of exon 6 to exon 8 due to loss of a donor splice site, resulting in a frameshift and the introduction of a stop codon after 81 amino acids, which corresponded to the WT short NFIX isoforms. However, the two\u2010nucleotide deletion in Nfix Del2 MEFs resulted in a frameshift and the introduction of a premature stop codon after 65 amino acids, and the 24\u2010nucleotide in\u2010frame deletion in Nfix Del24 MEFs predicted the loss of eight amino acids (QGSSPRMA).The differences in viability between the homozygous x7) Fig.\u00a0A. To strms Fig.\u00a0B\u2013D, butbp) Fig.\u00a0B,C. Nfiorm Fig.\u00a0B,C. In rms Fig.\u00a0D. ThereNfix Del2 and Nfix Del24 mutations on Nfix transcription, translation, and cellular localization, in vitro expression assays in COS\u20107 cells transiently transfected with N\u2010terminal\u2010FLAG\u2010tagged WT or mutant (Del2 or Del24) Nfix cDNA constructs that retain exon 7 were undertaken. Analysis by qRT\u2010PCR showed that there was no significant difference in the amount of Nfix Del2 or Nfix Del24 expression compared to Nfix WT, suggesting that these mutations affecting the C\u2010terminal part of the Nfix transcripts were not cleared by NMD mechanisms , as expected, compared to WT (55\u2009kDa) , whereas that of the NFIX Del24 protein was significantly increased (p\u2009<\u20090.01) compared to NFIX WT, thereby revealing differences in the stabilities and likely degradations of the mutant proteins  Fig.\u00a0B, thered p\u2009<\u20090.0 comparedd p\u2009<\u20090.0 comparedNfix Del2 and Nfix Del24 mutations on NFIX transcription factor function, given that NFIX is reported to activate GFAP expression, reporter constructs comprising the luciferase reporter gene downstream of the GFAP promoter were transiently cotransfected with WT or mutant Nfix cDNA constructs into COS\u20107 cells. WT NFIX activated the GFAP promoter and caused an approximately\u2009ninefold increase , viscera, and plasma biochemistry.Heterozygous and homozygous NfixDel2/Del2 mice were characterized by growth retardation and short stature when compared to Nfix+/+, Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice. Thus, at P1, there was no significant difference in the weights of Nfix+/+(1.5\u2009\u00b1\u20090.1\u2009g), Nfix+/Del2 (1.6\u2009\u00b1\u20090.0\u2009g), and NfixDel2/Del2 (1.5\u2009\u00b1\u20090.1\u2009g) mice . In addition, the NfixDel2/Del2 mice were visibly smaller than Nfix+/+ and Nfix+/Del2 mice at 2\u2009weeks . In contrast, the growth rates of the Nfix+/Del24 and NfixDel24/Del24 mice, and the Nfix+/Del140 and NfixDel140/Del140 mice were not significantly different from WT mice between 2 to 12\u2009weeks of age, irrespective of sex . Furthermore, visually, the Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice were indistinguishable from the Nfix+/+ mice at 12\u2009weeks .ice Fig.\u00a0, irrespeeks Fig.\u00a0A. In cosex Fig.\u00a0B,C. Fureks Fig.\u00a0B,C.NfixDel2/Del2 mice were also significantly shorter than Nfix+/+ and Nfix+/Del2 mice . In addition, Echo\u2010MRI analysis revealed a significant decrease in weight , even when normalized to body weight .The tail lengths, indicative of vertebral growth, of the es Table\u00a0A, even ks Table\u00a0B,C.A) of skeletons of 2\u20133\u2009weeks old mice revealed that >30% of NfixDel2/Del2 mice had kyphosis compared to <10% of Nfix+/+ and Nfix+/Del2 mice. Craniofacial measurements of the skulls of Nfix+/+, Nfix+/Del2, and NfixDel2/Del2 mice revealed that there was a significant reduction in the skull length of NfixDel2/Del2 mice compared to Nfix+/+ and Nfix+/Del2 mice . However, there were no significant differences in skull width, nasal bone length, and frontal bone length of NfixDel2/Del2 mice, compared to Nfix+/+ and Nfix+/Del2 mice , although parietal bone length of NfixDel2/Del2 mice was significantly different from that of Nfix+/Del2 but not Nfix+/+ mice . In contrast, radiological analyses of Nfix+/+, Nfix+/Del24, and NfixDel24/Del24 mice or Nfix+/+, Nfix+/Del140, and NfixDel140/Del140 mice revealed no skeletal abnormalities at 12\u2009weeks . MCT analysis of the lumbar and thoracic vertebrae also revealed NfixDel2/Del2 mice to have marked porosity at 2\u20133\u2009weeks  and revealed decreases in vertebral height  staining , although the lack of significant difference in the number of osteoclasts could be due to the low number of animals analyzed. To investigate whether the low BMC in NfixDel2/Del2 mice may be a result of abnormal osteoclast activity instead, the plasma concentrations of C\u2010terminal telopeptide (CTX), procollagen\u2010type\u20101\u2010N\u2010terminal propeptide (P1NP), and total alkaline phosphatase (ALP) activity, which are markers of bone resorption, bone formation, and bone mineralization, respectively, were therefore measured. NfixDel2/Del2 mice, when compared to Nfix+/+ and Nfix+/Del2 mice at 2\u20133\u2009weeks, had reduced CTX concentrations , thereby suggesting an overall abnormal bone turnover phenotype.Micro\u2013computed tomography (MCT) Fig.