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+{"text": "The bovine brachyspina syndrome is a recently reported malformation in the Holstein breed. The aetiology of this syndrome is unknown, but its occurrence following breeding between genetically related and phenotypically normal cattle may indicate that it is an autosomal recessively inherited disorder. Three cases are reported and compared to the originally reported case.Two Danish cases and a Dutch case are described. The calves were delivered following a slightly prolonged gestation period. Gross lesions consisted of growth retardation, significant shortening of the entire spine and long and slender limbs. Additionally, inferior brachygnatism and defects of several internal organs were recorded. The cases were diagnosed as having the brachyspina syndrome based on the presence of essential lesions. The parents of each case were genetically related and linked to the first reported case by a common ancestor.The findings support the hypothesis that the brachyspina syndrome in Holstein cattle is inherited autosomal recessively and illustrate some of the assumed phenotypical variation of this syndrome. The brachyspina syndrome may be an emerging disease in the Holstein breed. Recently, a new congenital lethal syndrome was reported in a Danish Holstein calf case DK1) [ [1]. TheHere, we report two additional cases from Denmark cases DK2 and DK3) obtained through the Danish Bovine Genetic Disease Programme [ and DK3 A female Holstein calf with a body weight of 10.8 kg Fig. . The heaThe volume of the thoracic cavity was reduced and almost occupied by an enlarged heart. The ribs radiated from the spine with non-parallel intercostal spaces. The number of ribs was reduced to 11 in each side, but they were difficult to separate due to the presence of extensive synostoses. The lungs had congenital diffuse atelectasis, were of reduced size and compressed by the heart. Low-grade hydrothorax and hydropericardium was found. The heart had a round shape due to dilation of the right ventricle and hypertrophy of the corresponding myocardium. A high interventricular septal defect was present. Truncus pulmonalis and aorta arose separately from the right ventricle (dextroposition of the aorta). The basis of aorta was dilated.The volume of the abdominal cavity was also reduced. The anterior part was occupied by an enlarged liver. Colon descendens had agenesia at the entrance to the pelvic cavity and a stenosis approximately 2 cm orally to the agenesia. The prestenotic segments were dilated due to accumulation of meconium, while the poststenotic segments only contained mucus. The kidneys had bilateral hypoplasia with a length of approximately 3 cm. The right uterine horn was hypoplastic. The adrenal glands and ovaries were present at their normal position.Multiple tissues were taken for histopathology and processed as previously reported . The tisThe calf was tested negative for bovine virus diarrhea virus (BVDV) by cell culture and for antibodies against BVDV by an ELISA technique .Examination of six-generation pedigree showed that the parents were genetically related through a common ancestor to the parents of the previously observed case (DK1) Fig. .This case was obtained retrospectively from the archive at the Department of Veterinary Pathobiology. This Holstein male calf was delivered stillborn in February 2003 at gestation day 299 by a two-year-old heifer. The calf had a body weight of 13.6 kg, mild inferior brachygnatism, and severely reduced length of the spine Fig.  due to wSLC35A3 gene, which causes the complex vertebral malformation syndrome (CVM) in Holsteins, and found homozygous normal.The calf was examined for BVDV and foetal antibodies with negative results. The calf was genotyped for the V180F mutation in the The pedigree was compared to pedigrees of the two other Danish cases. The parents were genetically related to the other cases Fig. .A stillborn male Holstein calf with a body weight of 10.5 kg was delivered in August 2006 at gestation day 288 by a 2-year-old heifer. The calf was severely growth retarded with significant shortening of the crown-rump length, thoracic kyphosis, and relatively long thin limbs. The length of the mandible was reduced  prior to processing. Vertebral lesions were characterized by widespread disturbed segmentation and organisation. Intervertebral disc development was mostly absent although protrusion of collagen from the periphery into the epiphyseal anlage was occasionally present. Consequently, adjacent vertebrae often share a common epiphysis with only one ossification centre. The growth zones were highly irregular towards the diaphyses with frequent abnormal arrangement of chondrocytes. The diaphyses were in some areas intersected by cartilaginous cores dividing the spongiosa into inlets of spongious bone. The appendicular skeleton was not examined. The kidneys were dysplastic with immature tubules, dilated tubules, intraluminal crystals and interstitial fibrosis.Multiple tissues were taken for histopathology, processed by routine laboratory methods and stained by haematoxylin and eosin. Bone tissues were decalcified by OsteomollSLC35A3 gene was performed after retrieving DNA from formalin fixed and paraffin embedded liver and heart tissue [The calf tested negative for BVDV by an antigen ELISA technique . Genotyping for the V180F mutation in the Pedigree analysis demonstrated that the parents of the Dutch case were genetically related to the three Danish cases Fig. .The gross morphology of the three cases  was in many aspects similar to the lesions found in the original case (DK1) , have deThe brachyspina syndrome and CVM are malformations occurring in the Holstein breed. Although these syndromes share several features as vertebral malformation, growth retardation and internal organ malformation, discrimination between these defects can be done morphologically. Calves suffering from the brachyspina syndrome have a mean body weight of 10.3 kg compared to a mean body weight of around 25 kg in CVM-affected calves . VertebrThe genealogical examination showed that the cases occurred in a familial pattern and that the parents of each calf were genetically related. A common ancestor to all parents (sire A) was found in generation VI Fig. . The occJSA performed the examination of the Danish cases and drafted the manuscript. KP examined the Dutch case and participated in the drafting. Both authors read and approved the final manuscript."}
+{"text": "Tumour cells were conjugated with hapten dinitrophenyl, mixed with Bacille Calmette Gu\u00e9rin and irradiated to 110\u2009Gy. Both disease free survival and overall survival were found to be correlated with intensity of evolving delayed type hypersensitivity to subcutaneous injection of unmodified melanoma cells. Patients with a delayed type hypersensitivity reaction of \u2a7e10\u2009mm had a median disease free survival of 17 months (mean 35 months) and a mean overall survival of 63 months (median not reached). In contrast, patients with a negative or weak delayed type hypersensitivity had a median disease free survival of 9 months , and a median overall survival of 16 months . Stage III patients with a positive delayed type hypersensitivity reaction had an improved disease free survival of 16 months and a mean overall survival of 38 months, whereas patients with a negative delayed type hypersensitivity had a median disease free survival of 7 months  and a median overall survival of 16 months . The adjuvant administration of autologous melanoma vaccine was associated with improved disease-free and overall survival to selected patients who successfully attained anti-melanoma reactivity as detected by positive delayed type hypersensitivity reactions to unmodified melanoma cells.This study evaluates the overall survival and disease free survival of melanoma patients that were treated with an autologous melanoma cell vaccine, administered as a post-operative adjuvant. Included are 43 patients with totally resected metastatic melanoma , with a median follow up of 34 months (6\u201362). The treatment consisted of eight doses of a vaccine made of 10\u201325\u00d710British Journal of Cancer (2002) 86, 1534\u20131539. DOI: 10.1038/sj/bjc/6600251www.bjcancer.comCancer Research UK\u00a9 2002  Patients suffering from malignant melanoma with metastases to regional lymph nodes, satellites and metastases in-transit are defined as stage III (The new American Joint Committee on Cancer (AJCC) classification) . FurtherMelanoma vaccines are an attractive alternative approach for an adjuvant treatment. It is true that their value in metastatic disease is limited: the response rates range only from 5 to 15%, and these positive responses are mainly limited to subcutaneous metastases (One approach of active immunotherapy has been developed by Berd and colleagues. Their vaccine is composed of autologous melanoma cells conjugated with a hapten-dinitrophenyl (DNP), irradiated and mixed with Bacille Calmete Gu\u00e9rin (BCG). They administered this vaccine as an adjuvant treatment to patients with AJCC stage III and stage IV resectable nodal disease. In comparison to control patients, this method has yielded improved disease free and overall survival  Twenty-eight patients with the modified AJCC stage III cutaneous malignant melanoma metastatic to the regional lymph node , five of them also having satellites and metastases in transit (stage IIIC); (2) 15 patients with cutaneous malignant melanoma metastatic to viscera (AJCC stage IV) or with metastatic non-cutaneous melanoma . For clinical details see Table 1To be eligible for our study, patients had to have undergone complete surgical excision of metastatic disease and be found disease free based on whole body CT scan performed less than 3 weeks prior to surgery. In addition, patients had to have a positive skin reaction to at least one out of four recall antigens: PPD, candidin, trychophytin, and tetanus toxoid. A positive reaction was defined as an erythema larger than 5\u2009mm in diameter. The study was approved by the Institutional Review Board and informed consent was obtained before any therapy was administered.Four of the patients received radiotherapy after surgery and prior to the initiation of the vaccinations. One of these received whole brain irradiation for a single brain metastasis, and the other three received radiation treatment to the abdominal wall or to cervical lymph nodes for close margins and for extracapsular lymph node invasion. We started the vaccine procedure for these four patients 1 month after their radiotherapy was completed. Although these patients received radiotherapy prior to their inclusion in our study, they remained immune competent, which we ascertained by their skin tests. After the resection of their liver metastases, two patients with ocular melanoma had received four courses of an intra-arterial hepatic infusion of fotemustine before we started the vaccine procedure. Four patients were referred to us with metastatic disease after they failed to respond to either low dose (one patient) or high dose (three patients) interferon-\u03b1 adjuvant therapy.6\u2009cells, cell suspensions were put into culture bottles with Dulbecco's Modified Eagle's Medium , 10% foetal calf serum (Gibco BRL), HEPES (1\u200a:\u200a500), pen-strep (1\u200a:\u200a100) and glutamin (1\u200a:\u200a100) and expanded to the required number, necessary for preparation of at least eight vaccine doses, of 10\u201325\u00d7106\u2009cells each. Culturing the cells resulted in the preferential selection of melanoma cells, so that the vaccine finally consisted of purified melanoma cells.Tumour specimens were procured fresh and sterile. For large tumour bulks, cells were extracted by mechanically or by enzymatic dissociation with collagenase and DNAse , frozen in a controlled rate freezer, and stored in liquid nitrogen in a medium containing 2.5% human albumin and 20% DMSO until needed. If the total cell yield was less than 200\u00d7103 in size, put on a 60\u2009mm2 plastic dish (Nunc), and then glued to the dish with drops of foetal calf serum (Gibco BRL). Culture medium was added to the dish 24\u2009h later. When cell growth was observed at the periphery of tumour pieces, we transferred the cell mass to a culture bottle. To assure melanocytic progeny, staining of slides covered by cultured cells was performed with monoclonal antibodies S-100 and HMB-45, using the alkaline phosphatase anti-alkaline phosphatase (APAAP) reaction . Positive staining of more than 50% of cells with at least one of these antibodies was required.When we were unable to dissociate the cells mechanically \u2013 as in the case of a small specimen or a dense connective tissue \u2013 tumours were cut into pieces of less than 1 mm6\u2009ml\u22121, mixed, incubated for 30 min and washed again. Prior to vaccination an appropriate amount of BCG was added, starting with a dilution of 1\u200a:\u200a50 and reaching 1\u200a:\u200a500\u20131000, according to the resulting granuloma at vaccination site.On treatment day, the cells were thawed, washed and irradiated to 110\u2009Gy. A sample was stained with Trypan blue and counted after irradiation. Conjugation of melanoma cells with DNP was performed by the method of 2 of intravenous cyclophosphamide. On days 4 and 5, patients were sensitised to DNP by applying 0.1\u2009ml of 2% DNP dissolved in acetone-corn oil (Sigma) topically to the inner aspect of the arm. On day 18, patients received a second dose of 300\u2009mg\u2009m2 cyclophosphamide. On day 21, the prepared vaccine was injected into three adjacent sites on the upper arm or thigh, avoiding limbs where lymph node dissection had been previously performed. Seven additional doses of the vaccine were administered at intervals of 21\u201328 days. Before injecting the second dose of the vaccine, a third dose of 300\u2009mg\u2009m3 of cyclophosphamide was administered i.v. Patients were evaluated periodically every 2 months. These evaluations included a physical examination, a complete blood count, and liver function tests. A chest X-ray was performed every 3 months and a total body CT scan was made every 6 months.6 unmodified melanoma cells irradiated to a dose of 170\u2009Gy. To avoid contamination with foreign proteins and chemicals, the cells were triple washed before injection. DTH was measured by the maximal diameter of erythema at 48\u2009h after the injection of the vaccine. Erythema smaller than 5\u2009mm was defined as a negative reaction. We arbitrarily chose the value of 10\u2009mm of erythema to discriminate between weak DTH (<10\u2009mm) and strong DTH (\u2a7e10\u2009mm). Patients were also skin tested to 3\u00d7106 unmodified autologous lymphocytes.Skin testing to evaluate delayed type hypersensitivity to autologous melanoma cells was performed by intradermal injection of 1\u20133\u00d710The analysis focused on the impact of DTH on OS and DFS. The OS and DFS were estimated by the Kaplan-Meier's method. Significance was calculated by the log rank test. To further validate the effect of DTH on survival, a Cox proportional hazards method was used to adjust for the following covariates: age , gender, time elapsing from surgery to the first vaccination , and stage . A relative risk (RR) of more than 1 denotes the increased risk of death or recurrence for patients with a negative or a weak DTH response as compared to patients with a strong positive DTH, calculated by the Cox model. The model was built for all patients (adjusted for stage) and for stage III patients only, because of the importance of this group.All of the patients that we entered into the study have been included in our analysis. Thirty-three (73%) of these patients completed full schedules; 11 (27%) of the patients suffered disease recurrence while on treatment. Median follow up period is 34 months (range: 6\u201362)vs DTH \u2a7e10\u2009mm (strong positive DTH). Twenty-six patients (60%) attained strong positive DTH whereas 17 patients (40%) had only weak or negative DTH. Patients who failed to attain a strong DTH had an increased risk of 15  3\u201376; P=0.001) to die of their disease and a relative risk of 4.5  to have a disease recurrence. Current patient status with respect to DTH response is shown in Figure 1Using the Kaplan-Meier method and the log rank test, the single parameter that was strongly correlated in a univariate analysis with overall survival (OS) and disease-free survival (DFS) was the DTH response. The Cox proportional hazards model, adjusted for age, gender, time from surgery and stage confirmed this finding  and the RR for recurrence was 4.5 .The vaccine was administered to 28 AJCC stage III patients whose median DFS was 12 months and median OS was 31 months. Patients that attained a DTH reaction >10\u2009mm had a median DFS of 16 months and a mean OS of 38 months . Patients with a negative or weak (<10\u2009mm) DTH had a median DFS of 7 months and a median OS of 16 months . This group is too small and heterogenous to be sub analysed. However, even for this small group, differences in OS and DFS according to patients' DTH status almost reach statistical significance. All eight patients with positive DTH are alive.One patient with ocular melanoma metastatic to the liver, who had intra-hepatic artery infusions of fotemustine, had extremely strong DTH reactions (erythema and oedema of >30\u2009mm in diameter); this patient has been disease-free for 47 months. Another patient, with rectal melanoma and metastatic mesenteric lymph nodes, had a DTH reaction of 45\u2009mm. She has been disease free for 54 months; her last DTH testing, done 36 months after the completion of full vaccination schedule, still revealed a 35\u2009mm erythema.No significant systemic toxicity was observed in any of the patients. Vaccination sites resolved leaving depressed atrophic scars.Stage III and IV melanoma patients face a grave prognosis. The overall 2-year survival for patients with lymph node metastases (stage III) is 29\u201338% and their median DFS is 12 months. Patients with distant metastases do even more poorly. Attempts towards improving the dismal prognosis of these patients by adjuvant conventional therapies, that is chemotherapy with DTIC, have not proven to be beneficial. This discouraging situation has stimulated researchers to seek different approaches, of which the use of immunotherapy is one of the most compelling. There are many examples supporting the suggestion that the immune system has an important role in the development of anti-melanoma reactivity. These examples include cases of spontaneous regression of primary and metastatic melanoma, and also the development of vitiligo in melanoma patients. These phenomena have been accompanied by various circulating anti-melanocyte antibodies and melanoma specific cytotoxicity conveyed by tumour infiltrating lymphocytes (Various vaccination strategies against melanoma are based on presenting melanoma-associated antigens (MAAs) or peptides to elicit specific cytotoxicity against tumour cells (P<0.001). A disadvantage of using autologous melanoma vaccines is that it is not always feasible to use the autologous tumour to prepare the vaccine: the tumour specimens may be small, containing only a small number of intact tumour cells. In an attempt to solve this problem, we cultured cells from the original tumours. Using these cell cultures allowed us to enlarge the number of cells available for vaccination. Moreover, by using cultured cells we could minimise or even avoid using tissue degrading enzymes, which might lead to changes in cell surface molecules. By passaging the cultured cells two to five times we assured that the vaccine would include only viable melanoma cells, and that any remaining undesirable enzymes from the dissociation procedure would be diluted and removed. Furthermore, culturing the tumour cells resulted in the preferred reproduction of melanoma cells, thus assuring that necrotic cells and associated non-tumour cells would be excluded from the vaccine.Theoretically, the advantage of using autologous melanoma cell vaccines is that the diverse antigens in the vaccine would be presented in the context of the patient's own Major Histocompatibilty Complex (MHC). This condition is mandatory for the recognition of cytotoxic T lymphocytes (CTLs) . A median DFS of 17 months for patients with a strong positive DTH reaction is significantly better than the median DFS of 9 months for the non-reactive patients . In stagThough we did not originally design our study to discern survival benefit for patients, over a median follow up period of 34 months we attained an overall cancer-related survival ratio of 76% , considerably higher than reported. It is important to note that patients eligible for adjuvant treatment with an autologous vaccine are in an unfavourable sub-group for their stage of disease. For a surgeon to recommend such treatment, stage III patients must have a macroscopic disease (Nb).The correlation between DTH and survival has been observed by the research groups of In conclusion, we found that the adjuvant administration of autologous melanoma vaccine was associated with the improved disease-free and overall survival of selected patients that successfully attained anti-melanoma reactivity as detected by positive DTH reactions to injected unmodified melanoma cells. Melanoma cell cultures permit this method to be used for patients with small tumour specimens and yield a purified tumour cell population. Obviously, our present study was carried out without any control groups. With this compromise, the study still supports the idea that an adjuvant vaccination strategy holds promise for the treatment of high risk melanoma patients."}
+{"text": "P\u2009<\u20090.05) as compared to controls. In parallel, the level of oxidative stress, as indicated by thiobarbituric acid reactive substances (TBARS), was increased in HFS rats by 1.2-fold in the liver in relation to controls and was negatively correlated with PON activity. Differential leukocyte counts in blood showed a significant change in lymphocytes and monocytes profile. In conclusion, these results show that PON1 activity is decreased in fructose-fed insulin-resistant rats on a high-salt diet, which may be associated with increased oxidative stress, leading to inflammation.Paraoxonase 1 (PON1) is a HDL-associated esterase/lactonase and its activity is inversely related to the risk of cardiovascular diseases. The aim of the present study was to evaluate the effect of a high-salt diet on serum PON1 activity in fructose-fed insulin-resistant rats. Adult male Fischer rats were initially divided into two groups. Control (CON), which received a normal salt diet and drinking water throughout the study; high fructose (HF), which received a normal salt diet and 20% fructose supplemented drinking water. After 10\u2009weeks, half of the animals from HF group were randomly switched to a high-salt diet and 20% fructose supplemented drinking water (HFS) for more 10\u2009weeks. Serum PON1 activity was determined by synthetic substrate phenyl acetate. HFS rats showed markedly decreased PON1 activity (HFS rats, 44.3\u2009\u00b1\u200914.4\u2009g/dL versus CON rats, 64.4\u2009\u00b1\u200913.3\u2009g/dL,  Diabetes is a long-term disorder affecting the inner walls of arteries and is characterized by endothelial dysfunction and oxidative modification of low density lipoprotein (LDL) which may induce atherosclerosis . In contAlthough the precise mechanism of PON1 effect is not yet completely known, it may possess anti-atherogenic and anti-inflammatory properties, resulting from its ability to destroy modified phospholipids and to prevent accumulation of oxidized lipids in lipoproteins -5. In diOur laboratory has recently reported that a hypercholesterolemic diet in rats cause a significant reduction of PON1 activity, associated with increased oxidative stress . Once raad libitum to induce insulin resistance. After 10\u2009weeks, HF animals were divided into 2 groups: those that continued to receive only 20% fructose in water and those switched to the high fructose regimen\u2009+\u20098% NaCl in the standard AIN93 diet (HFS). After 20\u2009weeks fasting rats were anesthetized and sacrificed. The experiments were approved by the institutional Ethics Committee with protocol number 068/2011 and were performed in accordance with the principles of the Brazilian College of Animal Experimentation [Twenty-nine Fisher male rats, weighing about 300\u2009g, obtained from the School of Nutrition of the Federal University of Ouro Preto, were housed in a temperature and humidity controlled room with a 12:12-h light/dark cycle (lights on at 06.00\u2009AM). Animals were initially divided into two groups. The control group (CON) received the standard AIN93 diet [entation .To determine the levels of serum components, blood samples were collected in test tubes and centrifuged. Serum total protein, albumin, total cholesterol, HDL-cholesterol and triglyceride were assayed with colorimetric or enzymatic methods using commercially available kits Labtest  # 99, 19, 76, 13 and 87, respectively. The atherogenic index was calculated as follows: total cholesterol - HDL cholesterol/HDL cholesterol.. mol-1. cm-1. The results were expressed in units per milliliter, where 1 U of arylesterase hydrolyzes 1\u2009mmol of phenylacetate per minute. Basal lipid peroxidation status was measured in the liver by TBARS assay [PON activity was measured by a spectrophotometric assay using phenyl acetate as substrate as described by Beltowski et al. . The enzRS assay .Panotic Fast which use essential components dyes of Romanowsky [A drop of blood was used to prepare blood smears, which were stained with manowsky . Counts -value of less than 0.05 was considered statistically significant.The normality of the sample distribution for each continuous parameter was tested with the Kolmogorov-Smirnov test. The significance of any differences in proportions or medians was tested with Kruskal-Wallis test, and in means analysis of variance (ANOVA) was used, followed by Dunns and Tukey test, respectively. Pearson\u2019s correlation coefficients were used to test the correlation among variables. A pA high-salt diet in fructose-fed insulin-resistant rats significantly decreased serum levels of total protein, albumin, total cholesterol and HDL-cholesterol after 20 wk of treatment in relation the control group as demonstrated in Table\u2009Increased oxidative stress was noted in the liver obtained from HFS rats, since TBARS levels were raised by 1.2 times (P\u2009<\u20090.05) as compared to controls  remained unchanged. Salt treatment caused a significant increase in the monocyte profile in HF and HFS rats as compared to the CON rats Table\u2009.In this study we observed a decrease in serum PON1 activity in insulin-resistant rats on a high-salt diet and an association among PON1, oxidative stress and inflammation, which can occur during the development of atherosclerosis.HDL is the most powerful independent negative predictor of cardiovascular events. The protective effects of HDL have been first attributed to its capacity to promote cellular cholesterol efflux from peripheral cells and deliver it to the liver for excretion, a process known as reverse cholesterol transport . FurtherIn our model, serum albumin and protein levels are reduced probably due to protein synthesis disruption in the liver as observed by Shinn and Moon . The damIn rats, unlike in humans and rabbits, plasma triglycerides and cholesterol are transported predominantly by HDL  and frucA previous study has shown that PON1 protects against atherosclerosis, by its ability to reduce macrophage foam cell formation, via reducing oxidative stress and stimulation of cholesterol efflux from macrophages . The preOur dietetic model was chosen due its relevance to human nutrition because it mimics a common Western diet, with high consumption of sugar drinks and salty foods and this study is the first one to show an association between paraoxonase status and salt overload in insulin-resistant rats. This supports the relationship between low PON1 activity and diseases with low antioxidant defense and excessive lipid peroxidation. Paraoxonase may perform protectively in atherogenesis by hydrolyzing some products of lipid peroxidation and consequently by limiting LDL oxidation and foam cell formation . This alIn conclusion, the present study indicates that a high-salt diet in fructose-fed rats leads to lowered serum PON1 activity, with deleterious effects on the antioxidant defense system. Furthermore, we suggest that a change in blood cells profile would reflect the oxidative stress detected. These findings we believe to be relevant in the field of human nutrition.HDL, High-density lipoprotein; LDL, Low-density lipoprotein; PMNs, Polymorphonuclear; PON, Paraoxonase1; ROS, Reactive oxygen species; TBARS, Thiobarbituric acid reactive substances.The authors declare that they have no competing interests.WCD and MES participated in the design of the study, interpretation of data and edited the manuscript. WCD, MOS, MS, MFD collected the data. RCS made substantial contributions in revising it critically for intellectual content. WGL contributed to the discussion of data and statistical analysis. MES coordinated the study. All authors read and approved the final version of the manuscript."}
+{"text": "Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy. The cellular sphingolipid signaling pathway is a highly conserved balanced system comprising ceramide and sphingosine with proapoptotic functions on the one hand, and sphingosine-1-phosphate (S1P) promoting cell survival and proliferation on the other hand 5 receptor show centrosomal localization in cells has raised speculations about a direct role of the kinases and their metabolites in cell cycle regulation The recent finding that SK-1, SK-2 and the S1P. Consequently, PKC has been investigated as a target for cancer therapy, and a myriad of drugs from small molecule inhibitors to antisense oligonucleotides has been evaluated for therapeutic efficacy in preclinical tumor models and oncology trials The serine/threonine kinase PKC is the major cellular target of tumor promoting phorbol esters and thus considered crucial for carcinogenesis The cell cycle is controlled by sequential activation of cyclin-dependent kinases and their cyclin substrates. p34cdc2, also named Cdk1, together with cyclin B1 constitutes the mitosis promoting factor (MPF) which is crucial for G2/M transition and execution of mitosis The therapeutic potential of targeting SK-1 and its product S1P in cancer has been reported in various preclinical studies . Ceramide levels were not altered in SK-1kd cells of all histotypes compared to wt  as an activator of PKC Inversely, in MDA-MB-231 cells overexpressing SK-1 phosphorylation of the same PKC substrates which were inhibited in SK-1 kd cells was increased compared to wt cells . This fu. Compared to SK-1 kd cells a similar degree of inhibition was achieved with >0.5 \u00b5M sphingosine, 0.01 \u00b5M CGP 41-251 or with 5 \u00b5M Ro 31-8220. From this we conclude that intracellular sphingosine and PKC are key players in controlling the clonal expansion of carcinoma cells.The cellular effects associated with PKC inhibition in tumor cells include decreased proliferation, cell cycle disruption and apoptosis Sphingolipids and PKC are implicated in the control of cytokinesis and mitotic exit By tendency, short term treatment with sphingosine, the SK-1 inhibitor SK I II, the PKC inhibitor Ro 31-8220, or with SK I II and sphingosine simultaneously  or sphinTo understand the molecular mechanism underlying endoreduplication and cytokinesis failure in MDA-MB-231 SK-1 kd cells, we examined the expression and phosphorylation of cyclin B1 and cdc2 by Western blotting. As shown in Decreased cdc2 activity may compromise cell cycle progression and cytokinesis as recently demonstrated using the cdc2-specific inhibitor RO 3306 Furthermore, we examined expression of the checkpoint kinase Chk1, which is essential during mitosis and cytokinesis to complete cell division. Chk1 expression is reduced in SK-1 kd cells, which may compromise spindle checkpoint function In addition, in a complementary viability assay we investigated the ability of sphingosine and the SK-1 inhibitor SK I II to sensitize MDA-MB-231 wt cells to taxol. As shown in These findings confirm that targeting SK-1 in carcinoma cells generates an unfavorable mitotic environment with the potential to increase the efficacy of taxane-based chemotherapy.We demonstrate that downregulation of SK-1 in various carcinoma cell lines leads to intracellular accumulation of sphingosine, a bioactive lipid with proapoptotic potential, whereas the level of ceramide was not affected. This effect was most pronounced in MDA-MB-231 cells. Since these cells exhibited the highest basal level of SK-1, one might speculate that this is necessary to keep the amount of the proapoptotic substrate in check. The relative concentrations of the sphingolipid metabolites S1P, sphingosine, and ceramide maintain a rheostat implicated in cell death and survival 2+-independent isoforms as major targets. The effect of CGP 41-251 on colony formation without detectable activity in the p-Ser-PKC substrate assay is likely due to inhibition of other survival kinases required for cell proliferation Interestingly, we found that in all three SK-1 kd carcinoma cell lines accumulation of sphingosine decreased PKC activity and this effect could be mimicked by the addition of exogenous sphingosine to cells. On the other hand, PKC activity was increased in cells overexpressing SK-1. The ability of sphingosine to inhibit PKC by interaction with its regulatory domain was first described by Hannun et al. low population using the SK I II inhibitor failed due to increasing toxicity over time.From all three carcinoma cell lines we established sublines in which SK-1 was stably downregulated by vector-based RNAi. Alternatively, we used a pharmacologic SK-1 inhibitor SK I II 5 receptor low cell population. However, despite an increasing number of cells with 4N DNA content, we could not find signs of increased endoreduplication. The ability of sphingosine and staurosporine to induce G2/M arrest was also described for other cell types Apart from the loss of PKC activity the SK-1 kd carcinoma cells may contain less S1P, a survival factor maintaining constant cross-talk with other major growth factors In addition to decreased cdc2 activity, expression of Chk1 was also reduced in MDA-MB-231 SK-1 kd cells. Since Chk1 delays entry of cells with damaged or unreplicated DNA into mitosis Altogether, our data demonstrate that downregulation of SK-1 in carcinoma cells leads to intracellular accumulation of sphingosine which inhibits PKC. Inhibition of PKC in SK-1 kd cells reduced proliferation and viability, and was accompanied by decreased cdc2 and Chk1 function, which compromised spindle checkpoint function and cytokinesis. More investigations are warranted to further delineate the intriguing connection between SK-1, PKC and the mitosis machinery.p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II), Taxol (paclitaxel), Aurora B antibody and TRITC-labeled phalloidin for actin staining were from Sigma-Aldrich . Antibodies against phospho-Ser\u2013PKC substrate, PKD/PKC \u00b5, phospho-cdc2(Tyr15), total cdc2, phospho-cyclin B1(Ser133), total cyclin B1, total Histone H3 and GAPDH were obtained from Cell Signaling . The antibodies against PKC \u0398, \u03bb, \u03b9, \u03b6 were from Transduction Laboratories . Synthetic peptides based on the C-terminal sequence of PKC \u03b1 (SYVNPQFVHPILQSAV), PKC \u03b5 (NQEEFKGFSYFGEDLMP) and PKC \u03b4 (KGFSFVNPKYEQFLE) were synthesized on an ARI 431 peptide synthesizer, coupled to keyhole limpet hemocyanin by glutaraldehyde and used to immunize rabbits. The detailed characterization of anti- PKC \u03b1, \u03b5, \u03b4 antibodies is described elsewhere Hyperfilm MP, nitrocellulose membranes, secondary horseradish peroxidase-coupled IgG, and enhanced chemiluminescence reagents were obtained from GE Health Care Systems GmbH . 2-. The breast carcinoma cell line MDA-MB-231 was obtained from the European Collection of Cell Cultures (ECACC) through Sigma-Aldrich, NCI-H358 lung and HCT 116 colon carcinoma cells were obtained from American Type Culture Collection . MDA-MB-231 and NCI-H358 were cultured in RPMI medium supplemented with 10% fetal bovine serum, 10 mM Hepes pH 7.4, and 100 units/ml each of penicillin and streptomycin. HCT 116 cells were cultured in McCoy medium containing 10% fetal bovine serum, 10 mM Hepes pH 7.4 and 100 units/ml each of penicillin and streptomycin. All cells were grown at 37\u00b0C in a humidified atmosphere containing 5% COR shRNA constructs from Sigma-Aldrich as described SK-1 downregulation in cells was performed using the All MISSIONEqual numbers of cells in 6-well plates were scraped in methanol containing internal C17-S1P and C17-sphingosine, C17-ceramide standards and subjected to lipid extraction and mass spectrometry as described AAG CAG TCG CAG TGA AGA TTG TAG, reverse: TTG CCT TCT CTC CTG TGA CCA TAG; human SK-1 forward: ACG CTC TGG TGG TCA TGT CTG, reverse: AGT TGG TCA GGA GGT CTT CAT TGG TTG CCT TCT CTC CTG TGA CCA TAG; 18 S rRNA forward: CGA TTC CGT GGG TGG TG GTG, reverse: CAT GCC AGA GTC TCG TTC GTT ATC. The IQTM5 Optical System Software  was used to analyze real time and endpoint fluorescence.The expression of Chk1, SK-1 mRNA was quantified using a BioRad iQ5-Cycler Detection System . The following primer sequences were used: human Chk1 forward: Cell homogenization, sample preparation and Western blotting was performed as described 4 cells were seeded in 96-well plates and allowed to adhere overnight. Thereafter, cells were treated for 48 h with the indicated inhibitors or vehicle (DMSO) before the MTT reagent was added for 5 h and the absorbance of formazan was measured using a SpectraMax M2 microplate reader .Cell viability was measured using the MTT cell proliferation kit  as described by the manufacturer. Briefly, 5\u00d710Cells were cultured in 60 mm diameter dishes at a density of 700 cells per dish in cell culture medium. After 24 h, cells were treated with CGP 41-251, Ro 31-8220, sphingosine or DMSO as vehicle control, and incubated for 14 days to allow colony formation. Then, cells were washed with ice-cold PBS, air dried, stained for 30 min with 2% crystal violet, washed with water, and air dried again. The number of colonies was counted using a ColCountTM . Only colonies containing more than 50 cells were evaluated.Cells were harvested by trypsinization, washed and fixed in 70% ethanol overnight at -20\u00b0C. Thirty minutes prior to analysis, cells were resuspended in PBS containing 50 \u00b5g/ml propidium iodide (PI) and 5 \u00b5g/ml RNase A. Samples were analyzed using a FACSCalibur flow cytometer and the Cell Quest software . In each sample, 10\u2032000 cells sorted for red fluorescence were analyzed.To analyze nuclear morphology, cells were grown on 12 mm glass cover slips (BD Biosciences), fixed with 4% paraformaldehyde and stained for actin using TRITC-labeled phalloidin and DAPI for nuclei. The anti-fading fluorescent mounting medium (Dako) was added and slides were covered with coverslips and analyzed by confocal laser scanning microscopy . Quantification of cells and measurement of the largest diameter of nuclei was performed using a Zeiss LSM Image Browser (Carl Zeiss MicroImaging GmbH). At least 30 cells in the field were counted and examined by three independent determinations.5 cells/ml in binding buffer containing 150 mM NaCl, 4 mM KCl, 2.5 mM CaCl2, 1 mM MgSO4, 15 mM HEPES. Cells were incubated with GFP-coupled annexin V (dilution 1\u22361000) and PI (dilution 1\u2236500), analyzed using a FACSCalibur flow cytometer and the Cell Quest software (Becton Dickinson Biosciences).The fraction of cells with fragmented DNA was quantified from the SubG1 peak as described under cell cycle analysis. In addition, apoptotic cells were detected by PI staining and surface binding of annexin V. To this end, cells were harvested by trypsinization, washed and resuspended at a concentration of 6\u00d710t-test was used. Statistical analyses of multigroup data were performed by one way analysis of variance (ANOVA) with Bonferroni correction. p<0.05 was considered statistically significant.Statistical analysis of data was performed with Graph Pad Prism 4 . Results are given as means \u00b1 SD. For two-group analyses Student\u2019s unpaired two-tailed Figure S1Transduction of carcinoma cell lines with SK-1 shRNA decreases SK-1 mRNA expression. (A) MDA-MB-231, NCI-H358, and HCT 116 cells were transduced with lentiviral SK-1 shRNA to downregulate SK-1 expression (SK-1 kd) or left untreated . SK-1 mRNA was measured by quantitative real-time PCR and data were normalized to 18S rRNA. Data are means \u00b1 SD (n\u200a=\u200a3); **p<0.01, ***p<0.001, compared to wt cells. (B) lysates were prepared from MDA-MB-231, NCI-H358, and HCT 116 wt and SK-1 kd cells, and analyzed for SK-2 by Western blotting using antibody against SK-2 (dilution 1\u22361000) or GAPDH (dilution 1\u22362000) as loading control. (C) cellular ceramide was determined in the genetically modified SK-1 kd carcinoma cell lines, in the respective wt cells. Lipid extractions were performed and sphingosine was quantified by mass spectrometry. Data are means \u00b1 SD. (C) endogenous sphingosine in the wt carcinoma cell lines upon treatment with the SK-1 inhibitor SK I II (10 \u00b5M) or DMSO as vehicle control for 24 h. Quantification was done as above. Data are means \u00b1 SD (n\u200a=\u200a3); *p<0.05, **p<0.01 compared to DMSO treated cells.(EPS)Click here for additional data file.Figure S2PKC isoenzyme expression in MDA-MB-231 cells. Lysates were prepared from MDA-MB-231 wt, SK-1 kd and SK-1 ov cells, and analyzed for PKC isoenzymes by Western blotting using specific antibodies against the various PKC proteins (dilution 1\u22361000) or GAPDH (dilution 1\u22362000) as loading control.(EPS)Click here for additional data file.Figure S3Inhibition of SK-1 and sphingosine treatment increases cells in SubG1 and with 4N and 8N DNA content. (A) MDA-MB-231 wt cells were seeded in 60 mm diameter dishes at a density of 3\u00d7105 cells per dish in cell culture medium. After 24 h, cells were treated with combination of SK I II inhibitor and sphingosine, DMSO was used as vehicle control. After another 24 h, cells were fixed, stained with propidium iodide and the DNA content was measured by flow cytometry using a FACSCalibur flow cytometer and the Cell Quest software for data processing. Data are means \u00b1 S.D. (n\u200a=\u200a3); *p<0.05, ***p<0.001 compared to the DMSO control values. (B) MDA-MB-231 wt cells were seeded in 60 mm diameter dishes at a density of 3\u00d7105 cells per dish in cell culture medium. After 24 h, cells were treated with the SK I II inhibitor or sphingosine, DMSO was used as vehicle control. After another 24 h and 72 h, cells were fixed, stained with propidium iodide and the DNA content was measured by flow cytometry using a FACSCalibur flow cytometer and the Cell Quest software for data processing. Data are means \u00b1 S.D. (n\u200a=\u200a3); *p<0.05, **p<0.01, ***p<0.001 compared to the DMSO control values.(EPS)Click here for additional data file.Figure S4Downregulation of SK-1 in MDA-MB-231 cells does not affect expression of Beclin 1 and Aurora B. Lysates were prepared from MDA-MB-231 wt and SK-1 kd cells, and analyzed for Beclin 1 (Atg 6) (dilution 1\u22361000), Aurora B (dilution 1\u22361000) and GAPDH (dilution 1\u22362000) as loading control by Western blotting using specific antibodies.(EPS)Click here for additional data file."}
+{"text": "Endocardiosis is the most common heart disease in Dachshunds and is therefore an important cause of cardiac morbidity and death. In recent years we have observed an increasing interest in the development of new genetic and genomic markers of heart disease. The discovery of miRNAs circulating in biofluids such as plasma or serum aroused researchers\u2019 interest in using them as potential biomarkers. In the present study we analysed the expression of 9 miRNAs described in literature as being involved in cardiovascular pathology in the plasma of dogs suffering from endocardiosis.Expression analysis using the Real-time PCR method revealed that two out of nine miRNAs were significantly downregulated: the expression of miR-30b differed between ACVIM stage B and stage A (control) dogs; the expression of mi-133b differed ACVIM stage C and stage A dogs. 5 miRNAs  showed a trend of downregulation in the ACVIM C group. Levels of miR-423 were the same in healthy and diseased dogs. Expression of miR-208a and 208b was not detected.miR-30b could be a potential biomarker of ACVIM stage B heart failure in Dachshunds with endocardiosis and miR-133b could be a potential biomarker of ACVIM stage C. The lack of expression or lack of significant changes in expression in 7 miRNAs which are potential biomarkers of heart diseases in humans proves that findings from human medicine are not always directly reflected in veterinary medicine.The online version of this article (doi:10.1186/s12917-014-0205-8) contains supplementary material, which is available to authorized users. Endocardiosis is the most common heart disease in Dachshunds and is therefore an important cause of cardiac morbidity and death. It is recognised mainly in middle-aged and older dogs. It is also referred to as myxomatous atrioventricular valvular degeneration, chronic degenerative valvular heart disease (CVHD), or chronic valvular fibrosis. Endocardiosis is characterised by progressive lesions affecting primarily the mitral valve, and less frequently the tricuspid valve. Affected valve leaflets are grossly shortened and thickened and have nodular areas along the free valvular edges. During disease progress, lesions extend and occupy larger areas of the valve surface, sometimes encompassing the chordae tendineae. In most cases atrioventricular valvulopathy occurs in the dog spontaneously and is age-related -3.In recent years significant development of new diagnostic methods allowing precise diagnosis of animal diseases as well as better understanding of their background has been observed. Genetics is a very important aspect of dog breeding and reproduction, and another significant reason why we have been observing a great interest in the development of new genetic and genomic markers of specific diseases. One group of such markers could be miRNA.miRNAs inhibit gene expression at the post-transcriptional level by directing the RISC complex to target mRNAs resulting in translational repression or cleavage . The traSeveral studies have recently discovered circulating miRNAs in blood, not only in platelets, nucleated blood cells, and erythrocytes, but also in plasma. Moreover, plasma miRNAs were found to be unexpectedly stable even under conditions such as boiling, long time storage at room temperature and low or high pH, whereas exogenously added synthetic miRNAs are quickly degraded because of RNAse activity in the plasma -7. This However, in veterinary medicine the situation is quite different\u2013there are only few reports about research on circulating miRNAs research in animal diseases. To our best knowledge there is only one report concerning circulating miRNA levels in dogs with heart disease. This study compares