\u00a0A, Alciaice Fig.\u00a0C\u2013E, alteks Fig.\u00a0B,C. MCTing Fig.\u00a0C, whichP21 Fig.\u00a0D,E, altNfix+/+  and inferior blade areas (p\u2009<\u20090.05) of the dentate gyrus .Mice homozygous for a null exon 2 te gyrus.18 Thesis Fig.\u00a0N,O of nsis Fig.\u00a0P,Q. Thisis Fig.\u00a0R or prosis Fig.\u00a0S in Nfiice Fig.\u00a0N,N' but ice Fig.\u00a0O,O'. Simice Fig.\u00a0P,P' wereice Fig.\u00a0Q,Q'. In ies Fig.\u00a0B,C.NfixDel2/Del2 mice, when compared to Nfix+/+ and Nfix+/Del2 mice, had raised plasma urea  and raised total bilirubin  at 2\u20133\u2009weeks, in males and females (Table\u00a0A), consistent with abnormal kidney and liver function, respectively. However, there were no significant differences in the plasma concentrations of sodium, potassium, chloride, total and corrected calcium, inorganic phosphate, aspartate aminotransferase, alanine aminotransferase, albumin, creatinine, and creatine kinase in the NfixDel2/Del2 mice compared to the Nfix+/+ and Nfix+/Del2 mice (Table\u00a0A). Moreover, histology of the kidneys and livers from NfixDel2/Del2 mice revealed no abnormalities compared to Nfix+/+ and Nfix+/Del2 mice . In addition, histological examination for liver inflammation  and liver fibrosis  revealed no hepatic abnormalities in the NfixDel2/Del2 mice compared to the Nfix+/+ and Nfix+/Del2 mice. The plasma biochemistry of Nfix+/+, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice at 12\u2009weeks was similar , suggesting normal kidney and liver function in these mice, and histological analysis of the liver, kidney, lung, and heart of these mice revealed no abnormalities .Plasma biochemical analysis revealed that es Table\u00a0A, consice Table\u00a0A. Moreoice Fig.\u00a0A. In adion Fig.\u00a0C.E.F ansis Fig.\u00a0D,G reveies Fig.\u00a0B,C.Nfix mouse models could be due to functional redundancy provided by the other members of the NFI gene family of transcription factors, we pursued RNA sequencing analysis to investigate differences in Nfia, Nfib, and Nfic gene expression in the MEFs derived from the Nfix+/+, Nfix+/Del2, NfixDel2/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice. RNA sequencing analysis identified that, compared to the mean of Nfix+/+ MEFs, Nfia transcripts were significantly altered  Nfix isoforms. This suggests that different frameshift mutations or in\u2010frame deletions affecting the C\u2010terminal part of the Nfix gene have different consequences on the transcripts and activity of the resulting proteins, thereby accounting for the different phenotypes in the three mouse models.Our study reports the phenotypic characterization of three CRISPR\u2010Cas9 generated mouse models with three allelic variants in  Redundant paralogs that are ubiquitously expressed in a partially overlapping manner and that recognize similar motifs may provide backup for one another in case of mutation by changing their expression pattern and acquiring new regulatory capabilities in order to compensate for the mutation. For example, NFIA, NFIB, NFIC, and NFIX have the same conserved N\u2010terminal DNA binding and dimerization domain that enables all four related genes to recognize the same consensus sequence present in the promoter region of genes expressed in almost every organ, including the brain, lung, liver, intestine, and skeleton. Nfia\u2212/\u2212 mice have CNS and kidney abnormalities and die perinatally,Nfib\u2212/\u2212 mice have CNS and lung anomalies and die at birth, while Nfic\u2212/\u2212 mice have only a mild phenotype involving abnormal tooth development of the incisors and molars. More recently, overlapping patterns of NFIA, NFIB, and NFIX expression have been reported in the brain. NFIA, NFIB, NFIC, and NFIX, which were previously shown to interact with each other as well as other cofactors, bind the same regulatory motif of promoters of genes, such as brain fatty acid binding protein (B\u2010FABP), GFAP, and inscuteable (INSC), and the NFIs or the ratio of the four NFIs have been shown to act either antagonistically or synergistically to regulate transcription in a promoter and context dependent manner. Moreover, knockdown of one NFI member can affect the expression levels of other NFI members, suggesting cross\u2010talks and possible compensation within the NFI family. NFIX was also recently shown to act sequentially after NFIA and NFIB during gliogenesis within