expression of miRNAs in the serum of Doberman Pinschers with dilated cardiomyopathy (DCM) in comparison to healthy control dogs.Therefore the aim of the present paper was to compare levels of miRNAs in the plasma of Dachshunds with endocardiosis and healthy control dogs. Moreover, dogs with endocardiosis were divided in two groups according to the ACVIM  classification scheme . The res24 client-owned Dachshunds were examined cardiologically in the Cardiology Service of the Small Animal Clinic, Faculty of Veterinary Medicine, School of Agriculture in Warsaw. Dogs included in the study were assigned to ACVIM B (n\u2009=\u20098), ACVIM C (n\u2009=\u20098) or ACVIM A\u2013unaffected control group (n\u2009=\u20098) according to the ACVIM  classification scheme. One sample of the ACVIM C group was removed from further analysis because of inconsistency of expression levels of miRNA compared to other samples. Study population characteristics are shown in Table\u00a0Nine miRNAs which have been previously described in literature as being involved in cardiovascular pathology in humans were selected for qPCR analysis Table\u00a0. The expUsing the same statistical tests the expression of miRNAs in ACVIM stage C and unaffected dogs was analysed. We observed a trend of downregulation in all examined miRNAs. The biggest differences were observed in case of miR-133b. The expression of this miRNA was -3.29 times lower in ACVIM stage C than in unaffected dogs. miR-29b and miR-423 remained almost unchanged but the other miRNAs were slightly downregulated. The mean fold change for each of them was as follows: -1.84 for miR-30b, -1.55 for miR-126, -1.82 for miR-125. Likewise in ACVIM stage B class the expression of miR-208a and miR-208b were not detected  can lead to congestive heart failure and the associated clinical signs. Since the molecular background of this disease is still far from fully elucidated, the search for any specific markers  of this disease would be of great value and importance.Although miRNA are currently intensively investigated in human medicine because of their diagnostic potential in many different conditions, there are only few reports related to circulating miRNA studies in animal diseases. One of these is the study about expression of miRNAs in serum of Doberman Pinschers with dilated cardiomyopathy (DCM). The authors compared the expression of 1368 miRNAs using miRNA microarrays. They identified 404 different miRNAs but the results did not show statistical significance after multiple testing correction (FDR) and Real-time PCR validation of 5 miRNAs which were differentially regulated without FDR correction showed no statistical differences . AnotherOne of the difficulties researchers face related to circulating miRNA studies is the impossibility of reliable large\u2013scale expression screening using the microarray method. Microarray analysis is not a suitable method for studying miRNAs expression in biofluids because of insufficient sensitivity which cannot ensure reproducible detection of samples with low miRNA levels . The resOur study included 24 client-owned dogs with endocardiosis classified into groups according to the ACVIM  classification scheme. The results revealed that among nine miRNAs that have been previously connected to heart disease in literature ). Since the blood for miRNAs analyses was taken during a routine veterinary examination and in accordance with the above mentioned legal act, a written ethical approval from the Local Ethical Committee before beginning the study was not necessary. However, before enrolling a dog into the study, an informed written consent for a full physical examination as well as additional diagnostic imaging tests and blood sampling via venepuncture was obtained from the owner and a high standard of care was adhered to throughout each examination. Study population characteristics are shown in Table\u00a024 client-owned dogs were examined cardiologically during routine veterinary examinations. The clinical picture of the animals included: animal history, clinical examination and echocardiographic examination. Dogs with recognized heart disease were classified according the ACVIM  classification scheme as stage B (asymptomatic)\u20138 dogs, stage C (mild to moderate heart failure)\u20138 dogs and stage A\u20138 dogs (the control group of unaffected dogs).A complete physical examination was performed, with a specific emphasis on thoracic auscultation. A standard transthoracic echocardiographic (TTE) examination was performed in all dogs with the Aloka 4000 ultrasound machine, equipped with a cardiology program and 2.5\u20137-megahertz (mHz) sector transducers according to previously published norms. All examinations were performed at rest without pharmacological restraint. Blood samples for basic morphologic and biochemical analysis as well as the transcription profile analysis were collected from the jugular or cephalic vein. The following laboratory tests were performed in all of the dogs: complete blood count, serum sodium, potassium, calcium, urea, creatinine, albumin, liver enzymes, and cholesterol. The dogs were classified into groups based on clinical signs of heart disease and the results of clinical examination. Echocardiography exam was used to diagnose endocardiosis.Blood samples were collected in EDTA-K3 probes . Within 2\u00a0hours the samples were centrifuged at 1300 RCF for 10\u00a0minutes at room temperature. Afterwards the 250\u00a0\u03bcl plasma aquilots were transferred to 1.5\u00a0ml tubes  and stored at\u201380\u00b0C until RNA isolation. Before extraction plasma samples were thawed on ice and centrifuged at 3000\u2009\u00d7\u2009g for 5\u00a0min at a room temperature to pellet any debris and insoluble components. An aliquot of 200\u00a0\u03bcl of plasma per sample was transferred to a new microcentrifuge tube and the total RNA including small RNAs, were purified according to the protocol supplied with miRCURY\u2122 RNA Isolation Kit\u2013Biofluids . In brief this procedures involves lyses of membranized particles using the provided Lysis Solution, precipitation of proteins and on column removing of any remaining impurities with provided Wash Solution. During isolation RNA Spike-in template mixture from miRCURY LNA\u2122 Universal RT microRNA PCR, RNA Spike-in kit  and carrier-RNA MS2 bacteriophage RNA (Roche Applied Science) were added as recommended by the manufacturer. RNA Spike-in kit was used for quality control of the RNA isolation and cDNA synthesis while MS2 RNA was used to minimize the loss of small RNA during isolation and the technical variation between replicates in further analyses. Total RNA was eluted by adding 50\u00a0\u03bcl of RNase-free water. The eluted RNA was stored at\u201380\u00b0C until further analysis.cDNA was synthesized using the miRCURY LNA\u2122 Universal RT cDNA Synthesis Kit II . For quality control of this process UniSp6 Spike-in was used. The cDNA was stored up to 5\u00a0weeks in -20\u00b0C. Directly before preparation of Real-time PCR reaction the cDNA products were subsequently diluted 40\u00d7. All analyses were performed on individual samples using a SYBR\u00ae Green master mix, Universal RT  as follows: polymerase activation at 95\u00b0C for 10\u00a0minutes; amplification (40\u00a0cycles) including denaturation at 95\u00b0C for 10\u00a0seconds, annealing at\u201360\u00b0C for 1\u00a0minute; melting curve including denaturation at 95\u00b0C for 0\u00a0seconds, annealing at 65\u00b0C for 15\u00a0seconds, continuous melting at 95\u00b0C for 0\u00a0seconds; cooling at 40\u00b0C for 30\u00a0seconds. All reactions were performed in triplicate and included the following controls: no template (RT NTC), no reverse transcriptase enzyme (no RT) and carrier MS2 RNA (carrier only). The primers are listed in Table\u00a0Initial qPCR data analysis was performed using MXPro software (Stratagene). Threshold cycle (Ct) for each miRNA was extracted by setting a threshold and automatically defined baseline. Furthermore, the data were analysed statistically using Exiqon GenEx qPCR analysis software . miR-16-5p was used as a housekeeping gene ,43. This"}
+{"text": "The isogenic lines carrying Jukkoku_sd1, IR8_sd1, d60, Jukkoku_sd1 plus d60, and IR8_sd1 plus d60 had considerably shorter culm lengths than Koshihikari by 19.2%, 22.8%, 26.0%, 45.1%, and 43.4%, respectively. The sd1 plus d60 lines showed additively reduced culms, indicating that the function of d60 was different from sd1. In contrast to the culm reduction, Jukkoku_sd1 showed productive merit with a panicle length of 2.5% greater than the origin. MiSeq next-generation sequencer was used to optimize a minimum scale to detect Jukkoku_sd1 in practical breeding. Mapping with the reference genome of Nipponbare gained the average depths of Koshihikari Jukkoku_sd1 and Koshihikari being 9.17 and 7.29, respectively. Comparing the vcf files of the entire genomes of Koshihikari Jukkoku_sd1 and the virtual Koshihikari revealed a G to T SNP at position 38,382,746 in the sd1 locus on chromosome 1 of Koshihikari, causing a loss-of-function mutation of GA20-oxidase.The influence of the semidwarfing gene The resulting semidwarf rice variety IR8 , developed in 1966, has dramatically improved rice yields and brought the Green Revolution to tropical Asia /(measurements of Koshihikari) \u00d7 100.sd1 at only 5\u00d7 rice genome coverage by using the MiSeq. The semidwarf gene sd1 (a loss-of-function mutation of the GA20-oxidase encoding gene) was transferred to Koshihikari by consecutive backcrosses to prepare a semidwarf Koshihikari named Koshihikari Jukkoku_sd1. The Koshihikari Jukkoku_sd1 backcross was used to detect single-nucleotide polymorphisms (SNPs) in Jukkoku-derived sd1 by NGS. Whole-genome analysis was conducted using Koshihikari Jukkoku_sd1 and Koshihikari. Genomic DNA was extracted from each cultivar by the CTAB (hexadecyltrimethylammonium bromide) method. Genomic DNA was tagged and fragmented to an average length of 500\u2009bp using the Nextera\u00ae transposome-based approach. After purification of the transposome with DNA Clean & Concentrator\u2122-5 , adaptors for fixation on the flow cell were synthesized at both ends of each fragment using a polymerase chain reaction (PCR). Then, the DNA fragments were subjected to size selection using AMPure XP magnetic beads . Finally, qualitative and quantitative measurements were performed using a Fragment Analyzer\u2122  and a Qubit\u00ae 2.0 Fluorometer  to prepare a DNA library for NGS. Aiming to achieve 5\u00d7 rice genome coverage, a MiSeq next-generation sequencer was used to simultaneously analyze five lines; namely, 4-5\u2009ng of five DNA libraries was applied in each MiSeq run. Clusters then were formed on the flow cells by bridge-PCR and each pair-end of a 250\u2009bp read was sequenced. Resulting sequenced reads were mapped using BWA software with the Nipponbare genome [http://samtools.sourceforge.net/).An advantage of genomics is the development of a next-generation sequencer that can decode DNA sequences at the giga level. Development of the next-generation DNA sequencer was advanced under the Affordable Care Act aims to realize societal implementation of medical genomics , 28. Thee genome  as a refsd1 (83 days to heading) and the latest heading panicles were observed in Koshihikari carrying Jukkoku_sd1 or Jukkoku_sd1 plus d60 (93 days to heading). However, the difference in the average number of days to heading was the largest between lines carrying IR8_sd1 (86.5 days) and those carrying d60 (90.5 days), but this 4-day difference was thought to be minor , the reduction in length between the third and fourth and between the fourth and fifth internodes was markedly large in lines carrying IR8_sd1 . Reductions in internode intervals were relatively uniform in lines carrying d60 , while percent differences were larger than those observed in lines carrying Jukkoku_sd1. In the sd1 plus d60 lines, marked reductions were observed in length between neighboring internodes, probably attributed to the additive effect of sd1 and d60  resulted in a reduction in culm length. The mean culm length of Koshihikari was 88.8\u2009cm, while those of lines carrying Jukkoku_ectively . Similar and d60 . The intsd1 was 16.2\u2009cm, which was solely 2.5% longer than that of Koshihikari. The panicle length of the line carrying Jukkoku_sd1 was longer than that of the original cultivar Koshihikari: not only the mean value but also the value including the standard deviation caused by circumstance. Therefore, there was certain merit in increasing the production of Jukkoku_sd1 as compared to other semidwarf genes.In contrast, as shown in 9\u2009bp from the total read number of 9.53 \u00d7 106 in Koshihikari Jukkoku_sd1, while a total read length of 1.92 \u00d7 109\u2009bp was obtained from a total read number of 6.13 \u00d7 106 in Koshihikari. By mapping the read sequences obtained by NGS using Nihonbare genomic DNA as a reference, sequence coverage rates of 93.5% and 88.5% were attained for Koshihikari Jukkoku_sd1 and Koshihikari, respectively, while average depths were 9.17 and 7.29, respectively. Furthermore, vcf files of the entire genomes were prepared and the whole-genome sequence of Koshihikari Jukkoku_sd1 was compared with that of the virtual Koshihikari genome. As a result, three reads were obtained, including Jukkoku_sd1 from Koshihikari Jukkoku_sd1, while three reads of Sd1 were obtained from Koshihikari. The Sd1/sd1 locus (Os01t0883800-01) was localized at positions 38,382,385\u201338,385,469 from the end of the short arm of chromosome 1 in the Koshihikari genome. The difference observed between the Koshihikari_Sd1/Jukkoku_sd1 alleles was only one SNP from G to T in exon 1 of the GA20-oxidase gene , suggesting that the negative effects of the semidwarf genes sd1 and d60 on panicle length were negligible  in the semidwarf lines than in Koshihikari , indicathihikari . Howevergligible . Ogi et  sd1 gene derived from Jukkoku [ sd1 locus was clearly cleaved into 613\u2009bp and 166\u2009bp fragments by PmaCI digestion on the SNP in Jukkoku_sd1, but there was no cleavage of the Sd1 locus in Koshihikari  in the defective GA20-oxidase gene, which had reduced the culm length by a loss-of-function mutation of GA synthesis, localized at position 38,382,746 from the end of the short arm of chromosome 1 of Koshihikari. This would be the minimum scale to detect Jukkoku_sd1 in practical breeding. In Japan, genetically modified organisms are not acceptable to consumers; thus the target SNP in Jukkoku_sd1 would be effectively tracked by using NGS of 5\u00d7 coverage scale in each backcrossed generation with Koshihikari. On the other hand, as the DNA sequence and function of sd1 have been deciphered [ Sd1.In this study, a MiSeq next-generation sequencer was used to achieve 5\u00d7 rice genome coverage; namely, 4-5\u2009ng of five DNA libraries was applied in one MiSeq run. Using this approach, the targeted gene mutation in Jukkoku_ciphered \u201318, 37, ciphered , are avad1 and d60) and also between sd1 loci of different origins (Jukkoku_sd1 and IR8_sd1). The effect on culm length was more pronounced in plants carrying d60 than in those carrying sd1 . The reduction in internode intervals was relatively uniform in lines carrying Jukkoku_sd1 or d60 (uniform reduction type), while reductions of the third and lower intervals were larger than in the upper intervals in lines carrying IR8_sd1 (sd1 than in those carrying Jukkoku_sd1 or d60. Although the function of d60 is not known, it is clearly distinct from that of sd1, as the additive double-dwarf effect of sd1 and d60 appeared in Jukkoku_sd1 plus d60 and IR8_sd1 plus d60, respectively. The results of this study demonstrated that d60 has a stronger effect than sd1 on culm length and exerts similar effects on other phenotypic traits as with sd1. Although many semidwarf genes are associated with a reduction in panicle length, d60 does not exert such negative effects on phenotypic traits of rice plants. Taken together, these results showed that d60 is useful for adding genetic diversity to semidwarf varieties and is thus of particular importance in the field of plant bleeding.The effects on phenotypic traits of rice differed between the two semidwarf genes (s IR8_sd1 . Thus, t"}
+{"text": "Approximately, fivefold increase in the concentration of Cu, threefold-elevated levels of Mn and a twofold increase in the concentration of Mg were found in the oral fluid of the periodontal patients compared to the controls. Cluster analysis confirmed the statistical significance of the differences in the level of metals in the oral fluid between the two groups in most cases, plus enabled the correct classification of the subjects into patients and controls. The relationship between concentrations of metals and periodontal disease may in the future serve to prevent the development of such disease.Recently, many studies have investigated the relationship between the level of metals in the body and various diseases. The objective of this study was to examine any possible influence of periodontal disease upon the concentration of metals in oral fluid and blood and to explore the usability of applying cluster analysis coupled with the analysis of selected elements in oral fluid, calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), zinc (Zn), cadmium (Cd) and lead (Pb), for effectively distinguishing people affected by periodontitis from healthy individuals. The quantification of eight metals in oral fluid and blood samples was performed by two inductively coupled plasma techniques\u2013inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). Most of the examined elements were detected at elevated concentration in the oral fluid of periodontal patients. However, the differences were statistically significant in the case of three metals: Cu, Mg and Mn ( Measurements of the concentration of various substances in the blood and urine form the basis of diagnostic tests and various scientific investigations. In recent years, researchers have been looking for other biological materials which have as much informative value as blood, but are cheaper and less troublesome in their collection. Examples of these types of materials are nails, hair  and oralThe determination of metals in biological fluids causes many problems. First of all, this type of material is characterized by a complex matrix, which may lead to numerous interferences during analysis. Moreover, many metals are present in biological materials at such low concentrations that highly sensitive analytical methods are required for their quantification .Elemental analysis of biological samples mainly involves spectroscopic methods among which most commonly used are inductively coupled plasma optical emission spectroscopy (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS). These techniques permit the carrying out of the simultaneous determination of numerous elements, and they also have good metrological parameters, such as low detection limits , 4.Mineral deficiencies cause specific human body dysfunctions . At the Periodontal diseases are mainly chronic inflammatory conditions of the tissues surrounding, supporting and protecting the teeth. Severe periodontitis can lead to the loosening and loss of teeth . The weaPrevotella intermedia, Porphyromonas gingivalis) [Due to high prevalence of periodontal diseases, the identification of biomarkers that can help in clinical evaluation of periodontal status is important. Therefore, many investigators have tried to identify markers specific to periodontitis. Traditionally, diagnosis was based on the measurement of specific biochemical parameters in blood or gingival crevicular fluid, but lately saliva has become a biological material of choice . Among mgivalis) \u201319. Saligivalis) . ConsideThe objective of this investigation was to study the usefulness of available methodology to determine the concentration of selected metals in the oral fluid and blood of patients with periodontitis and then compare them with those of healthy controls, by two techniques: ICP-MS and ICP-OES. The possible influence of periodontal disease upon the concentration of metals, calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), zinc (Zn), cadmium (Cd) and lead (Pb), in oral fluid and blood was examined. An attempt was also made to see if chemometric methods could distinguish people with periodontitis from healthy ones on the basis of the elemental levels in two biological materials.A total of 31 non-smoking patients  diagnosed with periodontal disease were included in the present study. The patients\u2019 mean age was 34.5\u00a0\u00b1\u00a010.4\u00a0years. Diagnosis of each case was carried out by medical experts, according to clinical symptoms and the Community Periodontal Index of Treatment Needs (CPITN). Subjects were selected from patients of the Department of Conservative Dentistry and Periodontology, Poznan University of Medical Sciences, Poland. The exclusion criteria included the following: clinically significant and diagnosed illness, alcohol and drug abuse and pregnancy. The gender- and age-matched control group consisted of 29 non-smoking volunteers  with healthy periodontal tissues (confirmed by dentists) and no known illnesses. All of them live in Poznan or cities of above 100,000 citizens. None of the subjects or controls was occupationally exposed to metals, and dietary supplements were not used by them. They had a typical Polish diet, mainly characterized by a high consumption of meat  and dairy products, and a low consumption of whole grain products, seafood, fresh vegetables and fruits. Informed written consent was obtained from each participant prior to commencement of the study. Each subject agreed to fill in a questionnaire recording age, gender and smoking habits.All procedures performed in the study were in accordance with the ethical standards of the institutional and national research committee and with the World Medical Association Declaration of Helsinki (version 2002). Ethical approval (Protocol Number nr 670/08) for the study was obtained from the Bioethics Committee of the Poznan University of Medical Sciences, Poland.The oral cavity was rinsed with deionized water immediately before the oral fluid collection. About 3\u00a0mL of unstimulated oral fluid was collected early in the morning, directly into special vessels . Samples of oral fluid were centrifuged at 4000\u00a0rpm (10\u00a0min). The blood samples (about 8\u00a0mL) were withdrawn from a forearm vein, using a stainless-steel needle surrounded by an inert plastic cannula, and transferred to plastic tubes without anticoagulant. Before digestion, all samples were stored at \u221280 \u00b0C.