the spinal cord, and NFIB was reported to be able to activate Nfix expression in vitro, thereby suggesting autoregulatory mechanisms within the NFI gene family. In this study, we have shown that the combination of NFI family expression might potentially influence the phenotypes of the Nfix mouse models. Nfia and Nfib, but not Nfic, change their expression pattern in order to possibly compensate for their respective Nfix Del2 and Del140 frameshift mutations in the unaffected Nfix+/Del2, Nfix+/Del140, and NfixDel140/NfixDel140 mice, while Nfia, Nfib, and Nfic expression was unaltered in the unaffected Nfix+/Del24 and NfixDel24/Del24 mice, thus suggesting that the in\u2010frame Nfix Del24 mutations might potentially be tolerated and are probably not as damaging as a frameshift mutation. Moreover, the lack of functional redundancy from the Nfix paralogs in the NfixDel2/Del2 mice as well as the presence of intermediate levels of aberrant mutant NFIX Del2 protein might possibly account for the more severe phenotype observed in the NfixDel2/Del2 mice.Cell autonomous, monoallelic, and stochastic variation in gene expression, as well as functionally redundant paralogs, could also account for phenotypic variability.31 RedNfixDel2/Del2 mice represent a mouse model for MSS in which patients commonly have: reduced growth rate; short stature; craniofacial defects; osteopenia with increased fracture rate and kyphosis that normally worsens in puberty and adolescence and that is possibly aggravated by decreased bone density; and anxiety and intellectual disability due to nonspecific rain abnormalities. Thus, the NfixDel2/Del2 mice had; short stature; reduced growth and TTM; kyphosis; shortened skull; marked porosity of the vertebrae; reduced BMC; shorter vertebrae height and femur length; reduced plasma CTX and P1NP concentrations but increased total ALP activity, indicative of abnormal bone function; and raised plasma urea and total bilirubin levels, suggestive of renal and hepatic dysfunction, which merits further investigation. Furthermore, NfixDel2/Del2 mice had enlarged anterior cingulate, somatosensory and retrosplenial cortices, and ventricles but reduced dentate gyrus . Moreover, the reduction in plasma CTX concentrations in the NfixDel2/Del2 mice may suggest abnormal osteoclast activity and function, which merits further investigation. Moreover, the paradoxical increased plasma ALP activity, which is a marker of bone turnover, in association with reduced plasma concentrations of CTX and P1NP, which are markers of bone resorption and bone formation respectively, in the NfixDel2/Del2 mice suggests additional extraskeletal origin for the raised ALP activity such as the kidneys or intestine, but not liver as mice, in contrast to humans, express little or no ALP in the liver, and a search for additional renal or intestinal abnormalities in MSS may be warranted. Thus, it seems possible that MSS patients may have renal, intestinal, and hepatic dysfunction, and that there may be more similarities with the NfixDel2/Del2 mice.us Table\u00a0. HoweverNfixDel2/Del2 mice have similarities and differences when compared to two previous homozygous Nfix\u2010deficient mouse models that had targeted deletions of exon 2 (Table\u00a0Nfix\u2010deficient mice (NfixlacZ/lacZ) were viable and had: growth retardation; an inability to fully open eyes; ataxic gait; feet\u2010clasping posture when lifted by their tail\u00a0indicating neurological abnormalities; gastrointestinal defects; brain malformations consisting of hydrocephalus and partial agenesis of the corpus callosum; defects in endochondral ossification, reduction in trabecular bone formation and calcification; thinning of cranial bones; kyphotic deformation of the spine; and early postnatal death between 3 and 4\u2009weeks of age. The other homozygous Nfix\u2212/\u2212 mice showed; failure to thrive and grow when on a standard lab chow diet; delayed eye and ear opening; leg\u2010clasping phenotypes indicating neuroanatomical defects; increased brain weight due to expansion of the cortex and entire brain along the dorsal ventral axis; aberrant neocortex, cerebellum, hippocampus, and spinal cord formation; and an abnormal ventricular cell population due to excessive generation of Pax6\u2010expressing ventricular cells with hydrocephalus. Liver and kidney phenotypes were not assessed in these two previously reported Nfix\u2010deficient mouse models, although it is important to note that NfixlacZ/lacZ mice had gastrointestinal defects. Importantly, the NfixDel2/Del2 mice are not Nfix\u2010deficient but instead have aberrant Nfix transcripts that escape NMD and lead to the production of mutant truncated NFIX