\u03bbCa\u00a0=\u00a0317.933\u00a0nm and \u03bbMg\u00a0=\u00a0422.673\u00a0nm. All other measurements were carried out using the Elan DRC-e  Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Mn, Fe, Cu, Zn, Cd and Pb were measured using masses of 55, 57, 65, 66, 111 and 208, respectively. The ICP methods were optimized and validated before analysis of the clinical samples. The applied analytical methods enabled the determination of each selected element with satisfactory accuracy , good precision (expressed by coefficient of variation in terms of repeatability was in range from 0.34 % for Ca to 2.74 % for Mn) and low limits of detection . The measured concentrations of metals of the reference material were in good agreement with the certified values. Detailed protocols of the measurement procedures were described previously [A microwave digestion system  equipped with eight high-pressure vessels was employed for the decomposition of samples. The Optima 2100 DV  Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES), equipped with axially viewed plasma, was used in the determination of Ca and Mg at a wavelengths of \u22121 of each analyte in 5 % HNO3, Perkin-Elmer Pure Plus, USA) and Multi-element Standard Solution VI  were used for the calibration and control of all the analytical processes. The daily test was performed with the use of Smart Tune Solution Std ELAN & DRC-e . Certified reference material for blood  was used for the accuracy evaluation. Certified reference material for oral fluid was not commercially available.All reagents used in this study were of analytical-reagent grade or the highest purity available. Ultrapure deionized water (20\u00a0M\u2126), obtained from WG-HLP deionizing system , was used throughout. Multi-element ICP-MS Calibration Std 3  from each subject, four replicate samples of certified reference material and a blank sample were directly introduced into digestion vessels and 6\u00a0mL of concentrated HNOp\u00a0<\u00a00.05). In the next step, a basic data analysis was performed to obtain the mean values, median, range and standard deviation. Outlying results were identified by Grubb\u2019s test and omitted in further calculations.The normality of the dataset was verified by the Kolmogorov\u2013Smirnov (with Lilliefors correction) and the Shapiro\u2013Wilk tests (F test (P\u00a0=\u00a095 %). In order to analyse, confirm and visualize the relationship among metals, a Pearson\u2019s (control group) or Spearman\u2019s (patients) correlation analysis was applied to the dataset as well as a principal component analysis (PCA), which allows reduction of the number of observed variables, without much loss of information. The data were normalized before analysis.Differences in the concentration of the various metals in oral fluid and blood between the different groups  were evaluated using a two-sided Mann\u2013Whitney test with a 0.05 significance level. Prior to that, the equality of variance was tested using the Snedecor The last step was to classify oral fluid samples based on their trace elements concentration by using hierarchical cluster analysis (HCA). Ward\u2019s hierarchical method of agglomeration and the squared Euclidean distance as a measure of the distance between the observations were applied. The cases included in cluster analysis were randomly selected from the study groups. The results were reported in the form of a dendrogram.Statistica 10  was used for the statistical analyses of data.In this study, the concentrations of eight elements were measured in two groups: 31 patients with periodontitis and 29 healthy volunteers. The elements were divided into two groups: essential elements, Ca, Cu, Fe, Mg, Mn and Zn, and toxic metals, Cd and Pb.\u22121, followed by Mg (9.9\u00a0mg\u00a0L\u22121) and Fe (1.0\u00a0mg\u00a0L\u22121). The other metals were present at relatively lower levels. The toxic metals had low concentrations, with the highest being that for Pb (15.8\u00a0\u03bcg\u00a0L\u22121). It is worth noting that Mn concentration in the oral fluid of patients (41.1\u00a0\u03bcg\u00a0L\u22121) was considerably higher compared to the value reported in another publication [\u22121. On the other hand, a study of Al-Rawi and Talabani [\u22121). It can be concluded that the concentration of Mn in the saliva varies widely. This is probably related to the effects of diet, to dietary supplements or to different places of residence.The concentrations of eight studied metals in the oral fluid and blood of periodontal patients and healthy subjects along with basic statistical parameters are given in Tables lication , where tTalabani  presente\u22121), Mg (5.2\u00a0mg\u00a0L\u22121) and Fe (0.9\u00a0mg\u00a0L\u22121) were present at the highest levels in saliva among the essential metals, while Pb (12.4\u00a0\u03bcg\u00a0L\u22121) emerged as the major contributor among the toxic metals. The high standard deviations observed in the concentration of metals are probably due to the biological spread between individuals.Similarly to the group of patients, in the control group, Ca , Mg (190 %) and Mn (273 %). The differences in salivary levels of these elements appeared to be related to periodontal disease.p\u00a0<\u00a00.05). The average levels of Ca, Cd, Mg, Mn and Pb were higher in the oral fluid of male patients, while in the cases of Cu, Fe and Zn, the mean concentrations were found to be higher in the oral fluid of female (Table \u22121 for women and 11.5\u00a0\u00b1\u00a05.3\u00a0mg\u00a0L\u22121 for men). However, no statistically significant differences were observed in the oral fluid concentrations of the metals between women and men from the patients\u2019 group. In the case of the control group, there were also no gender differences for metal concentrations , Ca (87.0\u00a0mg\u00a0L\u22121), Mg (37.1\u00a0mg\u00a0L\u22121), Zn (2.2\u00a0mg\u00a0L\u22121) and Cu (0.6\u00a0mg\u00a0L\u22121), were observed , Zn .2\u00a0mg\u00a0L\u22121The measured concentrations were compared to the results for healthy people reported in other studies , 22. Ther\u00a0=\u00a0\u22120.413 (Zn) to r\u00a0=\u00a00.336 (Ca). The results suggested that there were no strong linear correlations between the concentrations of metals (p\u00a0<\u00a00.05). The mean concentrations of all elements, with the exception of Cd, were statistically significantly different from each other in the blood and the oral fluid .In the next step, the concentration of metals in the examined biological samples was compared in order to confirm whether oral fluid can be used as alternative material for the assessment of elemental status. Samples of blood and oral fluid were collected simultaneously from nine patients with periodontitis. The correlation coefficient of each pair of selected elements in these body fluids was calculated and it ranged from r\u00a0=\u00a00.752), Mg (r\u00a0=\u00a00.540), and Cd (r\u00a0=\u00a00.455). Other notable correlations were Fe\u2013Mn (r\u00a0=\u00a00.499) and Pb\u2013Cd (r\u00a0=\u00a00.400). A quite similar pattern of mutual relationships between the elements was observed in the case of the control group. Strong positive correlations were again found between Ca and three metals: Mg (r\u00a0=\u00a00.782), Pb (r\u00a0=\u00a00.652) and Zn (r\u00a0=\u00a00.540). Additionally, some significant correlations were also observed between Mg and two elements: Pb (r\u00a0=\u00a00.456) and Zn (r\u00a0=\u00a00.384).The metal-to-metal correlations in the oral fluid of periodontal patients and controls were investigated, and the results were presented in Tables Accordingly, principal component analysis was employed to visualize the interactions between selected metals in the oral fluid of the two groups. Figure To examine whether it is possible to diagnose periodontal diseases on the basis of elemental content in oral fluid, hierarchical cluster analysis was performed. The results obtained are presented in the form of a clustering diagram in Fig. Traditional clinical methods of periodontal disease diagnosis include the evaluation of several parameters  and radiographic measurements. They have some limitations, so many scientists try to find valid biomarkers which allow not only early detection of periodontal diseases but also evaluation of treatment effects , 26. RecAdditionally, some studies have focused on a possible association between inorganic ions in oral fluid and periodontal disease. Khamees et al.  found thThe results obtained in our laboratory for the concentration of Mg in oral fluid were in good agreement with the results reported by other authors , 34, whop\u00a0<\u00a00.001), while Mg and Zn concentrations were lower (p\u00a0<\u00a00.001) in the group of patients than in controls. In our study, the periodontal group also had an increased concentration of all these metals in oral fluid (p\u00a0<\u00a00.05), but the differences were statistically significant for Cu and Mg.There have only been a few studies of the levels of elements other than main electrolytes (like Ca and Mg) in oral fluid. Mahmood et al.  measuredCluster analysis, due to a significantly different distribution of the eight elements in the oral fluid of those examined, enabled the correct division of the samples into two groups: patients and healthy subjects. Such a statistical approach has successfully been applied in distinguishing between drug-free people and drug abusers , the diaThe relationship between concentrations of metals , and periodontal disease, as also the possibility of diagnosis on that basis, may in the future serve to prevent the development of such diseases, as well as to treat existing ones.The present study was aimed at exploring the changes in the content of metals in the oral fluid and blood of patients affected by periodontal disease. The paper also examined the use of cluster analysis in the classification of samples of saliva based on the concentration of selected metals.p\u00a0<\u00a00.05) in the case of three metals: Cu, Mg and Mn.Salivary metal levels varied over a relatively wide range. Most of the examined elements were detected at elevated concentrations in the saliva of periodontal patients. However, the differences were statistically significant (The correlation study revealed that there were noticeably different mutual dependences of the selected elements in the saliva of two groups: patients affected by periodontitis and the healthy controls. There were fewer correlations between metals in the saliva of patients than in the controls.The results of cluster analysis indicate that the metal profiles of oral fluid in those with periodontal disease are different from the controls. The data obtained from this study can be a basis for the future development of diagnostic and prognostic biomarkers for periodontal disease."}
+{"text": "In modern casinos, multiline slot machines are becoming increasingly popular compared to traditional, three-reel slot machines. A paucity of research has examined how the unique presentation of near-misses and the use of a stop button in multiline slot machines impact erroneous cognitions related to the perception of skill and agency during play. Our goal therefore was to determine the prevalence of erroneous cognitions pertaining to near-miss outcomes and the usage of a stop button and then to see whether the stop button affected players\u2019 experiences of winning, losing and near-miss outcomes. We recruited 132 gamblers from a casino in Ontario. They played two versions of a slot machine simulator: one with a stop button and one without a stop button. We measured player\u2019s arousal  to wins, losses and near-misses during play. We predicted more robust physiological SCRs and longer PRPs to wins in the stop button game. We also predicted that near-misses encountered in the stop button game would trigger greater levels of arousal and frustration in players, as indexed by larger SCRs, and greater force applied to the spin\u00a0button to initiate the next spin. Erroneous cognitions pertaining to the stop button and near-misses respectively were assessed following play. Results showed that a small but meaningful percentage of players held erroneous cognitions about the stop button (13.6%) and near-misses (16%). Players depressed the spin button harder, and had larger SCRs for all outcomes when using the stop button. Players also paused longer for near-misses in the game involving the stop button. Our findings converge to suggest that the stop button encourages an erroneous perception of skill in some players, and consequentially impacts how such players perceive their outcomes in multiline slot machines. Slot machine gambling has been prevalent in Canada since its legalization in 1985  would nonetheless still hold erroneous cognitions about the stop button.player select this to-be-matched icon. When players encountered a near-miss, regardless of condition, they experienced negative affect. However, those in the personal agency condition experienced significantly more negative affect than when the computer selected the play icon  . In slots play, PRPs are operationally defined as the time between the onset of an outcome\u2019s delivery and the initiation of the next spin  were recruited from a local casino in the city of Brantford, Ontario. Participants were first asked to complete a pre-screen survey to ensure that participants: (1) Were over the age of 19, (2) had gambled on a slot machine at least once in the last 12\u00a0months, and (3) were not in treatment for problem gambling. Participants failing to meet these criteria were excluded from participating. Participants included experienced slot machine gamblers between the age of 19\u201386. Average age of participants was 55.88\u00a0years. Participants were excluded if there was any technical difficulty with the physiological data, survey data or if participants withdrew from the study.Slot Machine Simulator Participants played a five-reel multiline slot machine simulator modelled after a commercially available multiline slot machine game. The simulator was a music themed slots game called \u201cMagic Melody\u201d. Like the commercially available game on which it was patterned, losing outcomes  were followed by a complete absence of feedback . Winning outcomes (where the slot machine paid out more than the wager) were accompanied by animations highlighting the winning line(s), and auditory feedback in the form of winning jingles, with the length of the auditory feedback proportional to the win size. The simulator also displayed \u201cLosses Disguised as Wins\u201d or LDWs  and the player\u2019s initiation of the next spin, measured in milliseconds (ms) constituted the PRP for that outcome. The simulator was configured to send event markers to an ADInstruments Powerlab for each spin initiation (when the player would press the spin button), stop button deployment, and outcome delivery .Force A modified button  were recorded using two metallic electrodes attached to the index and ring finger on the participants\u2019 left hand. Participants were instructed to limit movement of their left hand while playing the slot machine simulator in order to minimize contamination of the SCR data. The electrodes fed into the Powerlab which amplified the participants\u2019 skin conductance signal. SCRs for each outcome were calculated by defining a 3\u00a0s window beginning 1\u00a0s after the last reel stopped moving . SCRs for each outcome were calculated by taking the peak skin conductance within this window and subtracting the skin conductance level value at the beginning of this window. As per the recommendations of Dawson et al.  as well as their gambling habits (e.g. gambling frequency in the last 12\u00a0months).Problem Gambling Severity Index The problem gambling status index . PGSI scores were derived by summing the responses on the nine items. Participants were stratified using the following mappings: 0\u00a0as\u00a0non-problem gamblers, 1\u20134 as low-risk, 5\u20137 as moderate risk and 8 or more as problem gamblers.General Gambling Related Erroneous Cognitions The Gambling Related Cognitions Scale .Stop Button Specific Erroneous Cognitions Participants rated an item designed to poll erroneous cognitions related to the stop button: \u201cUsing the stop button made wins more likely\u201d. Players answered the item using one of the following options . Participants also responded to four items patterned after Ladouceur and S\u00e9vigny ( S\u00e9vigny : (1) \u201cDoGame Experience Questionnaire For purposes external to the goals of the current study, players reactions to the games they had played were measured using the in-game version of the gaming experience questionnaire . The models of both laptops used were Lenovo G530-444625U. Participants first completed the demographic items, and the PGSI. Afterward participants were introduced to the slot machine simulator. Participants were shown the pay-table and told that they would be betting one credit on each of nine lines. They were told that they could win by lining up identical symbols on any one of the nine played lines, so long as the matching symbols were placed from left to right. They were told that there were two exceptions to this left-to-right rule: (1) the violin symbol which was a scatter symbol, and (2) the stereo triplet that occurred on reels 3, 4 and 5. They were told that 1000 credits ($10.00) had been preloaded into the machine and that they could keep whatever was left at the end of 500 spins . Participants who began with the stop button were shown how to use the stop button and were instructed to use it on every spin in that session. Skin conductance electrodes were attached at this point, and participants were instructed to keep their left hand still to the best of their ability while playing the slot machine. Following this instruction period, participants played the first block of 250 spins . A pop-up message occurred at the end of 250 spins, whereupon participants were instructed that they would now complete a short questionnaire (i.e. the in-game GEQ) before playing the second version of the game . The in-game GEQ was completed once more upon completion of the second block of 250 spins.Participants were detached from the skin conductance apparatus and then completed the post-game questionnaire battery including the GCRS and erroneous cognitions items specific to the near-miss and the stop button. For purposes unrelated to the current study, participants also completed the DASS-21. Participants were debriefed, given their winnings and a $25 Walmart gift card for participating.Of the 132 participants who had completed the PGSI, there were 26 non-problem gamblers, 63 low-risk gamblers (score of 1\u20134 on the PGSI), 19 moderate-risk gamblers and 24 Problem Gamblers (scoring 8 or greater on the PGSI). Seven participants withdrew from the study prior to completing all of the subjective measures leaving a sample of 125 participants for the subjective measures.Near-miss Erroneous Cognitions Frequencies pertaining to how players responded to \u201cNear-misses reflect my skill at this slots game, and that I was close to winning\u201d are shown in Table\u00a0r (123)\u00a0=\u00a0.792, p\u00a0<\u00a0.001).r (123)\u00a0=\u00a0.193, p\u00a0=\u00a0.031), as was the near-miss win imminence item (r (123)\u00a0=\u00a0.209, p\u00a0=\u00a0.02). Each item was also significantly related to the subscales of the GRCS as shown in Table\u00a0Crucially, the near-miss as a skill item was correlated with PGSI scores  strongly disagreed with the statement, 46 (36.8%) disagreed with this statement, and 19 (15.2%) neither agreed or disagreed. By contrast, a concerning total of 15 participants (12%) agreed with this statement, and 2 (1.6%) participants strongly agreed with this statement.r\u00a0=\u00a0.667, p\u00a0<\u00a0.001).We also calculated a composite score based on the items from Ladouceur and S\u00e9vigny , designer\u00a0=\u00a0.240, p\u00a0=\u00a0.007), indicating a relationship between problem gambling status and erroneous beliefs pertaining to the stop button. Composite scores were also significantly correlated with the illusion of control, predictive control and interpretive bias scales of the GRCS  than those who preferred the no stop button game (M\u00a0=\u00a0.87), t(123)\u00a0=\u00a04.194, p\u00a0<\u00a0.001. As the question format was dichotomous, the remaining 91 respondents preferred the game without the stop button.Thirty-four of the 125 respondents preferred the game with the stop button feature. Independent t-tests revealed that those who preferred the stop button more strongly endorsed the idea that the stop button made wins more likely (\u03b72).Of the 132 participants, seven dropped out prior to completing both slot machine tasks. One participant\u2019s force data and one participant\u2019s SCR data was not recorded leaving samples of 124 for force, 124 for SCRs and 125 for PRPs. In analyzing our physiological measures, we partitioned our analysis in two ways. Firstly, we performed separate repeated measures analyses of variance (ANOVAs) for skin conductance (SCRs), force, and post-reinforcement pauses. The repeated factors were outcome type  and button use . We then conducted a more restricted version of the analyses above in which the outcomes measured were limited to the three types of losing outcomes that result in no credit gains. Post-hoc analyses were conducted using Fisher\u2019s LSD procedure with alpha set at .01. Violations of sphericity were tested using Mauchly\u2019s test, and in the event of significant violations we applied Greenhouse-Geisser corrections to the degrees of freedom which are reported in the analyses below. We made an a priori decision not to look at gambling status effects because no effects of gambling status had been shown to influence these in-game measures in our previous studies. For all repeated measures analyses effect sizes were measured using partial eta squared \u00a0=\u00a06.550, p\u00a0=\u00a0.012, (\u03b72\u00a0=\u00a0.051). Overall, SCR magnitudes were on average greater for participants when they used the stop button (M\u00a0=\u00a0.163) compared to when they played without the stop button (M\u00a0=\u00a0.147). A significant effect of outcome was also observed, F\u00a0=\u00a037.863, p\u00a0<\u00a0.001 (\u03b72\u00a0=\u00a0.235), but there was no stop button use by outcome interaction \u00a0=\u00a01.006, p\u00a0=\u00a0.380). As shown in Fig.\u00a0p values <.001). Stereo triplet near-misses triggered larger SCRs than regular losses, gramophone near-misses, LDWs, small wins and gramophone wins . The lack of interaction indicates that using the button did not preferentially inflate the SCRs on any particular outcome.on M\u00a0=\u00a0.17. A signAs can be seen in the left panel of Fig.\u00a0F\u00a0=\u00a04.951, p\u00a0=\u00a0.028, (\u03b72\u00a0=\u00a0.039) and a main effect of outcome, F\u00a0=\u00a028.695, p\u00a0\u2264\u00a0.001 (\u03b72\u00a0=\u00a0.189). There was also a significant outcome by button use interaction, F\u00a0=\u00a04.699, p\u00a0=\u00a0.013 (\u03b72\u00a0=\u00a0.037). Average SCRs following losing outcomes are displayed in the right panel of Fig.\u00a0F\u00a0=\u00a029.837, p\u00a0<\u00a0.001, (\u03b72\u00a0=\u00a0.193) with stereo near-misses having more robust SCRs than losses (p\u00a0<\u00a0.001) and gramophone near-misses (p\u00a0<\u00a0.001). Similarly, in the stop button condition the effect of outcome was also highly significant, F\u00a0=\u00a014.738, p\u00a0<\u00a0.001 (\u03b72\u00a0=\u00a0.106) with stereo triplet near-misses significantly larger than either gramophone near-misses (p\u00a0<\u00a0.001) or losses (p\u00a0<\u00a0.001) in the stop button condition. As can be seen in the right panel of Fig.\u00a0When analyzing SCRs for outcomes that resulted in no credit gain , there was a main effect of button use, Force Applied to the Spin-Button A repeated measures ANOVA revealed a significant main effect of button use F\u00a0=\u00a021.39, p\u00a0<\u00a0.001, (\u03b72\u00a0=\u00a0.148). Greater force was applied to the spin-button in the stop button condition (M\u00a0=\u00a0.416) compared to the no stop button condition (M\u00a0=\u00a0.333). A main effect of outcome was also observed, F\u00a0=\u00a016.468, p\u00a0<\u00a0.001 (\u03b72\u00a0=\u00a0.118), however, there was no button by outcome interaction, F\u00a0=\u00a01.494, p\u00a0=\u00a0.212). Fisher\u2019s LSD Post-hoc comparisons indicated that players elicited greater magnitudes of force following LDWs compared to regular losses (p\u00a0=\u00a0.001) as well as gramophone near-misses (p\u00a0=\u00a0.004). Stereo wins triggered more force than any other outcome (largest p\u00a0=\u00a0.008). Crucially, as shown in the left panel of Fig.