protein, which is representative of MSS. Interestingly, MSS patients are heterozygous for NFIX mutations, and this contrasts with Nfix+/Del2 mice, which are normal, while developmental, skeletal, cranial, neural, hepatic, and renal abnormalities are observed in NfixDel2/Del2 mice, which could account for their reduced viability. However, phenotypic differences between organisms are not uncommon and can be attributed to allelic variation, modifier genes, genetic variations, genetic background, environmental conditions, and reduced sensitivity of assays, such as behavioral assays, in animal models versus in patients. For example, the autosomal dominant disorder spondyloepimetaphyseal dysplasia, Missouri type (SEMDMO) in humans, is due to a heterozygous matrix metalloproteinase 13 (MMP13) missense F56S mutation, whereas heterozygous Mmp13+/\u2212 mice deleted for exons 3, 4, and 5 have normal growth plates, but the homozygous Mmp13\u2212/\u2212 mice have defects in growth plate cartilage and delayed endochondral ossification.Our ) Table\u00a0. Thus, hNfix mouse models with three different targeted mutations in exon 7 of the Nfix gene, which are representative of the most frequent NFIX mutations observed in MSS patients. The three mouse models, although being on the same genetic background, have differing phenotypes and viability. While the NfixDel2/Del2 mice have some similarities to previously reported Nfix deficient mouse models, they also have a number of other phenotypes that are consistent with MSS. Further studies of the NfixDel2/Del2 mice will help better understand the role of NFIX mutations that result in dominant\u2010negative NFIX proteins and give rise to MSS, as well as provide useful resources for testing potential future treatments.In summary, in this study we report three Kreepa G. Kooblall: Conceptualization; data curation; formal analysis; investigation; methodology; validation; writing \u2013 original draft; writing \u2013 review and editing. Mark Stevenson: Supervision; writing \u2013 original draft; writing \u2013 review and editing. Michelle Stewart: Data curation; methodology; project administration; writing \u2013 review and editing. Lachlan Harris: Conceptualization; data curation; formal analysis; investigation; methodology; validation; writing \u2013 review and editing. Oressia Zalucki: Conceptualization; data curation; formal analysis; investigation; methodology; validation; writing \u2013 review and editing. Hannah Dewhurst: Data curation; formal analysis; investigation; methodology; validation; writing \u2013 review and editing. Natalie Butterfield: Data curation; formal analysis; investigation; methodology; validation; writing \u2013 review and editing. Houfu Leng: Data curation; formal analysis; investigation; methodology; validation; writing \u2013 review and editing. Tertius A. Hough: Data curation; investigation; methodology; validation; writing \u2013 review and editing. Da Ma: Data curation; formal analysis; investigation; methodology; software; validation; writing \u2013 review and editing. Bernard Siow: Conceptualization; data curation; formal analysis; investigation; methodology; software; validation; writing \u2013 review and editing. Paul Potter: Writing \u2013 review and editing. Roger D. Cox: Writing \u2013 review and editing. Stephen D.M. Brown: Writing \u2013 review and editing. Nicole Horwood: Supervision; writing \u2013 review and editing. Benjamin Wright: Data curation; formal analysis; investigation; methodology; software; validation; writing \u2013 review and editing. Helen Lockstone: Conceptualization; data curation; formal analysis; investigation; methodology; software; validation; writing \u2013 review and editing. David Buck: Software; supervision; writing \u2013 review and editing. Tonia Vincent: Supervision; writing \u2013 review and editing. Fadil M. Hannan: Conceptualization; writing \u2013 review and editing. J.H. Duncan Bassett: Conceptualization; funding acquisition; supervision; writing \u2013 review and editing. Graham R. Williams: Conceptualization; funding acquisition; supervision; writing \u2013 review and editing. Kate E. Lines: Supervision; writing \u2013 original draft; writing \u2013 review and editing. Michael Piper: Conceptualization; funding acquisition; supervision; writing \u2013 review and editing. Sara Wells: Resources; writing \u2013 review and editing. Lydia Teboul: Conceptualization; data curation; formal analysis; investigation; methodology; resources; supervision; validation; writing \u2013 review and editing. Raoul C. Hennekam: Conceptualization; funding acquisition; supervision; writing \u2013 review and editing. Rajesh V. Thakker: Conceptualization; funding acquisition; resources; supervision; writing \u2013 original draft; writing \u2013 review and editing.https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/jbm4.10739.The peer review history for this article is available at Data S1. Supporting Information.Click here for additional data file.Data S2. Supporting Information.Click here for additional data file."}
+{"text": "Radix glycyrrhizae) is a plant root extract widely used in various applications, including cosmetics, food supplements, and traditional medicine. It has a long history of medicinal use in different cultures due to its diverse pharmacological properties. Licorice has traditionally been used for treating gastrointestinal problems, respiratory infections, cough, bronchitis, arthritis, and skin conditions. In recent years, the potential therapeutic benefits of licorice for oral health have gained significant interest. This paper aims to provide a comprehensive review of the effects of licorice extracts and their bioactive components on common oral diseases such as dental caries, periodontitis, halitosis, candidiasis, and recurrent aphthous ulcers. The chemical composition of licorice has shown the presence of several bioactive compounds such as glycyrrhizin, glabridin, isoliquiritigenin (ISL), and licochalcone exhibiting various pharmacological activities, including anti-inflammatory, antimicrobial, antioxidative, and immunomodulatory effects. Interestingly, in certain patients, licorice has shown a promising potential to inhibit the spread of viruses, prevent biofilm formation, reduce inflammation, boost immune responses, alleviate pain, and exert antioxidative effects. In this review, we provide a brief overview of the current understanding of licorice\u2019s therapeutic benefits in the treatment of oral ailments, emphasising its potential as an alternative treatment option for oral diseases. Further research is warranted to explore its efficacy, safety, and clinical applications using placebo-controlled clinical trials.Licorice ( Radix glycyrrhizae) is obtained from the roots of Glycyrrhiza glabra L. (G. glabra), a small, flowering, bushy perennial plant of the family Fabaceae that is native to Western Asia, North Africa, and Southern Europe [Glycyrrhiza uralensis Fisch, referred to as Chinese licorice, which is a perennial herb of the family Fabaceae native to Asia [Licorice root  on oral health and disease. The search was also extended to include reference lists of original and review articles. The search was limited to studies published between 1973 and 2023 in the English language. Unpublished data were not included. Two reviewers assessed the quality and characteristics of the selected studies. The search used a combination of keywords, including \u201clicorice\u201d, \u201cGlycyrrhiza glabra\u201d, \u201coral health\u201d, \u201coral disease\u201d, and \u201coral lesions\u201d.A search of the literature was conducted using four databases: PubMed, Google Scholar, ScienceDirect, and Web of Science, to find relevant articles on the effects of licorice : antimycobacterial, uterine relaxant and analgesic, antitussive activity.Licochalcone: anticancer, antimalarial.Liquiritigenin: corticosteroid activity, antimicrobial.Glycyrrhizic acid (GL): antiulcer, antiallergic, antiviral activity, antihyperglycemic.18-\u03b2-glycyrrhetinic acid (GA): memory-enhancing activity, corticosteroid activity, antiviral activity, immuno-stimulating activity.Glycyrrhizin: corticosteroid activity, antiallergic, hepatoprotective, anti-inflammatory, antiviral activity, antihyperglycemic, immuno-stimulating activity.Licocoumarin: Uterine relaxant and analgesic.These activities are not exclusive to the chemical components listed, and some activities are listed under different components due to their various potential effects .A summary of the chemical bioactive composition, structure, and function is demonstrated in GL and glabridin can be extracted from licorice using different solvents, such as water, methanol, ethanol, acetonitrile, and chloroform. Some studies have shown that the optimum condition for this extraction would be under 50 \u00b0C, and it takes around 60 min . The enzThe active ingredients of licorice show wide biological activities that can boost the body\u2019s immunity and protect against different diseases. It has antibacterial, antiviral, anti-inflammatory, analgesic, and antioxidant properties, as well as many others . DiffereGlycyrrhizic acid (GL) has been shown to have potential immunomodulatory properties by inhibiting the spread of viruses and disassembling their particles . Its antStaphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans . This occurs due