\u00a0p\u00a0=\u00a0.002), gramophone near-misses (p\u00a0<\u00a0.001) and regular losses (p\u00a0<\u00a0.001), and equivalent amounts of force as small wins, and gramophone wins. F3.68, 43.040\u00a0=\u00a01F\u00a0=\u00a020.388, p\u00a0<\u00a0.001 (\u03b72\u00a0=\u00a0.142), and a main effect of outcome, F\u00a0=\u00a022.532, p\u00a0<\u00a0.001, (\u03b72\u00a0=\u00a0.155) were revealed. However, there was no button by outcome interaction. The main effect of button use was attributed to players applying more force to initiate the spin button in the stop button condition (M\u00a0=\u00a0.410) compared to the no stop button condition (M\u00a0=\u00a0.325). As illustrated in the right panel of Fig.\u00a0p\u00a0<\u00a0.001), or after regular losses (p\u00a0<\u00a0.001). Gramophone near-misses were not significantly different from regular losses (p\u00a0=\u00a0.942).When restricting the analysis to just the losing outcomes , a main effect of button 1.423, 17.029\u00a0=\u00a022Post-reinforcement Pauses The repeated measure ANOVA for PRPs observed no significant main effects of stop button use F\u00a0=\u00a0.937, p\u00a0=\u00a0.335. However, there was a significant main effect of outcome, F\u00a0=\u00a0455.039, p\u00a0<\u00a0.001 (\u03b72\u00a0=\u00a0.786), while the button by outcome interaction fell just short of significance following a Greenhouse Geisser correction F\u00a0=\u00a02.82, p\u00a0=\u00a0.06. The main effect of outcome is shown in the left panel of Fig.\u00a0me, F1.28, 159.458F\u00a0=\u00a05.118, p\u00a0=\u00a0.01, (\u03b72\u00a0=\u00a0.040). Simple effect analyses revealed that there was a main effect of outcome only in the stop button condition, F\u00a0=\u00a06.245, p\u00a0=\u00a0.007, (\u03b72\u00a0=\u00a0.048) and not the no stop button condition. As shown in the right panel of Fig.\u00a0p\u00a0=\u00a0.820), the stereo near-misses had longer PRPs than both losses (p\u00a0=\u00a0.008) or gramophone near-misses (p\u00a0=\u00a0.011) in this stop button condition.The restricted analysis of PRPs for the three types of full losses , showed that the main effects of both button use and outcome were not significant. Crucially however, there was a significant button use by outcome interaction, In the present study, players played two separate games on a slot machine simulator: one involving the use of the stop button, and the other without the use of a stop button. We sought to assess whether two distinct structural slot machine features, the stop button and near-miss outcomes, would perpetuate a unique set of erroneous cognitions and amplified emotional responses related to the presence of such erroneous cognitions.not influence the outcomes on the machines. Critically, our composite \u201cstop button skill\u201d score  when using the stop button. It would seem that they literally played the game \u201charder\u201d. One interpretation of this pattern of results is that that players may be exerting a strategy  to stop the reels in a winning combination, presumably by \u201cprecision\u201d timing and effort. If players were merely using the stop button to reveal the outcome faster, there is no reason to assume they would pull the stop button especially hard. If, however, players were attempting to stop the reels at a given outcome, then they would likely pull hard to get the reels to stop spinning at a winning combination. The arousal and effort used to stop reels at a winning combination could then bleed over into the initiation of the next spin, causing players to initiate spins with greater force in the stop button condition than the no-stop button condition. Thus the finding of greater SCRs and greater force in the stop button condition may constitute in-game evidence of the erroneous cognitions we saw using the post-game questionnaires where at least some gamblers thought that using the stop button was related to skill at slots. Although in our simulator , stop buttons merely reveal the outcome faster, our force results suggest that players feel they can impact the outcomes of the games by pulling at just the right time. As such our force and SCR results may actually reflect the illusion of control during play.Another notable aspect of our force measures is that unlike SCRs, force pulls appeared to be quite sensitive to the amount of credits gained. Whereas the losses, LDWs and small wins, and even the large gramophone win all led to roughly equivalent SCR responses  than a slot machine simulator game housed in a realistic cabinet where players push on a spin/stop button located on the front panel. Additionally, our measures involving skin conductance where participants had electrodes attached to their fingers of their non-dominant hand, and were instructed not to move said hand, could affect how absorbed players become in slots play. Such playing conditions are novel and unnatural for any slot machine player. Also the rather standard limitation for studies of this type, namely that players were not using their own money certainly applies here. Despite this, we were still able to detect robust changes in participants\u2019 physiological and behavioural reaction patterns to different outcomes and show clear effects of both stop button use, and reactions to near-misses. It may be that the robust effects shown would be even more prominent in more naturalistic settings.The current study makes two novel contributions: First we move beyond the classic near-miss effects that have been demonstrated in simplistic, three-reel slot machines. Here we show that near-misses can dramatically affect player frustration and arousal even despite the complexity of the winning (and losing) symbol alignments in multi-line games. This extension is important given that multiline games appear to be the games of choice for seasoned gamblers (Livingstone et al."}
+{"text": "Staphylococcus aureus strains and their role in the pathogenesis of mastitis is lacking.Superantigens (SAgs) represent a diverse family of bacterial toxins that induce V\u03b2-specific T cell proliferation associated with an array of important diseases in humans and animals, including mastitis of dairy cows. However, an understanding of the diversity and distribution of SAg genes among bovine  Staphylococcus aureus strains and their role in the pathogenesis of mastitis is lacking. Population genomic analysis of 195 bovine S. aureus isolates representing 57 unique sequence types revealed that strains encode 2 to 13 distinct SAgs and that the majority of isolates contain 5 or more SAg genes. A genome-scale analysis of bovine reference strain RF122 revealed a complement of 11 predicted SAg genes, which were all expressed in vitro. Detection of specific antibodies in convalescent cows suggests expression of 7 of 11 SAgs during natural S. aureus infection. We determined the V\u03b2 T cell activation profile for all functional SAgs encoded by RF122, revealing evidence for bovine host-specific activity among the recently identified RF122-encoded SAgs SElY and SElZ. Remarkably, we discovered that some strains have evolved the capacity to stimulate the entire T cell repertoire of cattle through an array of diverse SAgs, suggesting a key role in bovine immune evasion.Superantigens (SAgs) represent a diverse family of bacterial toxins that induce V\u03b2-specific T cell proliferation associated with an array of important diseases in humans and animals, including mastitis of dairy cows. However, an understanding of the diversity and distribution of SAg genes among bovine  Staphylococcus aureus produces a family of at least 26 distinct superantigens (SAgs), including the staphylococcal enterotoxins (SEs) SEA to -E, SEG to -J, and SER to -T; the staphylococcal enterotoxin-like toxins (SEls) SElK to -Q, -U, -V, and -X to -Z; and toxic shock syndrome toxin 1 (TSST-1)  , 4. The  , 4. Theor cells \u20137. TakenS. aureus is a common cause of bovine mastitis, an infection of the milk-secreting tissue of the udder, which represents a huge economic problem for the dairy industry worldwide  and was the first animal-associated isolate to be fully sequenced  for the presence of all 26 known members of the S. aureus SAg family (We examined 195 bovine selw and selx were found in 100% (195/195) and 79% (150/195) of isolates analyzed, respectively. Previous studies identified selw to be inactivated in a large number of human S. aureus isolates examined, due to the lack of an ATG start codon , consistent with data from previous reports  was less prevalent than the egc cluster, found in 10 of the 57 STs analyzed, primarily in association with CC133 and CC151. The plasmid-borne SAg genes sed, sej, and ser were identified together in 4 strains, consistent with the presence of a pIB485-like plasmid as described previously in human strains (sely and selz genes are distributed in a lineage-specific manner (CC151 and CC9 for sely and CC151 for selz), and the SAg genes sea, seb, seh, selk, selp, and selq were randomly distributed across the diversity of STs examined, consistent with horizontal gene transfer. The set and ses genes were not found in any S. aureus genomes examined, suggesting that they are not important in bovine pathogenesis.Consistent with data from previous studies, rt codon , 22. How reports . The egc strains . The selS. aureus strains examined contained at least 2 and up to 13 SAg genes. The majority of bovine STs analyzed (31/57) encode 5 or more SAgs, with CC151 isolates, such as RF122, generally encoding more SAgs (up to 13) than other bovine S. aureus strains. Fewer than half of the STs (n = 26) contained selw and selx only. An important example is the bovine reference strain Newbould 305, which has been the focus of a number of studies  revealed a complement of 11 SAg genes and 2 SAg pseudogenes  in the genomic island vSa\u03b2 containing allelic variants of the SAg genes seg, sei, selo, seln, and selu and a pseudogene of selm. Spread out across other parts of the genome, RF122 also contains selw , selx, sely, and selz , rSEGbov, and rSElObov were not reactive with any of the samples tested  induced CD8+ CD25+ FOXP3+ regulatory T cells that strongly suppress the activation of effector T cells   .S. aureus strain RF122 to facilitate plasmid-mediated expression of each SAg in isolation by its native S. aureus strain. S. aureus RF122-1, a TSST-1-deficient derivative of RF122, was constructed previously by allele replacement of the tst gene with a tetracycline resistance cassette , resulting in the sequential mutants RF122-2 to RF122-7 and the final SAg-deficient derivative RF122-8 . Finally, to limit Hla-mediated toxicity for T cells, we constructed hla mutants in the parent strain RF122 and SAg-deficient derivatives, resulting in strains RF122t-\u03b1 and RF122-8\u03b1, respectively (Table S3). The mutants were validated to rule out that spurious mutations accrued during in vitro passage that impact secreted virulence proteins . Analysis of the mitogenicity of stationary-phase and mid-exponential-phase culture supernatants of RF122 and RF122-8 confirmed the loss of all detectable mitogenic activity .In order to examine the mitogenicity of each of the 11 identified SAgs, we constructed a SAg-deficient mutant of cassette . In turnbov, TSST-1bov and SElXbov were described previously . To examine the mitogenicity of RF122-encoded SAgs for bovine T cells, culture supernatants of RF122-8\u03b1 containing pALC2073::SAg constructs and recombinant SAg proteins were used to stimulate bovine peripheral blood mononuclear cells (PBMCs), and proliferation was measured by using a thymidine incorporation assay  activated bovV\u03b2 subfamilies 24, 28, and X . For SElY, three positions varied between the bovine allele from RF122 (ST151) and the human allele from MSA2020 (ST121) . In particular, the glutamic acid residue at position 19 was identified in the SElY allele of ST151 and other cattle isolates  but not in any of the SElY variants of human origin. For SElZ, four positions varied between the bovine allele from RF122 (ST151) and the human allele from MSA1695 . Of note, the glycine residue at position 106 of RF122 SElZ was found in all but one of the bovine SElZ variants analyzed and was absent among the majority of human variants (6/8).bov activates bovV\u03b26 and -24 but not humV\u03b26 and -24, TSST-1bov activates only bovine V\u03b24 and -24, SEIbov activates bovV\u03b216 and humV\u03b25 and -6 but not the equivocal variants in the opposite species, and SElNbov activates bovV\u03b23, -16, and -24 and humV\u03b27 and -8 but not the equivalent human or bovine subgroups. It is important to note that with the exception of SECbov and SElXbov, the human V\u03b2 profiles described here were determined in previous reports in response to stimulation with SAgs derived from human S. aureus strains  and bovV\u03b210, -28, and -X (absent in human) . However strains , 12. It S. aureus bovine mastitis, preliminary experimental infections of bovine mammary glands were carried out using RF122 and RF122-8 over a course of 21 days. Seven healthy dairy cows in their 1st to 4th lactation were enrolled in two groups of 4 and 3 cows and challenged with wild-type (WT) RF122t and the SAg-deficient strain RF122-8, respectively. There were no differences observed between the groups in terms of somatic cell counts, milk yields, and core body temperatures . S. aureus was isolated from the mammary glands of all animals during the trial; taken together with the milk quality and somatic cell counts, these data indicate that SAgs are not required to establish subclinical mastitis. The group infected with wild-type RF122 exhibited clinical mastitis at least once, in three out of the four animals infected during the course of the study  class II molecules in multiple ways, contributing to immune evasion. Our findings contribute to the understanding of staphylococcal SAg diversity and provide a comprehensive analysis of the bovine T cell response to SAgs. In addition, we report examples of toxins that contribute to the capacity of in vivo work was done after local ethical review, under the oversight of the Kalamazoo IACUC, and in accordance with local, state, and federal animal welfare regulations. Bovine venous blood was taken under the authority of a UK Home Office project license (PPL 604394) within the terms and conditions of the regulations of the UK Home Office Animals (Scientific Procedures) Act of 1986 and the code of practice for the housing and care of animals bred, supplied, or used for scientific purposes. Human venous blood was taken from healthy donors in accordance with a human subject protocol approved by the National Research Ethics Service (NRES) Committee of South East Scotland under research ethics committee reference number 11/AL/0168. Volunteers were recruited by a passive advertising campaign within The Roslin Institute (University of Edinburgh), and written consent was given by each volunteer before each sample was taken.All S. aureus strains were grown in tryptone soya broth (TSB) or brain heart infusion (BHI) broth  shaken at 200 rpm or on tryptone soya agar (TSA)  at 37\u00b0C for 16 h unless otherwise stated. E. coli strains were grown in Luria-Bertani (LB) broth  shaken at 200 rpm or on LB agar  at 37\u00b0C for 16 h unless otherwise stated. Media were supplemented where appropriate with 150 \u03bcg/ml X-gal , 50 \u03bcg/ml ampicillin, and 10 \u03bcg/ml erythromycin or chloramphenicol . For growth curve analysis of S. aureus, strains were cultured overnight in 5 ml BHI broth  in triplicate. After 12 h, strains were subcultured at a dilution of 1:100 into 30 ml fresh BHI broth in 250-ml Erlenmeyer flasks and placed in a shaking incubator at 37\u00b0C at 200 rpm. Absorbance readings were measured at 600 nm  using a spectrophotometer  over a period of 12 h, and a growth curve was determined.S. aureus genomes using BLASTn with a minimum alignment of 90% nucleotide identity averaged across the entire gene sequence using the Blastable script (https://github.com/bawee/blastable). Representative genomes with unique sequence types and SAg contents were selected, and a core genome alignment was built using Parsnp (S. aureus RF122 genome sequence (GenBank accession number AJ938182) as a reference.The sequences of characterized staphylococcal SAg genes were obtained from the NCBI GenBank database . SAg homologs were identified in publicly available whole-genome sequences of bovine and representative human g Parsnp . The assg Parsnp . Nucleotg Parsnp , 41. A mg Parsnp . BRIG  and stationary-phase (12-h) cultures using the RNeasy miniprep kit  according to the manufacturer's instructions except for an added lysis step with resuspension of the bacterial pellet in Tris-EDTA (TE) buffer with 100 \u03bcg/ml lysostaphin and incubation at 37\u00b0C for 20 min. RNA was treated with Turbo DNase . A total of 0.5 \u03bcg mRNA was analyzed for gene transcription using the same protocol as the one outlined by Wilson et al. . Deletion of the hla gene in RF122 resulted in a reduction in the hemolytic titer, indicating that these strains are less toxic than the wild type. Analysis of the profiles of secreted and cell wall-associated (CWA) proteins of WT and mutant strains revealed no unexpected differences .Gene deletion constructs of SAg genes in RF122 were performed by using constructs prepared in plasmid pMAD  see Tab, 44. TheS. aureus mid-exponential-phase (OD600 = 0.6) and stationary-phase (12-h) cultures grown in BHI medium. Cells were centrifuged at 4,000 \u00d7 g, and supernatant fractions containing secreted proteins were removed and concentrated with Amicon Ultra-15 centrifugal filter units with a 10-kDa molecular weight cutoff (MWCO) according to the manufacturer's instructions . To extract CWA proteins, pelleted cells were washed with 1 ml phosphate-buffered saline (PBS) , resuspended in 1 ml lysis buffer  containing 200 \u03bcg/ml lysostaphin  and protease inhibitors , and incubated at 37\u00b0C for 20 min. Samples were centrifuged at 6,000 \u00d7 g for 20 min, and CWA proteins were recovered from the supernatant fraction. Protein preparations were separated on 10% SDS-PAGE gels, stained overnight at room temperature with Coomassie blue (Severn Biotech), or transferred to nitrocellulose membranes  for Western blot analysis. The membrane was incubated with primary antibody for 1 h with a 1:2,500 dilution of anti-SEC  or for 2 h with a 1:2,000 dilution of rat antiserum specific for rTSST-1, rSElL, or rSElXbov. The membrane was incubated with secondary antibody for 1 h at a dilution of 1:2,500  or 1:1,500  and visualized by enhanced chemiluminescence (ECL).Secreted and CWA proteins were extracted from E. coli DH5\u03b1 cells. The resulting pALC2073::SAg plasmids were isolated from DH5\u03b1 cells and transformed by electroporation into an intermediate electrocompetent strain of S. aureus, RN4220. Subsequently, the plasmids were reisolated and transformed into the SAg-deficient strain RF122-8. S. aureus strains were made competent as described previously (5\u2032 oligonucleotides to amplify RF122-borne SAg genes for cloning into the expression plasmid pALC2073 were designed to prime upstream of the predicted ribosome binding site (RBS) with a KpnI site incorporated to facilitate cloning . The 3\u2032 primer was designed to include the stop codon of the gene with a SacI site incorporated (Table S2). PCRs were carried out with 10 ng RF122 genomic DNA (gDNA) and 100 nmol forward and reverse primers, as listed in Table S2 in the supplemental material, using 1 U Vent polymerase  according to the manufacturer's instructions. PCR products were cloned into the Strataclone pSC-B plasmid , and inserts were released by digestion with SacI and KpnI for 3 h at 37\u00b0C, purified by gel extraction, ligated with digested pALC2073 plasmid DNA using T4 DNA ligase, and transformed into eviously . RF122-8http://www.cbs.dtu.dk/services/SignalP/), and 3\u2032 primers were designed to include the stop codon of the gene . The cloning procedure was performed as outlined above for pALC2073, and ligated constructs were transformed into E. coli DH5\u03b1 or XL1-Blue (for pQE30-Xa constructs) cells. pET constructs were isolated from DH5\u03b1 cells using the QIAprep spin miniprep kit  and transformed into E. coli BL21(DE3) cells. BL21 or XL1-Blue cells containing expression constructs were cultured in Luria broth containing 50 \u03bcg/ml ampicillin  and induced in the mid-exponential phase of growth (OD600 = 0.6) with 1 mM isopropyl-\u03b2-d-1-thiogalactopyranoside (IPTG)  for 4 h. Cells were recovered by centrifugation at 8,000 \u00d7 g and disrupted by using a French press, and His-tagged recombinant proteins were purified by affinity chromatography on a Ni-nitrilotriacetic acid (NTA) nickel affinity column . Proteins were dialyzed by using Spectra/Por Float-A-Lyzer tubing with an 8,000 to 10,000 MWCO .5\u2032 primers for cloning into the pET15b  or pQE30-Xa  plasmid were designed to anneal immediately after the signal peptide coding region, as predicted by the Signal P 3.0 server  was incubated with 10 ml of blocking buffer containing 5% (wt/vol) skimmed milk powder  in PBST  overnight at 4\u00b0C. The membrane was then incubated for 2 h with a 1:1,000 dilution of pooled bovine convalescent-phase serum in PBST with 1% (wt/vol) skimmed milk and washed three times with PBST. Secondary antibody  was added at a concentration of 400 ng/ml for 1 h at room temperature. The blot was washed again. Immunoreactivity was visualized by chemiluminescence from ECL.SDS-PAGE and Western blotting were carried out on SAgs overexpressed in d-glucose  and 20.5 g trisodium citrate  added to 1 liter of double-distilled water [ddH2O]). The buffy coat was isolated by spinning the blood at 1,500 \u00d7 g for 15 min with no break, and PBMCs were then isolated by using Ficoll Paque plus  according to the manufacturer's specifications. PBMCs were adjusted to a concentration of 1 \u00d7 106 cells/ml in complete cell culture medium  and stimulated at least in triplicate with the concentrated total protein S. aureus supernatant fraction or recombinant protein. Culture medium and 50 \u03bcg/ml concanavalin A were used as negative and positive controls, respectively. Proliferations of bovine and human PBMCs were assessed by a [3H]thymidine incorporation assay as described previously (6 cells) by using Tri reagent  according to the supplier's instructions or by using the RNeasy plus kit  according to the manufacturer's instructions. First-strand cDNA was generated from 0.5 \u03bcg of RNA using a Power SYBR green RNA-to-CT 2-step kit or a high-capacity RNA-to-cDNA kit and Power SYBR green PCR master mix . The reverse transcription reaction was performed with a 20-\u03bcl volume according to the manufacturer's specifications. Bovine V\u03b2 subfamily-specific qRT-PCRs were carried out as described previously  as described previously . Human Peviously . Total Reviously . Human Veviously , 46.S. aureus grown overnight were inoculated 1:50 into fresh TSB and grown until an OD600 of 1.1 was reached. Staphylococci were diluted in TSB to obtain an inoculum of 5 \u00d7 107 CFU/ml. Inocula were determined by CFU enumeration following serial dilution, plating on TSA, and growth at 37\u00b0C. Animals were challenged via teat dip immersion twice daily (22-mm immersion) until a score of 1 or higher for milk appearance or udder evaluation was observed and the animal developed an intramammary infection twice within a 5-day period. Following infection, animals were observed for a total of 3 weeks. Somatic cell counts (SCCs) and cultures were performed twice a week. Udder and milk clinical scores and milk yield and milk conductivity data were collected at each milking, which was performed twice daily.Adult cows (Holstein) in their 1st to 4th lactation at 92 to 174 days in milk (DIM) were used in this study. Cultures of t test with Welch's correction if required. Tests were unpaired and two tailed, and significant differences were considered when the P value was <0.05.All statistical analysis was performed in GraphPad Prism 7. Fold change enrichment data were analyzed by using the Student"}