to inhibiting biofilm formation, preventing yeast\u2013hyphal transition, reducing toxin production, and decreasing the production of \u03b1-hemolysin [Helicobacter pylori [Licorice aqueous extract, ethanol extract, and supercritical fluid extract have powerful inhibitory effects on both Gram-positive and Gram-negative bacteria, including emolysin . Licochar pylori .Glycyrrhizin is a well-known anti-inflammatory ingredient that has been shown to enhance the duration of plasma recalcification and to extend thrombin and fibrinogen coagulation time in vitro, making it the first plant-based thrombin inhibitor , by reduG. glabra polysaccharide fractions have shown immuno-stimulating activity, boosting the immune response [Glycyrrhizin is also capable of activating macrophages. response . Glycyrrresponse . Both coresponse .Licorice\u2019s inhibitory effects on the functions of cyclooxygenase-2 (COX-2), high mobility group box 1 protein (HMGB1), and gap junction/connexin raise the possibility that some of the plant\u2019s components may be useful for treating pain . PainfulG. glabra exhibit potent 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging action, as well as the capacity to stop the peroxidation of microsomal lipids [Significant antioxidant activity is present in licorice phytochemicals. Licorice prevents neutrophils from producing reactive oxygen species (ROS) at the site of inflammation. The licochalcone B and D found in l lipids ,28.By increasing mitochondrial permeability transition, GL has been found to support the proapoptotic pathway, which, in particular, encourages tumour cells to undergo apoptosis . Twelve Because of its many biological advantages, such as its anti-inflammatory, antioxidative, and antibacterial activities, licorice has been extensively researched . This isG. glabra. The first property investigated was the antiadherent property of glycyrrhizin that inhibits the glucosyltransferase activity of Streptococcus mutans, which is required for biofilm formation [Studies carried out in recent decades have confirmed the anticariogenic role of natural compounds extracted from licorice, specifically glycyrrhizin and triterpenoid saponin glycosides in ormation . Severalormation ,32,33. Oormation , but addG. glabra gum paint at a 10% concentration experienced a considerable reduction in gingival bleeding, probing pocket depth, and attachment loss [G. glabra have been shown to have strong antiadhesive effects against Porphyromonas gingivalis  [The use of herbal formulations is seen as an attractive alternative to conventional antibiotics. Thus, the use of licorice in periodontal therapy has been studied. Licorice extract demonstrated potent anti-inflammatory effects by inhibiting inflammatory cytokines ,36. A reent loss . A comprent loss . As theygivalis) .C. albicans infection [G. glabra against C. albicans have previously been reported [C. albicans [C. albicans; it was found that glabridin and licochalcone A both blocked the yeast\u2013hyphal transition. Additionally, they claimed that nystatin and each of these substances could work together to combat C. albicans [Several animal studies have been conducted investigating the antifungal effect of licorice compounds. In one study, glycyrrhizin treatment increased mice\u2019s resistance to nfection . The antreported . In an ialbicans . Anotheralbicans . Investialbicans .Various studies have been published on the effectiveness of licorice in reducing aphthous ulcers\u2019 healing time and controlling pain. Using a mouthwash with a deglycyrrhizinated licorice extract for two weeks seemed to reduce discomfort and hasten the healing of aphthous ulcers . The effDue to its antiviral activity, the potential use of licorice in reducing the severity and duration of herpes simplex virus (HSV) infections has been investigated. It has been demonstrated that by creating an environment that was resistant to HSV1 replication, glycyrrhizin possessed its anti-HSV1 action . A cliniThe effect of licorice on xerostomia studied in haemodialysis patients in a randomised controlled trial (RCT) has shown that only the licorice mouthwash offered subjective xerostomia alleviation. Clean water and licorice-containing mouthwash have both improved the objective measurement of salivary flow rate . This imA Japanese study has shown that patients with OLP who tested positive for HCV have shown clinical improvement in OLP after the intravenous administration of glycyrrhizin ,57. IntrP. gingivalis, Prevotella intermedia, and Solobacterium moorei and thus potentially manage halitosis [P. gingivalis bacterial growth has been studied. There are some safety concerns about the available commercial mouthwashes, necessitating the search for more natural ingredients [It has been shown that licorice extract and the two isolates, licoricidin and licorisoflavan, can serve as natural components that have the ability to decrease the production of bacterial volatile sulphur compounds by alitosis . Compareredients .G. glabra extract can be used effectively in the prevention and treatment of oral mucositis following radiation and chemotherapy. It is advantageous in two ways: first, there are no interruptions of their chemotherapy or radiation; second, their food intake is not significantly impacted, allowing patients to maintain their nutritional state [w/v) was used as a mouthwash before and right after each session of radiation therapy [In cancer patients, particularly those with head and neck cancer, al state . In an Ral state . Anotheral state . A clini therapy .Glycyrrhiza inflata by causing apoptosis via the mitochondrial pathway [Isoliquiritigenin (ISL) is a flavonoid with a significant therapeutic potential for treating adenoid cystic carcinoma and the potential to be used as a cancer chemotherapeutic agent . In anot pathway .Licorice was suggested as a treatment for OSF because it demonstrated antifibrotic effectiveness in human fibroblast cell lines .2 alone, licorice extract had a much greater inhibitory impact against Enterococcus faecalis  [G. glabra was added [E. faecalis compared to G. glabra and Ca(OH)2, despite the reduction in the number of colonies in all types of irrigation [The usefulness of licorice as a root canal irrigant and medication has only been studied in a small number of in vitro investigations. In one of these studies, it was demonstrated that, compared to Ca(OH)aecalis) . Furtheras added . A recenrigation .An illustration of the effects of licorice on oral health is shown in In the past, in the United States, the production of several dietary supplements, including licorice, was not strictly controlled. A 100 mg/day maximum limit for glycyrrhizin consumption was suggested by the European Union based on investigations with human volunteers. The Dutch Nutrition Information Bureau warns against exceeding a daily glycyrrhizin intake of 200 mg, or 150 g of licorice candy. Licorice fluid extracts have a glycyrrhizin content of 10\u201320% and typically include 200\u2013800 mg. About 2% of frequent users ingest more GA than the recommended daily limit of 100 mg .For most applications, a high level of GL in the blood with fewer side effects is recommended. A dosage of 200 mg powder of deglycyrrhiznated licorice (DGL) dissolved in 200 mL of warm water used as mouthwash four times daily has been suggested . GlycyrrAlthough licorice root is a natural, bushy herb that is frequently used as a flavouring agent in food and beverages as well as for therapeutic purposes, it can have certain systemic negative effects when ingested in high quantities or used for extended periods of time. The Food and Drug Administration (FDA) has designated licorice as \u201cGenerally Recognised as Safe\u201d if used carefully by those who are not allergic to glycyrrhizin. Licorice consumption in excess can result in hypertension, hypokalaemia, rhabdomyolysis, respiratory problems, muscle paralysis, hyperparathyroidism, abrupt renal failure, and encephalopathy. The World Health Organisation (WHO) states that licorice at a dose of 100 mg per day can be consumed without risk. Due to the antiplatelet and anticoagulant properties of licorice, individuals who take anticoagulants for cardiovascular or cerebrovascular illnesses along with herbal treatments containing licorice may experience a risk of excessive bleeding . GlycyrrLicorice raises blood pressure by inhibiting the 11-beta-hydroxysteroid dehydrogenase enzyme. This enzyme is involved in metabolising the hormone cortisol, which controls blood pressure. When licorice blocks this enzyme, the body\u2019s cortisol levels rise, which causes sodium and water retention; as a result, blood pressure is increased .Licorice can make the body lose potassium, which is needed for healthy neuron and muscle function. The increased activity of the hormone aldosterone, which controls the body\u2019s sodium and potassium levels, is the reason for potassium loss. As licorice prevents aldosterone from being broken down, sodium and water are retained, raising blood pressure, while potassium is expelled in urine ,80.Licorice can impair the adrenal gland\u2019s ability to produce hormones like cortisol and aldosterone. Men\u2019s testosterone levels may drop as a result of this interference, which could result in symptoms including weariness, erectile dysfunction, and a reduced libido. Licorice can raise oestrogen