+{"text": "The Interferon regulatory factors (IRFs) are a family of transcription factors that play pivotal roles in many aspects of the immune response, including immune cell development and differentiation and regulating responses to pathogens. Three family members, IRF3, IRF5, and IRF7, are critical to production of type I interferons downstream of pathogen recognition receptors that detect viral RNA and DNA. A fourth family member, IRF9, regulates interferon-driven gene expression. In addition, IRF4, IRF8, and IRF5 regulate myeloid cell development and phenotype, thus playing important roles in regulating inflammatory responses. Thus, understanding how their levels and activity is regulated is of critical importance given that perturbations in either can result in dysregulated immune responses and potential autoimmune disease. This review will focus the role of IRF family members in regulating type I IFN production and responses and myeloid cell development or differentiation, with particular emphasis on how regulation of their levels and activity by ubiquitination and microRNAs may impact autoimmune disease. Interferon regulatory factors (IRFs) are a family of transcription factors that regulate many aspects of innate and adaptive immune responses\u2014including driving anti-viral responses, responding to pathogens to drive pro-inflammatory responses and regulating immune cell differentiation . ComprisThis review will address the role of IRF family members in regulating type I IFN production and responses and myeloid cell development or differentiation. Specifically, it will focus on providing an update on how regulation of their levels and activity by microRNAs or ubiquitination may impact IFN-driven autoimmune disease.The type I IFN system comprises 13 subtypes of IFN-\u03b1, in addition to IFN-\u03b2, IFN-\u03b5, IFN-\u03bb, and IFN-\u03b8 , 7. The Irf\u2212/\u2212 MEFs suggested IRF1 was non-essential for induction of IFNs in response to cytosolic viruses  or Newcastle disease virus (NDV)  . This imus (NDV) . In addius (NDV) , 40. In us (NDV) . IRF5 isalbicans . Such prIFNA1 promoter, while IRF3 and IRF5 cooperatively activate this promoter  and monocytes has also been reported. Whilst principally known for its role in proinflammatory gene induction, IRF8 also reportedly takes part in a second phase of interferon induction in dendritic cells in response to Newcastle Disease virus (NDV) which triggers IFN induction via activation of RIG-I dependent pathways . The rolCanonical type I IFN signaling occurs following binding of IFN to the ubiquitously expressed type I IFN receptor (IFNAR), comprising two transmembrane proteins, IFNAR1 and IFNAR2 . This reInterestingly, the long-held paradigm that IFN\u03b1-driven tyrosine phosphorylation of both STAT1 and STAT2 is a prerequisite for interaction with IRF9  has receThe role of IRFs in infection, protective immunity and primary immunodeficiencies has been reviewed extensively elsewhere in this focused issue . Given tSTING or TREX1 (which both work to regulate IFN-\u03b2 production) drive monogenic forms of IFN-driven disease (interferonopathies) have suggested that dysregulation of these pathways may contribute to interferon driven diseases such as SLE or Sjogren's syndrome  in patients with initially high ISG scores, whereas the effects of Rontalizumab were greatest in patients with low to moderate levels of ISGs , 96. Anisyndrome . Indeed,syndrome \u2013101. Morsyndrome . Whethersyndrome , remainsIn addition to regulating IFN production IRFs have important roles in regulating immune cell development and differentiation . Whilst Hematopoietic stem cells give rise to both the myeloid and lymphoid arms of hematopoietic lineage. Myeloid cells derive primarily from the Common Myeloid Progenitor (CMP) whereas the lymphoid arm derive from the Common Lymphoid Progenitor (CLP). The CMP can give rise to all types of myeloid cells, including monocytes, neutrophils and most types of dendritic cells (DCs). A unique subset of DCs, termed plasmacytoid DCs derive from CLP. IRFs play an integral role in both DC and monocyte development. DCs are essential for antigen presentation and act as the bridge between innate and adaptive immune responses. They comprise four main subsets of DCs\u2014conventional DCs (cDCs), plasmacytoid DCs (pDCs), monocyte-derived DCs, and Langerhans cells. Conventional DCs in mice are further sub-grouped into cDC1 and cDC2 subsets with different markers for human and murine counterparts .+ T cell effector function and expansion. High expression of IRF8 in combination with E2-2 and Bcl11A are required for development of pDCs, which secrete high amounts of type I IFN in response to stimulation. IRF1 and IRF2 also appear to be important in regulating DC subset development\u2014Irf\u2212/\u2212 mice show a loss of splenic and epidermal DCs   , 110 whend TGF-\u03b2 . In addind TGF-\u03b2 . Irf8\u2212/\u2212d organs , 114. IRd organs . A recend organs , indicatLike DCs, macrophages play an important role in sensing pathogens, initiating innate immunity, and cross-talking with the adaptive immune system to generate an appropriate immune response. Like DCs and T cells, subsets of macrophages with differing functions have been identified . Bet al to work in concert with IRF4 to induce M2 polarization  targeting.Ubiquitination, like phosphorylation, is a reversible process regulated by E3 ligases that add ubiquitin chains to targets and de-ubiquitinases that remove these chains . UThe activity of IRF proteins is tightly controlled through both ubiquitination and SUMOylation. In general, ubiquitination and phosphorylation of IRFs are integrally linked, with one modification often being a pre-requisite for the other to take place . For exaRegulation of IRF3 activity by ubiquitination or other ubiquitin like modifiers such as SUMO or ISG15, is highly complex, and most likely is highly dependent on context and cell type. IRF3 stability is regulated by K48-linked ubiquitination by TRIM21 promoting proteasomal degradation post TLR-stimulation in order to turn off and limit responses . Indeed,Similar to IRF7, IRF3 is also regulated by other ubiquitin-like modifiers: addition of SUMO and another modifier interferon stimulated gene 15 (ISG15) to on the N terminal DBD works to sustain IRF3 levels by protecting these sites from ubiquitination. Ubc9 for example SUMOylates IRF3  whilst SIRF5 stability is also regulated by ubiquitination. K63-linked ubiquitination of IRF5 by Pelino-1 for example positively regulates M1 polarization downstream of TLR4/IFN-\u03b3. This study also linked the Pellino-1-IRF5 axis to regulation of glucose intolerance in obesity, with BMDMs from mice lacking Pellino-1 showing improved glucose intolerance when fed a high-fat diet . Work frInterestingly, ubiquitination of IRF1 is linked with stability and seems to be required for IL-1-induced expression of the chemokines CXCL10 and CCL5, thus promoting inflammatory cell recruitment . The E3 As to whether other IRF proteins that are involved in regulating IFN production or downstream signaling pathways are regulated by ubiquitin-like post-translational modification remains to be determined. Given the fact that type I IFNs themselves rapidly induce expression of both E3 ligases [particularly the TRIM family ] that tamicroRNAs (miRs) are important regulators of gene expression in a whole host of cellular processes and immune responses , 164. Thper se\u2014for example in breast cancer cells, miR-762 targets IRF7, inhibiting proliferation and invasion in a matrigel assay , IRF9 levels and activity are critical in mediating STAT1/STAT2 driven responses. A number of microRNAs have been published that target IRF9 directly. miR-373 for example is upregulated by Hepatitis C virus (HCV) and targets IRF9 and JAK1 in order to turn off and limit anti-viral defense mechanisms . Our ownRegarding a role for microRNAs in targeting IRFs to influence myeloid cell development or differentiation, one would expect that targeting IRF4, IRF8, or IRF5 would directly influence these events. Indeed, as mentioned above, miR-302a targets IRF5 to influence M1/M2 levels in response to viral infection . miR-125miR-125a expression decreased in SLE monocytes and identified a novel target, IL-16, which regulates CXCL10 expression in lung epithelial cells and helps drive lung inflammation in an autoimmune context (Given the numerous roles microRNAs play in fine tuning TLR and IFN responses, it is not surprising that the dysregulation of these molecules has been implicated in SLE. To date numerous examples of dysregulated SLE associated microRNAs have been identified \u2013189. Bes context . Given tNumerous mechanisms exist to control the innate immune response and myeloid cell differentiation in order to prevent inflammatory and autoimmune disease. As IRF family members are critical in this respect, tight regulation of their levels and activity is one mechanism of maintaining tolerance to self-antigens such as self-nucleic acids. But in different diseases it appears individual IRFs have greater or lesser involvement [reviewed in ]. For exRegarding potential targeting strategies: Ubiquitination of IRFs is a rapid and versatile way to regulate both levels and activity of IRFs, whereas epigenetic targeting of IRFs by microRNAs can fine tune IRF expression levels. Both work in concert to tailor immune responses appropriately. However, many questions remain regarding the IRFs and how they are regulated as it pertains to IFN biology: for example\u2014what role do IRFs play in IFNAR-independent induction of ISGs? Is it possible that different combinations of STATs and IRFs can replace the canonical ISGF3 transcriptional complex? What role does regulation of availability of IRFs by microRNA targeting play in this process? And finally, can we target E3 ligases to fine tune IRF function and levels? Answering these questions will undoubtedly contribute to our understanding regarding how IRFs contribute to the pathology of autoimmune diseases such as SLE, but its biggest impact will be in explaining the following: firstly how we can improve on current IFN-targeting strategies\u2014i.e., will JAK inhibition provide enhanced efficacy compared with IFNAR targeting strategies? And secondly, potentially uncover additional new therapeutic targets\u2014be they modulators of E3 ligase activity or RNA-targeting strategies. As central regulators of monocytes function and IFN biology, addressing these questions promises to have a big impact in IFN-driven autoimmune disease.The author confirms being the sole contributor of this work and has approved it for publication.The author declares 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": "Lactobacillus amylovorus (LA), Lactobacillus mucosae (LM) or probiotic Escherichia coli Nissle 1917 (EcN) in infection of gnotobiotic piglets with Salmonella Typhimurium (ST). Transcription of HMGB1 and Toll-like receptors (TLR) 2, 4, and 9 and receptor for advanced glycation end products (RAGE), TLR4-related molecules , and adaptor proteins (MyD88 and TRIF) in the ileum and colon were measured by RT-qPCR. Expression of TLR4 and its related molecules were highly upregulated in the ST-infected intestine, which was suppressed by EcN, but not LA nor LM. In contrast, HMGB1 expression was unaffected by ST infection or commensal/probiotic administration. HMGB1 protein levels in the intestine measured by ELISA were increased in ST-infected piglets, but they were decreased by previous colonization with E. coli Nissle 1917 only. We conclude that the stability of HMGB1 mRNA expression in all piglet groups could show its importance for DNA transcription and physiological cell functions. The presence of HMGB1 protein in the intestinal lumen probably indicates cellular damage.High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can be actively secreted by immune cells after different immune stimuli or passively released from cells undergoing necrosis. HMGB1 amplifies inflammation, and its hypersecretion contributes to multiple organ dysfunction syndrome and death. We tested possible immunomodulatory effect of commensal  High mobility group box 1 (HMGB1) is an intracellular nuclear DNA-binding protein that can be produced by innate immune cells or released from cells undergoing necrosis  and partGram-positive and Gram-negative bacteria induce different inflammatory cytokine patterns  and theiIn contrast to DAMPs, pathogen-associated molecular patterns (PAMPs) are molecular structures typical for microorganisms . Both PASalmonella enterica serovar Typhimurium (S. Typhimurium) commonly causes gastroenteritis  in humans and pigs [S. Typhimurium can also cause life-threatening invasive diseases in immunocompromised individuals [Salmonella enterica against extracellular antibiotics and facilitates disease relapse [Closely related human and pig anatomy, genetics, physiology , and higand pigs  but humaand pigs . S. Typhividuals . The int relapse ,34,35. T relapse ,37. One  relapse . Lactobacillus spp. are Gram-positive facultative anaerobes that create an abundant bacterial group in human and pig microbiota in the distal small intestine and colon [Lactobacillus spp. produce organic acids with antimicrobial properties and some species also produce other antimicrobial compounds, such as bacteriocins and H2O2 [Lactobacillus spp. are typically beneficial for the host, care should be taken with their application in immunocompromised hosts [L. rhamnosus GG, L. casei Shirota, and L. acidophilus LB, are widely used probiotics [Escherichia coli that includes both pathogenic [E. coli Nissle 1917 (EcN) is anti-diarrheic in humans [nd colon ,40 . A snd colon . Moreoveand H2O2 . Despiteed hosts  and all ed hosts . Some laobiotics , and comobiotics ,47. Anotthogenic  and probthogenic  strains.n humans  and pigsn humans . This efn humans . Salmonella Typhimurium strain LT2 is a well-characterized laboratory strain [Gnotobiotic animals have, in contrast to conventional animals, simple and defined microbiota . Their ly strain . It was y strain , but it y strain . Salmonella Typhimurium, signaling via TLR2, TLR4, TLR9, and RAGE, and the possible influence of previous colonization of the piglets with commensal Gram-positive lactobacilli Lactobacillus amylovorus and Lactobacillus mucosae or probiotic Gram-negative E. coli Nissle 1917. Knowledge of innate immune regulations and possibilities to modify TLRs signaling pathways can be helpful for the development of advanced therapeutic procedures to ameliorate health problems in the S. Typhimurium infections, especially in life-threatened immunocompromised individuals. Our work aimed to describe intestinal HMGB1 release after infection of gnotobiotic piglets with Gram-negative enteric pathogen S. Typhimurium (ST); (iii) GF piglets colonized with L. amylovorus (LA); (iv) LA piglets infected with S. Typhimurium (LA+ST); (v) GF piglets colonized with L. mucosae (LM); (vi) LM piglets infected with S. Typhimurium (LM+ST); (vii) GF piglets colonized with E. coli Nissle 1917 (EcN), and (viii) EcN piglets infected with S. Typhimurium (EcN+ST).Eight groups of gnotobiotic piglets were used in the experiments \u2014(i) germL. amylovorus (LA) and L. mucosae (LM) nor probiotic E. coli Nissle 1917 (EcN) influenced TLR4 transcription in the ileum of the gnotobiotic piglets   D. Statisin (LBP) E. In conin (LBP) F, where in (LBP) G showed in (LBP) H, and alin (LBP) I also diS. Typhimurium infected groups  compared to GF group. MD2 showed statistically significant upregulation in LM+ST only . In contrast, LBP expression  only. TLR2 did not show any obvious pattern , but only in the group precolonized with L. mucosae was this upregulation statistically significant , showed increased expression of CD14 mRNA, but it was statistically significant in the LA+ST group only  showed MyD88 mRNA statistically significant upregulation  except the EcN+ST piglets (L. amylovorus or L. mucosae (The relative expression of TLR4 was upregulated in mesenteric lymph nodes (MLN) of the groups infected with nificant A. MD-2 wnificant B. The group only C. No staoup only D. All Sagulation E. In congulation F. TLR2 m piglets G. TLR9 s piglets H. RAGE w mucosae I. The mRNA relative expressions of TLR4 and its related molecules, TLR2, TLR9, and RAGE in the ileum, colon, and MLN were summarized in p \u02c2 0.01) between the control GF and EcN groups only  were studied by immunofluorescence . In plasma, much lower concentrations were detected compared to ileal contents and no statistically significant differences were found in any group  is highly immunomodulatory compound that can induce endotoxin shock ,62, whicLactobacillus spp. are abundant in pig distal small intestine and colon [L. amylovorus and L. mucosae, and the probiotic E. coli Nissle 1917. We anticipated that the actions of two Lactobacillus strains would differ. It was reported that L. amylovorus modulation of TLR4 signaling prevented damage of an epithelial cell line and intestinal explants infected with enterotoxigenic E. coli K88 [L. mucosae express a typical strain-specific mucus-binding protein (Mub), which mediates its binding to mucus in vitro [E. coli Nissle 1917 has a semi-rough LPS (incompletely synthesized LPS chain) that determines its serum sensitivity and prevents the immunocompetent host from developing bacteremia from E. coli Nissle 1917 [nd colon ,76. Prevnd colon ,77, intend colon , and connd colon . We attecoli K88 ,79, wherin vitro . E. colisle 1917 .Salmonella Typhimurium and the host is in the distal ileum [E. coli [S. Typhimurium infection in gnotobiotic piglets up-regulated ileal mRNA expression of both members of TLR4/MD-2 complex and its co-receptor CD14 and LBP, with the exception the piglets previously associated with E. coli Nissle 1917 . This increase in TLR4 expression coincided with an upregulation of chemokine IL-8, pro-inflammatory cytokine TNF-\u03b1, and regulatory cytokine IL-10 in S. Typhimurium-infected piglets, but not in the piglets associated with L, amylovorus, L. mucosae or E. coli Nissle 1917, as we described elsewhere [E. coli Nissle 1917 suppressed the increased cytokine expression induced by S. Typhimurium infection. The main site of cross-talk between al ileum . It is a[E. coli ,82. S. Tlsewhere . Thus, pSalmonella enterica serovar Infantis and serovar Typhimurium, with an incompletely synthesized LPS chain, both protected gnotobiotic piglets against subsequent infection with virulent S. Typhimurium strains F98 [S. enterica serovars [E. coli Nissle 1917 [S. Typhimurium. Therefore, they prevented the excessive detrimental production of the inflammatory cytokines [It was shown that the avirulent rough LPS ains F98  and LT2 ains F98 , respectains F98 ,84, actiserovars ,84, as wsle 1917 , disruptytokines . All theytokines ,83,84, wS. Typhimurium or association to other bacteria used before the challenge with S. Typhimurium. We concluded that the signaling in the Salmonella infection was via the MyD88 regulatory pathway. TLR4 is only one of the Toll-like receptors that use both MyD88 and TRIF adaptor molecule pathways ,85. MyD8E. coli and E. coli-derived LPS in pigs by CD14 neutralization antibodies resulted in decreased levels of pro-inflammatory cytokines IL-1\u03b2, IL-6, IL-8, and TNF-\u03b1, and lower granulocyte activation in a pig model of sepsis [S. Typhimurium in the ileum, but the downregulation was not statistically significant. It is possible that TLR2 and TLR9 play opposite roles during S. Typhimurium infection in the gnotobiotic piglets, as was found in mice [E. coli Nissle 1917 inhibited the up-regulation of TLR9 mRNA expression after S. Typhimurium infection. Similarly, E. coli Nissle 1917 ameliorated dextran sulfate-induced colitis and decreased the pro-inflammatory cytokine response in wild-type mice, but not in TLR2 or TLR4 knockout mice [E. coli Nissle 1917 immunomodulation of host TLR2 and TLR4 signaling. Concordantly with our findings of induced inflammatory cytokines reported elsewhere [S. Typhimurium showed upregulation of TLR2 and TLR4 in the ileum that resulted in the expression of pro-inflammatory cytokine IL-1\u03b2, IL-6, and TNF-\u03b1 mRNA expressions 2 days post-infection [The CD14 is well known about as co-receptor of TLR4, but much less is known its participation as co-receptor of TLR2  and TLR9f sepsis . TLR2 mR in mice . Consistout mice , which slsewhere , 4-week-S. Typhimurium differed in their mRNA expression of inflammatory response-related genes, including TLRs [E. coli Nissle 1917-associated groups showed significantly increased TLR9 mRNA expression, which has not been previously reported.The colon has a different histological structure and physiological role than the ileum . The micing TLRs . In the The MLN are the main site of antigen presentation to combat bacteria that are translocated via M-cells, enterocytes, and dendritic cells or have penetrated autonomously through the disrupted intestinal barrier ,96. The Salmonella-infection, except in piglets previously innoculated with the probiotic E. coli. MD-2 enables TLR-2 to recognize LPS and enhances TLR2-mediated responses to both Gram-positive and Gram-negative bacterial, including peptidoglycan, lipoteichoic acid components, and LPS [Salmonella was probably discriminated in the cooperation of MD-2, CD14, TLR4 but also TLR2. The TLR2 signaling confirmed increased MyD88 mRNA expression that is the adaptor molecule participating in the TLR2 signaling pathway [In our experiments, gene expression of the receptors was lower in the MLN than either ileum or colon. In the MLN, expression of CD14, TLR2, and MyD88 all increased in response to  and LPS . It seem pathway .8 CFU S. Typhimurium serovar DT104 did not show any differences in CD14, TLR2, and MyD88 mRNA expression in the MLN two days post-infection [L. amylovorus, which was not found in the MLN, in contrast to L. mucosae that was found in a low number of CFUs in part of the piglets [In contrast to our results, four-week-old conventional piglets orally infected with 10nfection . RAGE mRnfection ,102. The piglets . Even in piglets . The eluE. coli O55-infected gnotobiotic piglets, but not in infected piglets that thrived [S. Typhimurium-infected ileum [Clostridium perfringens type C [HMGB1 is the endogenous ligand of TLR2, TLR4, and TLR9 . It is a thrived . Gnotobied ileum . Lastly,s type C . TherefoS. Typhimurium-infected piglets only to simplify imagination about the HMGB1 localization and migration changes in the S. Typhimurium infection. HMGB1 in the mesenteric lymph nodes of the GF piglets was colocalized mainly with the nucleus, but it was spread into the cytoplasm in the Salmonella-infected ST group. Posttranslational modifications of HMGB1 determine its localization, migration, and biological activity. HMGB1 protein stays in the nucleus, but its hyperacetylation , hyperphS. Typhimurium-infected groups suggest that secretion was in response to the infection. Lower intestinal HMGB1 levels in the intestine of infected piglets who has been inoculated with E. coli Nissle 1917 (EcN+ST) confirmed the anti-Salmonella properties of this probiotic bacteria [Salmonella-infected piglets were not from de novo synthesized HMGB1.HMGB1 translocated from the nucleus to the cytoplasm and was secreted from cells; nevertheless, the expression of HMGB1 mRNA was not upregulated 16 hrs after the stimulation . The higbacteria . The HMGS. Typhimurium within 24 hrs. This stability of the HMGB1 mRNA expression in all groups underlines the importance of HMGB1 for DNA transcription and other physiological cell functions and indicates that increased intestinal HMGB1 levels likely originated from formerly synthetized and actively secreted HMGB1 or passively released HMGB1 from damaged cells. However, the presence of HMGB1 protein in the intestinal lumen probably more attests spontaneous release after cellular damage than induced secretion as it was possible to suppress with the prior association of the gnotobiotic piglets with probiotic E. coli 1917. Future bacteria-driven immunomodulatory studies are needed to modify the signaling pathways and prevent or dampen the sepsis. In conclusion, HMGB1 mRNA transcription was not activated by the infection of the gnotobiotic piglets with All animal experiments were approved by the Animal Care and Use Committee of the Czech Academy of Sciences, protocol No. 117/2012, on 24 April 2012. E. coli Nissle 1917 (EcN) was originally donated to the collection by U. Sonnenborn  and Salmonella enterica serovar Typhimurium strain LT2 (ST) by O. L\u00fcderitz . Commensal pig lactobacilli L. amylovorus strain P1 (LA) and L. mucosae strain P5 (LM) were isolated and characterized as we described elsewhere [8 CFU/m; in PBS.All bacterial strains were from a collection of microorganisms at the Laboratory of Gnotobiology . A probiotic lsewhere . Fresh bBacterial colonization of the gnotobiotic piglet intestine and translocation into liver, spleen, lungs, and blood we described elsewhere .n = 6); (ii) ST ; (iii) LA ; (iv) LA+ST ; (v) LM ; (vi) LM+ST ; (vii) EcN , and (viii) EcN+ST . Each piglet group was composed of three independent hysterectomies and bred separately from other groups in a fiberglass gnotobiotic isolators. The bacteria were supplied in 5 mL of a milk diet, and the control GF piglets received 5 mL of milk without any bacteria. At the end of the experiment, the piglets were humanely euthanized by exsanguination via cardiac puncture under isoflurane anesthesia.Gnotobiotic piglets were obtained and bred in gnotobiotic isolators as described elsewhere . Fifty-f260/A280 \u2265 2.0 as measured in 10 mM Tris-HCl buffer pH 7.5) were reverse transcribed by QuantiTect Reverse Transcription kit (Qiagen) according to manufacturer\u2019s instructions. 20 \u03bcL of the synthesized cDNA was 1/10 diluted by PCR quality water , and these PCR templates were stored at \u221225\u00b0C until the following real-time PCR.Total RNA from the terminal ileum, transverse colon, and mesenteric lymph nodes was isolated and transcribed as described elsewhere . Approxi2 \u03bcL of the PCR template was added to 18 \u03bcL of the FastStart Universal Probe Master (Roche Diagnostics) containing 100 nM LNA (lock nucleic acid) probe  and 500 nM each of the forward and reverse primers \u2014CT\u2212\u0394 method [Ten minutes\u2019 initial heating at 95 \u00b0C was followed by 45 cycles at 95 \u00b0C for 15 s and 60 \u00b0C for 60 s were incubated and measured in duplicates on an iQ cycler with iQ5 Optical System Software 1.0 . Cq for genes of the interest was normalized to Cq for \u03b2-actin and cyclophilin A reference genes and the relative mRNA fold change expressions of the genes of interest were calculated by 2T method  by GenExMesenteric lymph nodes were embedded in Tissue-Tek , snap-frozen in isopentane cooled in liquid nitrogen vapor, and stored at \u221270 \u00b0C. 5-\u03bcm acetone-fixed cryosections on SuperFrost/Plus slides  were kept at \u221240 \u00b0C until labeling. The sections were incubated with 10% normal rabbit serum  in a humid chamber for one h at RT. Labeling by anti-HMGB1 rabbit polyclonal antibodies  was performed overnight at 4 \u00b0C. The sections were incubated with secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies), for 2 h at RT. The sections were subsequently embedded in ProLong Gold Antifade Reagent (Life Technologies) and examined under an Olympus BX 40 microscope with a Olympus Camedia C-2000 digital camera . Control sections without primary antibody were treated in the same way. The colocalization of HMGB1 and nuclei was analyzed by ImageJ software .g for 30 min at 8 \u00b0C, and supernatants were filtered through a 0.2 \u03bcm nitrocellulose filter . A citrated blood sample was spun at 1200\u00d7 g for 10 min at 8 \u00b0C. A protease inhibitor cocktail  was added to the lavage supernatants and plasma. Both supernatants and plasma were aliquoted, immediately frozen, and stored at \u221245 \u00b0C. The HMGB1 levels were quantified in duplicates by ELISA kit  according to manufacturer\u2019s instructions. Optical densities were measured at 450 nm and 620 nm with an Infinite M200 Microplate reader and the results were evaluated with Magellan 6.3 software .40 cm of the distal small intestine (labelled here as ileum) was filled with 2 mL of Dulbecco\u2019s Phosphate Buffered Saline (DPBS), gently kneaded, and irrigated. The obtained intestinal lavages were spun at 2500\u00d7 p \u02c2 0.05, ** p \u02c2 0.01, and *** p \u02c2 0.001 by GraphPad 6 software  and statistical significances of differences depicted in figures by asterisks.Differences of the groups to the control GF group in parameters with normal distribution were evaluated with one-way ANOVA with Dunnet\u2019s multiple comparisons post-hoc test. The statistical evaluations were performed at *"}
+{"text": "Furthermore, the molecular weights of XR MWL were increased, possibly because of condensation of the lignin during the xylose production. A study on antioxidant activity showed that XR lignin had better radical scavenging ability than that of 2,6-Di-tert-butyl-4-methyl-phenol (BHT) and CC MWL. The results suggested that the lignin in xylose residue, showing great antioxidant properties, has potential applications in food additives.Xylose residue (XR), after diluted acid treatment of corncob, consists of cellulose and lignin. However, structural changes of XR lignin have not been investigated comprehensively, and this has seriously hindered the efficient utilization of lignin. In this study, corncob milled wood lignin (CC MWL), and xylose residue milled wood lignin (XR MWL) were isolated according to the modified milled wood lignin (MWL) method. The structural features of two lignin fractions were thoroughly investigated via fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), thermogravimetric analysis (TGA) and two dimensional nuclear magnetic resonance (2D NMR) spectroscopy techniques. XR MWL with higher yield and lower bound carbohydrate contents presented more phenolic OH contents than CC MWL due to partial cleavage of  Efficient exploitation of lignocellulose for producing bio-fuels, bio-chemicals, and bio-materials can decrease the consumption of fossil fuels . Xylose 31P nuclear magnetic resonance (31P NMR) spectroscopy and two dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC NMR) spectroscopy, determined structural properties of two lignin samples. Furthermore, the antioxidant activity was investigated, because of the close relation between the typical structure and possible application. Comparative characterizations of lignin form reaction stages in the corncob biorefinery will not only facilitate more effective deconstruction of lignocellulose, but also enable faster development of bio-fuel, bio-chemicals, and bio-materials production from byproduct lignin.In this work, a modified milled wood lignin (MWL) preparation was successively isolated from two feeds (corncob and xylose residue). All kinds of systematic techniques containing fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), thermogravimetric analysis (TGA), quantitative The xylose residue and corncob were obtained from Shandong Longlive Corporation . The samples were firstly washed several times by distilled water, and then dried at 55 \u00b0C overnight. The dried samples were milled to a size under 40 meshes. Then the two samples were extracted with a 2/1 (v/v) toluene/ethanol mixture for 8 h. Chemical components of the extractive-free materials were analyzed by employing the national renewable energy laboratory (NREL) procedure [2 vessels (500 mL) by a planetary ball mill . Each sample was ground for 5 h with 10 min breaks between 10 min working. To prepare the MWL, the milled sample was extracted by 96/4 dioxane/H2O mixture for 48 h, and then centrifuged. The dioxane was distilled off under reduced pressure. The crude lignin was precipitated in 3 volumes of deionized water. The crude lignin was washed by the 0.01 M HCl solution, and lyophilized. The relatively pure lignin fractions were dissolved in 2/1 (v/v) dichloromethane/ethanol. The insoluble precipitate was washed with diethyl ether, and then centrifuged, and finally freeze-dried to obtain purified lignin [The two MWL fractions were separated according to the procedure of MWL isolation ,10. The d lignin .31P NMR experiment, 20 mg MWLs were dissolved in 1.6/1 pyridine/deuterated chloroform with a total volume of 500 \u03bcL. The cyclohexanol was the internal standard. Before NMR analysis, the mixture was reacted with 100 \u03bcL 2-chloro-1,3,2-dioxaphospholane [6 [2.The associate carbohydrates were analyzed according the typical procedure of NREL . FTIR spspholane . For theolane [6 . Thermal2O. The DPPH concentration was 25 mg/mL in the ethanol solution. Then the MWL solution was mixed to 3.9 mL DPPH solution at 25 \u00b0C for 0.5 h. The measure of absorbance was conducted at 517 nm. The DPPH radical-inhibiting activity was calculated: DPPH radical-inhibiting activity (%) = (A0\u2212A1)/A0The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals inhibiting capacity of MWL was estimated according to the typical protocol . Typical0 is the absorbance of sample without lignin, and A1 is the absorbance of lignin sample. The 50% of DPPH radical-inhibitation activity was defined as IC50. The radical scavenging index (RSI) was defined as the inverse of IC50. The measurement was conducted in triplicate.Where AThe carbohydrates and lignin in the corncob and xylose residue were presented in Mw) and number-average (Mn), and the polydispersity index (PI: Mw/Mn) are shown in The values of the weight-average (\u22121 are characterized as the O\u2013H stretch vibration and hydrogen bond in the lignins. Corncob (CC) MWL contained stronger O\u2013H stretching vibrations than XR MWL. The absorption band at 2940 cm\u22121 is originated from the stretch vibration of methyl and methylene in the lignin. C\u2013H stretching of methoxyl groups is present at 2847 cm\u22121. The absorbance band of 1699 cm\u22121 corresponds to unconjugated carbonyl and carboxyl group stretching. The representative bands at 1590, 1506, and 1456 cm\u22121 are attributed to the aryl ring stretching of lignin. We found that the typical structure of lignin did not alter after acid pretreatment processes. The peaks at 1326 cm\u22121 correspond to syringyl and noncondensed guaiacyl ring breathing with a C\u2013O stretch [\u22121 is attributed to guaiacyl ring breathing with a C=O stretch. The peaks at 1160 cm\u22121 in the spectra were observed, which indicates the existence of an ester bond with p-hydroxyphenyl units of grass lignin, indicating the occurrence of an H\u2013typed structure. The 1120 cm\u22121 band corresponds to an aromatic C\u2013H in plane deformation. The intensity of the 1030 cm\u22121 C\u2013O\u2013C band was decreased after acid pretreatment, indicating that a partial ether bond was cleaved during the diluted acid treatment [The change of functional group of two lignin fractions was determined by FTIR according to studies ,11, and  stretch . The banreatment .2D-HSQC NMR were required for qualitative and quantitative understanding of the lignin structure. The side chain (\u03b4C/\u03b4H 50.0\u201395.0/2.00\u20135.50) and aromatic regions (\u03b4C/\u03b4H 100.0\u2013150.0/5.50\u20138.50) of the two MWLs are shown in To study the structural aspects of complex lignin, quantitative p-counmaroylated \u03b2-ether units A (\u03b3-pCA) (p-hydroxycinnamyl (sinapyl/coniferyl) alcohol. This finding is consistent with the eucalypt MWL [p-hydroxyphenyl (H) are clearly distinguished. Except to the signals mentioned above, the correlations of the p-coumaric acid (pCA) and ferulic acid (FA) are found. Furthermore, the aromatic C3\u2212H3 (\u03b4C/\u03b4H 104.7/7.03) and C2\u2032,6\u2032\u2013H2\u2032,6\u2032 (\u03b4C/\u03b4H 103.9/7.30) signals exist in The lignin isolated from corncob contains a considerable number of \u03b2-ether unit (A), with some detectable phenylcoumaran (C) . The sig (\u03b3-pCA) . Furtherlypt MWL . Signals\u2013O\u20134 aryl ether after diluted acid pretreatment [The calculated S/G ratios were 1.48 and 1.12 for CC MWL and XR MWL, respectively. XR MWL had a lower S/G ratio than that of CC MWL. Lignin fractions in the middle lamella contained relatively more G units; whereas lignin in the secondary wall S2 had relatively more S units. We believe that lignin from xylose residue was easily isolated from the middle lamella after acid treatment. In addition, the \u03b2\u2013O\u20134 of CC MWL (37.10/100Ar) was more than in the XR MWL (9.22/100Ar). The results were attributed to partial cleavage of \u03b2reatment .31P NMR is regularly used to quantify different hydroxyl groups  in the lignin structure [tructure . The intAfter diluted acid pretreatment, the content of aliphatic OH groups decreased from 2.73 mmol/g to 2.0 mmol/g, owing to the cleavage of ether bonds. The content of condensed OH greatly increased from 0.08 mmol/g to 0.16 mmol/g, indicating that some monophenols were condensed during the production of xylose process. Total phenolic OH increased from 2.05 mmol/g to 2.30 mmol/g. Furthermore, the carboxylic group increased significantly from 0.11 mmol/g to 0.29 mmol/g, which may be due to occurrence of an oxidation reaction in the diluted acid pretreatment or the ball milling process .The thermal properties are significant for thermochemical conversion of isolated lignin or raw biomass. The trend of the thermal conversion of CC MWL and XR MWL was similar, but there some differences shown in The antioxidant properties of lignins in raw corncob and treated xylose residue are compared to the commercial antioxidant, BHT. The DPPH inhibitory effects of these fractions were shown in In conclusion, the structures of CC MWL and XR MWL were studied in detail. In XR MWL, less bound carbohydrate content and relatively high isolation yields were found. In addition, the molecular weight of XR MWL was higher than that of CC MWL, indicating that condensation was the main side reaction during the xylose manufacture. Furthermore, the residual weight of the XR MWL was higher than CC MWL. Compared with CC MWL, XR MWL contains more phenolic hydroxyls and carboxyls, suggesting that cleavage of \u03b2\u2013O\u20134, and carboxyl formation were main structural changes. The XR MWL showed good antioxidant activity, which was higher than that of BHT. This suggested that the lignin in xylose residue could show potential application as a natural antioxidant."}
+{"text": "The lignin can compete for binding cellulase enzymes with cellulose fibers and decrease the accessibility of enzymes to carbohydrates. The competitive adsorption of cellulase to lignin mainly depended on the chemical structure of lignin. The post-pretreatment can decrease the lignin content and modify the lignin structure of pretreated substrates, which reduced the lignin inhibition on enzymatic saccharification. Therefore, the post-treatment by modifying the lignin structure would attract considerable attention for weakening the cellulase\u2013lignin interactions.P. amarus, 15.4% for AO-P. amarus and 21.4% for AC-P. amarus, respectively, which were 1.4\u20133.5 times of alkali pretreated P. amarus.Three modified lignins, including sulfonated lignin (SL), oxidized lignin (OL), and carboxylated lignin (CL), were prepared from alkali lignin (AL) and their structures and physicochemical properties were characterized using FTIR, NMR, XPS analysis, zeta potential, and contact angle, respectively. Compared to AL, three modified lignin preparations exhibited the decrease in contact angle by 61\u201370% and phenolic hydroxyls content by 17\u201380%, and an obvious increase of negative charges by about 21\u201345%. This was mainly due to the drop of condensation degree and the incorporation of carboxylic and sulfonic acid groups into modified lignins. Langmuir adsorption isotherms showed that the affinity strength between cellulase and modified lignins significantly reduced by 54\u201380%. Therefore, the 72\u00a0h hydrolysis yield of Avicel with SL, OL, and CL was 48.5, 51.3, and 49.4%, respectively, which was increased 8\u201315.3% than that of Avicel with AL, 44.5%. In the enzymatic hydrolysis of bamboo biomass, the glucose yield at 5 d was 38.5% for AS-The post-treatment can weaken the nonproductive adsorption between lignin and cellulase proteins and improve the enzymatic saccharification efficiency. This study will provide a conceptual combination of pretreatment technologies and post-pretreatment by modifying lignin structure for reducing the cellulase\u2013lignin interaction. Biochemical conversion of lignocellulosic biomass to sugar-based compounds has been devoted for the production of biofuels and platform chemicals such as 5-hydroxymethylfurfural and furfural . A crucip-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, through the linkage of various interunit linkages, such as \u03b2-O-4\u2032, \u03b2-5\u2032, \u03b2-\u03b2\u2032, 5-5\u2032, 5-O-4\u2032, and \u03b2-1\u2032 [Lignin is a cross-linked polymer and consists of different phenylpropane, such as and \u03b2-1\u2032 . In addiand \u03b2-1\u2032 . Therefoand \u03b2-1\u2032 , but alsand \u03b2-1\u2032 , 9. Alth2+ and Mg2+, might also occupy the active sites of lignin and form lignin\u2013metal complex, which blocked the interaction between cellulase and lignin [How to reduce the cellulase\u2013lignin interaction for enhancing the enzymatic saccharification efficiency has been widely studied. The anionic surfactant and hydrophobic proteins, as lignin blocker, had been reported to be effective to weaken or eliminate the nonproductive adsorption by blocking the exposed sites of lignin surfaces, thereby decreasing the cellulase activity loss , 11. Ligd lignin , 14. Thed lignin , 16. It d lignin . Lignin d lignin , 19. In d lignin \u201322. ThesThe primary goal of this work is to modify the surface properties and structure of residual lignin through introducing functional groups, which have potential for reducing the cellulase\u2013lignin interaction. Modified lignin preparations, including sulfonated lignin (SL), oxidized lignin (OL) and carboxylic lignin (CL), were prepared from alkali lignin (AL), and their structural properties were thoroughly investigated by FTIR, 13C, 2D HSQC NMR, and XPS analysis. The phenolic hydroxyl group content, the amount of negative charges and contact angle of lignin preparations were measured, respectively. The Langmuir adsorption isotherm was used to characterize enzyme affinity to lignin preparations. Finally, the effect of modified lignins on the enzymatic digestion of cellulose were evaluated and compared; whether the lignin modification as post-treatments can work on lignocellulosic biomass was also investigated.13C and 2D HSQC NMR were used to characterize the chemical structure of lignin preparations. The fingerprint region (800\u20131800\u00a0cm\u22121) of FTIR spectra, corresponding to the stretching vibrations of different groups in lignin preparations, is displayed in Fig.\u00a0\u22121 observed in all samples were the aromatic skeletal vibrations, implying that the core of lignin structure was not altered significantly during the modified process. However, the spectra also showed some changes in the peaks and the absorption intensities. The peak at 1708\u00a0cm\u22121 characteristic of non-conjugated carbonyl groups is much more intense in AL than OL. And even such a band was not present in SL and CL. It could be due to the carboxyl or ester linkage of C\u03b3 in AL side chain was partially cleaved during the modification process. The absorption band of 1649\u00a0cm\u22121, assigned to conjugated carbonyl groups, was identified in SL, OL, and CL. The intensity of 1272\u00a0cm\u22121 corresponding to the C\u2013O stretching of G type lignin had not been significantly changed after modifications. The signal intensity of p-hydroxyphenyl units (H unit) at 834\u00a0cm\u22121 was reduced in modified lignin, compared with that of AL, indicating the dissolving of H-type lignin. As compared to AL, OL, and CL, the successful introduction of the sulfonic groups into SL can be justified by the stronger bands at 1043\u00a0cm\u22121 (S=O stretching vibration) and 536\u00a0cm\u22121 (C\u2013S stretching vibration).The FTIR, 13C NMR spectra  units occurred as two signals at 128.0 and 129.2\u00a0ppm (C-2/C-6); and the lignin sulfonation, oxidation and carboxylation can reduce the level of H-lignin unit. The transformation of above signals indicated that lignin polymer was dissociated and the modification can improve the hydrophilicity and solubility of lignin due to altering the relative proportions of lignin subunit . Moreover, the bands of condensed aromatics (140\u2013123\u00a0ppm) became weak and narrow in SL, OL, and CL compared to those in AL, implying that fewer condensation reactions could occur between free lignin units during the modification. The signal intensities between 50 and 90\u00a0ppm corresponding to lignin interunit linkages had no changes, suggested that the modification could occur on the surface of lignin moieties. The decreasing signal intensities for \u2013OCH3 can be seen at 56.5\u00a0ppm, implying that the modification reactions could cause demethoxylation and demethylation in AL. In addition, the aliphatic COOR at 178\u2013168\u00a0ppm are more abundant in CL, followed by OL and SL. In the carboxylation reaction, the H atom in lignin hydroxyl was substituted by the \u2013OCH2COOR, which could be responsible for the most abundant aliphatic COOR.In the tra Fig.\u00a0 and the tra Fig.\u00a0 of ligniositions , 26. As \u03b4C/\u03b4H 50\u201395/2.5\u20136.0) and aromatic regions (\u03b4C/\u03b4H 95\u2013160/5.5\u20138.50) and correlation signals are assigned in Table\u00a0To further structural investigation, the 2D HSQC NMR was used to analyze the structural units and different interunit linkages of lignin polymers. The HSQC spectra consisted of the side chain (O-4\u2032 aryl ether (A) and methoxyl groups (\u03b4C/\u03b4H 55.4/3.72) are the most prominent signals observed in all spectra. The signals at \u03b4C/\u03b4H 71.6/4.88 (A\u03b1), \u03b4C/\u03b4H 82.8\u201385.7/4.13\u20134.46 (A\u03b2(G) and A\u03b2(S)), and \u03b4C/\u03b4H 59.5\u201359.7/3.40\u20133.63 (A\u03b3) belong to the C\u03b1-H\u03b1, C\u03b2-H\u03b2, and C\u03b3-H\u03b3 correlations of the \u03b2-O-4\u2032 ether substructures, respectively. As shown in Fig.\u00a0A\u03b1 and A\u03b2 were still detected and no changes occurred in all samples. However, it was interesting that by comparison with AL, three modified lignins exhibited the absorption of intense signals at \u03b4C/\u03b4H 59.5\u201363.5.7/3.40\u20133.83 (A\u03b3). This phenomenon suggested that the AL has been modified at the acylated \u03b3-carbon in \u03b2-O-4\u2032 aryl ether linkages of the side chains. And \u03b2-\u03b2\u2032, \u03b2-1\u2032and \u03b2-5\u2032 substructures were not also observed in the spectra of modified lignin, suggesting that the surface modification of lignin could not cause the condensation between lignin units. Aromatic regions of the HSQC spectra can give basic correlation signals of the p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units. Specifically, the C2,6-H2,6 correlations signals of S and C\u03b1-oxidized S (T\u2032) units were shown at \u03b4C/\u03b4H 103.8/6.69 and \u03b4C/\u03b4H 106.4/7.31. In Fig.