levels in women, which can result in mood swings, sensitive breasts, and irregular menstruation .Glycyrrhizin, the key ingredient in licorice, can harm the liver if ingested in significant quantities over an extended period of time. Although it is uncommon, this effect can happen in people who ingest significant doses of licorice supplements or teas .Other side effects were associated with gastrointestinal, skin and subcutaneous tissue, and hepatobiliary disorders . It is iWhen used frequently or in excessive doses, licorice might have certain unfavourable dental effects. The licorice ingredient glycyrrhizin in particular might result in the following dental problems:When taken in its natural state, licorice root can offer certain advantages for dental health. However, licorice candies and other licorice-flavoured goods that are high in sugar can contribute to dental decay. Sugar is metabolised by oral bacteria to create acid, which can erode tooth enamel and cause cavities .Over time, especially if ingested in large quantities, licorice sweets and other dark-coloured foods can discolour the teeth and tongue temporarily and result in a brownish appearance. This discolouration could last longer and appear darker if licorice is used in combination with tobacco products .If one routinely ingests licorice, it is crucial to take precautions to preserve their dental health. This can involve using fluoride toothpaste, brushing and flossing often, and going to the dentist for routine examinations. The acid produced can be neutralised, and food residues can be washed away by drinking water .Licorice should not be used by patients who have uncontrolled hypertension. Additionally, the primary active ingredient in the root, GA, can cause what seems to be pseudohypoaldosteronism . This caDeutch and colleagues have conducted a comprehensive review summarising recent research in this area . SimilarEuphorbia kansui (Gan Sui), or Flos genkwa (Yuan Hua), according to the theories of traditional Chinese medicine. These four plants, taken along with licorice, may weaken liver function and cause cardiac toxicity [The pharmacokinetics and pharmacodynamics of many drugs may vary when taken simultaneously with glycyrrhizin or GA. In general, due to their potential drug interactions, GZ, GA, or related drugs should be used with caution when combined with other medications . It has toxicity .Licorice root powder ;Licorice root extract ;Licorice lozenges ;Licorice mouthwash ;Licorice tea .It is necessary to keep in mind that regional restrictions and the availability of particular licorice products could vary. For advice on where to find licorice goods in the KSA, it is usually preferable to speak with a medical expert or a nearby merchant. It is also crucial to make sure that any licorice items you buy are safe to eat and from reliable suppliers.Health food stores (Atara Shops): some health food stores in the KSA may carry licorice root powder, licorice root extract, licorice lozenges, and other licorice products.Pharmacies: licorice root extract, licorice lozenges, and licorice mouthwashes may be available at pharmacies in the KSA.Supermarkets: licorice tea may be available at some supermarkets in the KSA, particularly in the health food or tea sections.Online retailers: there are several online retailers that sell licorice products, including licorice root powder, licorice root extract, licorice lozenges, licorice mouthwash, and licorice tea, that are local or can be shipped to the KSA.The availability of licorice products in the KSA may vary depending on the region and local regulations. However, listed below are a few places where you may be able to find licorice products:In conclusion, licorice possesses numerous biological advantages, resulting in extensive research on its potential therapeutic applications. Studies have investigated the use of licorice and its bioactive components in the prevention and treatment of different oral diseases. Licorice has shown anticariogenic effects, anti-inflammatory effects, and the ability to reduce discomfort and promote healing in some oral conditions when used within mouthwashes or as bio-adhesive patches. Overall, licorice holds promise and power as a natural therapeutic agent for various oral diseases. However, more clinical trials and research are necessary to validate its effectiveness, explore its full therapeutic potential, and establish standardised treatment protocols. These studies may encourage different pharmaceutical companies to manufacture a wider range of licorice forms. Undoubtedly, this should be conducted in a proper, well-controlled manner."
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