\u00a02,6-H2,6 in S unit tended to reduce in the modified samples compared to that of the control AL, indicating a decrease of S units in the surface of modified lignins, which was due to the cleavage of \u03b2-O-4 linkage. Similarly, the C5-H5, C2-H2, and C6-H6 correlations in G units, at \u03b4C/\u03b4H 115.1/6.72, \u03b4C/\u03b4H 111.0/7.02, and \u03b4C/\u03b4H 119.0/6.77, respectively, were observed in very low intensities for three modified lignins. It is worth mentioning that p-hydroxyphenyl (H) units exhibited the strong signal for C2,6-H2,6 at \u03b4C/\u03b4H 127.5/7.23 in others lignin, while it was weak in OL due to that oxidation treatment can result in less H units than that of other lignin samples. The above findings to aromatic ring units of lignin are consistent with FTIR analyses. In addition, the C2-H2, C6-H6, and C7-H7 correlations signals of ferulic acid (FA) were clearly observed at \u03b4C/\u03b4H 110.8/7.35, 123.1/7.16, and 144.4/7.42 in the spectra. Alkali lignin showed a higher proportion of FA, but the intensity of FA signals could be weakened after the oxidized and carboxylic reaction, and it disappeared after sulfonated reaction. Simultaneously, the relatively strong signals at the \u03b4C/\u03b4H 129.8/7.52, 143.9/7.51, and 115.0/6.24, corresponding to p-coumarates (pCA) were presented in all lignin samples except for SL; however, in comparison with AL, pCA units of oxidized (OL) and carboxylic lignin (CL) samples showed a slightly decrease. It had suggested that p-coumaric and acid ferulic acid are associated with the lignin monomer through either C\u2013C or ether linkages and the carboxylic groups were not in free but in ester form [In the side-chain regions, the \u03b2-ter form \u201331. Thister form .Fig.\u00a032DThe XPS analysis can provide some knowledge about the elemental composition and functional group abundances on the near-surface regions of materials , 34. As p-coumaric, acid ferulic acid, sulfonate groups, and aliphatic COOR, and a decrease of condensation degree and S-lignin, G-lignin unit in the modified lignins. The results were further indication that the sulfonated, oxidized, and carboxylation modification of lignin could be used to adjust lignin chemical functionality and surface hydrophilicity. Zeta potential values of all lignin samples were measured and are compared in Table\u00a0The total concentration of free phenolic groups in all lignins using the F\u2013C method is listed in Table\u00a0R\u2009=\u2009K\u2009\u00d7\u2009\u0393m) of cellulase on AL that represented the binding strength of lignin to cellulase enzymes was 0.253\u00a0L/g lignin, which was two to threefolds higher than those on SL (R\u2009=\u20090.083\u00a0L/g), OL (R\u2009=\u20090.089\u00a0L/g) and CL (R\u2009=\u20090.104\u00a0L/g). It was noticed that the binding strength of AL (R\u2009=\u20090.253\u00a0L/g) was even higher than that of Avicel (R\u2009=\u20090.133\u00a0L/g), suggesting that AL would get more chance to adsorb cellulase enzyme than cellulose. Consequently, after modification reaction, the maximum adsorption capacity of lignin toward enzymes was significantly reduced. Specifically, AL (\u0393m\u2009=\u200942.78\u00a0mg/g lignin) had stronger adsorption capacity, which can adsorb about 7.9\u201324% more protein than modified lignins. And the maximum adsorption capacity of modified lignins decreased in the order OL\u2009>\u2009CL\u2009>\u2009SL, among which sulfonic acid groups caused greater decrease in the binding strength than the oxidized and carboxylate groups. This was probably because the introduction of sulfonic groups could consume more phenolic hydroxyls and bring more negative charges in lignin moieties to some extent.To examine how the modification in AL interfered the cellulase\u2013lignin interaction, the cellulase adsorption on lignin samples and Langmuir adsorption isotherms of cellulase enzymes were analyzed and are summarized in Fig.\u00a0\u22121), thus the zeta potential values of modified lignin were increased from 3.5\u00a0eV (AL) to \u2212\u200921.5\u00a0eV (OL), \u2212\u200927.3\u00a0eV (CL) and \u2212\u200945.9\u00a0eV (SL), which increased the electrostatic repulsion between enzyme and lignin and prevented the nonproductive binding. And with the consumption of phenolic hydroxyl groups in the sulfonation, oxidation, and carboxylation, the content of phenolic hydroxyls decreased by 49, 17, and 80%, respectively, comparing to AL. This meant the decrease of binding strength between cellulase and modified lignins, because the cellulase adsorption on lignin correlated positively with phenolic hydroxyl content [We believe that the decrease of cellulase\u2013lignin interaction was related to the structure and physicochemical properties of modified lignins. As well known, the hydrophobic, electrostatic, and hydrogen-bonding interactions could mediate the nonproductive adsorption of cellulase onto lignin. Alkali lignin had a contact angle of 98.7\u00b0 and the binding strength of 0.253\u00a0L/g lignin. The modification led to a decrease in contact angle of lignin by 61\u201370%, due to the drop in the degree of condensation and lignin subunits, such as syringyl (S) and guaiacyl (H), and the more exposed carboxylic groups. This can make the lignin more hydrophilic, thereby decreasing the nonproductive binding for cellulase. In addition, the functional groups introduced into AL also had a much greater impact on the surface charge of lignin moieties and the formation of hydrogen bonds. According to XPS analysis  and C13 NMR 178\u2013168\u00a0ppm), a low content of carboxylic groups was introduced to the CL and OL surface, and the abundant of sulfonic acid groups also was incorporated into SL in the FTIR  was extracted with 10% (w/w) sodium hydroxide at 70\u00a0\u00b0C for 3\u00a0h, followed by sulfonation, oxidation and carboxylation post-treatment. As seen in Fig.\u00a0P. amarus almost was not degraded by enzymatic cellulase due to the strong recalcitrance. The 5\u00a0days hydrolysis of alkali pretreated P. amarus (A-P. amarus) only obtained the glucose yield of 11.1%. However, when alkali pretreatment was followed by the sulfonation, oxidation and carboxylation post-pretreatment, glucose yield was increased to 38.5% (AS-P. amarus), 15.4% (AO-P. amarus), and 21.4% (AC-P. amarus), respectively. The corresponding xylose yield was also increased from 26.2 to 53.9, 29 and 30.9%, respectively. And the enhancing effects caused by sulfonation post-treatment were greater than oxidation and carboxylation post-pretreatment. The results suggested that the post-pretreatment by modifying lignin can enhance glucose and xylose release in the enzymatic hydrolysis of lignocellulosic biomass. We believe that the increase of glucose and xylose yield after post-treatment can most likely be attributed to the decrease of lignin inhibition on enzymatic hydrolysis. In addition, it is important that alkali pretreatment is still considered as the appropriate method for developing the combined pretreatment technologies for reducing lignin inhibition on enzymatic hydrolysis. Because alkali pretreatment can decrease the lignin content, the sulfonation, oxidation, and carboxylation post-treatment can reduce the nonproductive adsorption of cellulase on lignin, which could contribute to the decrease of lignin inhibition together.To verify whether lignin modifications as post-treatment technologies can work on lignocellulosic biomass, bamboo  was obtained from Yunnan, China and its glucan, xylan, and lignin contents were 41.7, 15.7 and 37.5%, respectively. Cellulase (UTA-8) from Trichoderma reesei with the filter paper activity of 100\u00a0FPU/mL and the \u03b2-glucosidase activity of 71\u00a0IU/mL, was kindly donated by Youteer Biochemical Co., Ltd. (Hunan) and used in subsequent enzymatic saccharification of cellulose. Celluclast 1.5\u00a0L (Sigma 2730) from Sigma-Aldrich (Shanghai) exhibited the protein content of 40\u00a0mg/mL and was employed in experiments of cellulase adsorption.Alkali Lignin, a mixture of different herbaceous plants, such as corn stover, bamboo and straw, were supplied by Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai), which was obtained by NaOH pretreatment and then purified by an acidulation precipitation method. The chemical composition of AL was as follows (dry weight basis): 90.9% acid insoluble lignin, 3.67% acid soluble lignin, 0.1% glucan, 0.05% xylan, 2.19% ash, and 2.79% others. Microcrystalline cellulose  was purchased from Sigma-Aldrich (Shanghai). The bamboo (2O2 of 2\u00a0mL/g lignin and FeSO4 (16\u00a0mM) were added in the solution and the mixture was reacted at room temperature (22\u2009\u00b1\u20091\u00a0\u00b0C) and 80\u00a0rpm in a water bath for 24\u00a0h. Carboxylated Lignin (CL) was prepared through the reaction between hydroxyl groups and sodium chloroacetate, as described previously [Three modified lignins were prepared from AL. Sulfonated Lignin (SL) was obtained by treating 10.0\u00a0g AL with 30\u00a0mL NaOH solution (0.8\u00a0M) and 1.5\u00a0mL formaldehyde in 70\u00a0\u00b0C for 90\u00a0min. Sodium sulfite of 2\u00a0g was mixed with the above solution to carry out sulfonation reaction at 95\u00a0\u00b0C and 3\u00a0h . Oxidizeeviously . The sol13C and HSQC NMR spectra of lignins were acquired with a Bruker AVIII 400\u00a0MHz spectrometer. The sample (80\u00a0mg) was placed in NMR tubes with 0.5\u00a0mL dimethyl sulfoxide-d6. 2D HSQC spectra were recorded on the same spectrometer using 128 scanning time, a 2.6-s delay time between transients, and a 1.5-s relaxation time. X-ray photoelectron spectroscopy (XPS) analyses was conducted on an Escalab 250Xi spectrometer , with non-monochromatic Al K alph , under a high vacuum of 2\u2009\u00d7\u200910\u22129 mbar and at room temperature. The content of free phenolic group in lignin samples were estimated by the Folin\u2013Ciocalteu (F\u2013C) method using phenol as standard [\u03b8) values were obtained from measurements at three points in each lignin surface. The measurements of Zeta potentials were analyzed using a Malvern Zetasizer Nano ZS instrument. Lignin preparations  were incubated in 0.05\u00a0M citrate buffer (pH 4.8) for 1\u00a0h, followed by determination of zeta potential. The reported result of each sample was the average of three trials.The lignin preparations were analyzed using a 710 FTIR spectrophotometer . The standard , 40, 41.\u0393m, mg/g lignin) and affinity constant were estimated by nonlinear regression of adsorption data according to the Langmuir isotherm equation (\u0393\u2009=\u2009\u0393mKC/(1\u2009+\u2009KC)), where \u0393 is the amount of adsorbed enzyme (mg/g substrate), C the amount of free enzyme in solution (mg/mL) and K the Langmuir constant (mL/mg enzyme). And the distribution coefficient (R\u2009=\u2009\u0393m\u2009\u00d7\u2009K\u00a0L/g), represented binding strength, was also analyzed.The adsorption isotherm of enzyme on lignin preparations was studied with different concentration cellulases from 0.01 to 2\u00a0mg/mL . Lignins2SO4 as eluent at the flow rate of 0.6\u00a0mL\u00a0min\u22121, as described before. The glucose yield was defined as the percentage of the glucose content in hydrolysate based on the theoretical glucose available in the cellulose. And protein concentration in supernatant was determined by Bradford assay, and calculated based on the total protein concentration. The relative enzyme activity at 72\u00a0h of enzymatic hydrolysis was also measured using the filter paper assay [Enzymatic hydrolysis was conducted at 50\u00a0\u00b0C and 150\u00a0rpm in the 0.05\u00a0M sodium acetate buffer (pH 4.8) for 72\u00a0h, with a solid substrate loading of 2% (w/v) Avicel. The dosage of cellulase (UTA-8) was 10\u00a0FPU/g glucan. To investigate the effects of lignin compounds  on enzymatic hydrolysis, the 4\u00a0g/L lignin sample  or the mixture of 2\u00a0g/L AL with 2\u00a0g/L  were added into pure cellulose, respectively, prior to the addition of cellulase. Enzymatic saccharification with addition of AL only was used as the control. Aliquots (0.2\u00a0mL) were withdrawn from the supernatant at 2, 4, 6, 10, 24, 48 and 72\u00a0h of hydrolysis. Quantitative analyses of glucose were measured by an Agilent 1260 series HPLC equipped with Aminex HPX-87H column at 60\u00a0\u00b0C column temperature, using 5\u00a0mM Her assay .P. amarus was extracted at 70\u00a0\u00b0C for 3\u00a0h with 10% NaOH (w/w) based on the dry weight of biomass, in a solid to liquid ratio of 1:10 (w/w). After washing with water, the solid residue was subjected to sulfonation, oxidation and carboxylation post-treatment; the post-treatment condition was the same as that of the AL. The effects of combination of alkali pretreatment and post-treatment were evaluated based on the subsequent glucose and xylose release in the hydrolysate. Enzymatic hydrolysis of pretreated substrates was conducted at 50\u00a0\u00b0C, pH 4.8 and 150\u00a0rpm for 5\u00a0days, with a substrate loading of 5% (w/v) and a cellulase (UTA-8) loading of 20 FPU/g glucan. The glucose and xylose yield were defined as weight percent yield of glucose and xylose with respect to the total glucan and xylan in the bamboo biomass. The compositions of alkali pretreated P. amarus (A-P. amarus) were 51.8% glucan, 17.5% xylan, and 35.1% lignin. The combination with alkali pretreatment and sulfonation, oxidation and carboxylation post-treatment of P. amarus was defined as AS-P. amarus and AO-P. amarus and AC-P. amarus, respectively. After post-treatment, AS-P. amarus contained 50.9% glucan, 13.1% xylan, and 23.4% lignin; and AO-P. amarus 54.4% glucan, 15% xylan, and 27.1% lignin; and AC-P. amarus 60.9% glucan, 14.8% xylan, and 21.6% lignin, respectively."}
+{"text": "Reproductive function in women with end stage renal disease generally improves after kidney transplant. However, pregnancy remains challenging due to the risk of adverse clinical outcomes.We searched PubMed/MEDLINE, Elsevier EMBASE, Scopus, BIOSIS Previews, ISI Science Citation Index Expanded, and the Cochrane Central Register of Controlled Trials from date of inception through August 2017 for studies reporting pregnancy with kidney transplant.Of 1343 unique studies, 87 met inclusion criteria, representing 6712 pregnancies in 4174 kidney transplant recipients. Mean maternal age was 29.6\u2009\u00b1\u20092.4\u2009years. The live-birth rate was 72.9% . The rate of other pregnancy outcomes was as follows: induced abortions , miscarriages , stillbirths , ectopic pregnancies , preeclampsia , gestational diabetes , pregnancy induced hypertension , cesarean section , and preterm delivery was 43.1% . Mean gestational age was 34.9\u2009weeks, and mean birth weight was 2470\u2009g. The 2\u20133-year interval following kidney transplant had higher neonatal mortality, and lower rates of live births as compared to >\u20093\u2009year, and\u2009<\u20092-year interval. The rate of spontaneous abortion was higher in women with mean maternal age\u2009<\u200925\u2009years and\u2009>\u200935\u2009years as compared to women aged 25\u201334\u2009years.Although the outcome of live births is favorable, the risks of maternal and fetal complications are high in kidney transplant recipients and should be considered in patient counseling and clinical decision making.The online version of this article (10.1186/s12882-019-1213-5) contains supplementary material, which is available to authorized users. Women with end stage renal disease have impaired fertility due to disruption of hypothalamic gonadal axis. Pregnancy is therefore rare in women on dialysis with very low incidence of conception ranging from 0.9 to 7% . Since tPregnancy in a kidney transplant recipient continues to remain challenging due to risk of adverse maternal complications of preeclampsia and hypertension, and risk of adverse fetal outcomes of premature birth, low birth weight, and small for gestational age infants . AdditioData on clinical outcomes of pregnancy in kidney transplant recipients is limited from case reports, single-center studies, and voluntary registries. The usefulness of the voluntary registries is further limited due to underreporting and incomplete data capture \u20138. To thWe performed a systematic review and meta-analyses reported according to PRISMA guidelines for studies exploring incidence and outcomes of pregnancy in women with kidney transplant Fig.\u00a0. We searn\u2009>\u200910 pregnancies) that explored the pregnancy, maternal, and fetal outcomes among women \u226518\u2009years, and who received a kidney transplant. Studies of patients with multiple organ transplants, studies that analyzed the teratogenic effects of mycophenolate or sirolimus, and non\u2013English language studies were excluded. Titles and abstracts of all identified citations were screened independently by two reviewers (S.S. and T.G.), who discarded studies that did not meet all inclusion criteria. The same reviewers independently screened the abstracts of all eligible studies. If eligibility was indeterminable from the abstract, the study was included in the full-text screen. All disagreements were adjudicated by the principal investigator (S.S).We considered observational studies , case series, and case reports  using standard data extraction forms. Data elements were then rechecked for accuracy by all the three data extraction team members. When more than one publication of a similar patient population existed with more than 25% overlap, publication with higher number of pregnancy events and the most complete details was included. Disagreements in data extraction and quality assessment were resolved in consultation with an arbitrator (T.G.) and primary investigator (S.S.). For each included study, the following data was extracted: country of location, years of data collection, number of kidney transplant recipients, number of pregnancies, mean maternal age, mean interval between kidney transplant and pregnancy, pregnancy outcomes , maternal outcomes , fetal outcomes ; and graft outcomes . To maintain consistency across extracted data, the number of pregnancies was used as a denominator for the outcomes of live births, miscarriages, induced abortions, stillbirths, neonatal deaths, preeclampsia, pregnancy induced hypertension, and gestational diabetes mellitus. The number of live births was used as the denominator for the outcome of preterm deliveries, and cesarean section. Preterm was defined as babies born alive before 37\u2009weeks gestation.Patients characteristics were reported as frequencies. The pregnancy incidence was reported for women per 1000 live births. For each study, estimates were expressed as prevalence and 95% confidence intervals (CI). Prevalence estimates from individual studies were pooled using a random-effects model. Heterogeneity across included studies was analyzed formally using Cochran Q (heterogeneity 2) and I2 statistics. For binary outcomes, the DerSimonian-Laird method was used, and for continuous outcomes, a weighted average methodology was used to calculate the pooled estimates and 95% CI. Two-sample test of proportions was used to compare the pooled incidence for each analysis to the most recent United States (US) general population incidence.  We deter(Table\u00a01). Overall, there were 6712 pregnancies in 4174 kidney transplant recipients. Mean maternal age was 29.6\u2009\u00b1\u20092.4\u2009years and mean interval between kidney transplant and pregnancy was 3.7\u2009years.Among the 4134 citations that were retrieved, 136 full-text articles were reviewed and 87 were selected to be included in the final study cohort , miscarriages rate was 15.4% 95% CI, 13.8\u201317.2), induced abortions rate was 12.4% 95% CI, 10.4\u201314.7), stillbirths rate was 5.1%  and rate of ectopic pregnancies was 2.4% . In our study cohort of kidney transplant recipients, live birth rates were higher as compared to the US general population (72.9% vs. 62%) and favorable across all geographic regions , cesarean section was 62.6% , gestational diabetes was 5.7% , and pregnancy induced hypertension was 24.1% . , 16 Pree . Th. Th.The overall acute rejection rate during pregnancy among 822 kidney transplant recipients was 9.4% , which was comparable to the US mean of 9.1%.  Rates ofOutcomes were also stratified by interval of <\u20092\u2009years, 2\u20133\u2009years, and\u2009>\u20093\u2009years between pregnancy and kidney transplant  and this trend was consistent throughout the globe . Our stuOur study highlights the significantly higher risk of maternal and fetal complications in women with kidney transplants. About a quarter of women developed preeclampsia, and the rates of preeclampsia were almost six fold higher as compared to the general US population (21.5% vs. 3.8%) . VanneveWe found significant differences in rates of gestational diabetes mellitus between various geographical location, for example rates were as high as 8.9% in Europe and as low as 1% in Oceania. Although, the increased rate of gestational diabetes in kidney transplant patients can be well explained by the use of immunosuppressive medications like steroids and calcineurin inhibitors, the striking differences between rates of gestational diabetes according to geographic location also highlights the importance of predisposition to diabetes due to ethnicity. Unfortunately, it was not possible to evaluate the differences in immunosuppressive medications as usually they are individualized to the needs of the patients and transplant center protocol .Rates of stillbirth and neonatal mortality were significantly higher in our study as compared to the general population. While prior studies have not reported higher rates of neonatal mortality and stillbirths in kidney transplant recipients, the current study finding is highly significant. Possible reasons could be prematurity, preeclampsia or presence of other risk factors like hypertension, proteinuria, and serum creatinine of 1.5\u2009mg/dl or higher \u201328. WhilThe optimal time to conception after renal transplant continues to remain an area of contention. The ideal time of conception in women with renal transplant is between 1 and 2\u2009years after transplantation according to guidelines by American Society of Transplantation, whereas European best practice guidelines recommend delaying pregnancy for a period of 2\u2009years after transplantation , 31. In A significant strength of our study is that it involves a large number of pregnant renal transplant recepients from all around the globe, thus providing us with information about pregnancy outcomes for a heterogenous population. Additionally, we have analyzed region specific outcomes and identified outcomes which may require intensive management pertaining to that region. This will help in making future region specific guidelines for follow up and management of pregnancy in kidney transplant recpients. The following limitations should be considered when interpreting the findings of our study. We examined pregnancy outcomes over several decades in the present study. While it is expected for the outcomes to change due to improvement in obstetric care in kidney transplant recipients over the course of time, subgroup analysis for studies from 2000 to 2017 showed consistent results. There were inconsistencies in the definition of live birth rate amongst different studies that may have affected the results. Reporting bias may have affected the miscarriage rate. We were unable to account for differences in socioeconomics, and healthcare conditions among the different geographic regions. Due to lack of individual patient data, we were not able to assess pregnancy outcomes in relation to immunosuppression regimens.This meta analysis of pregnancy outcomes in 6712 pregnancies in 4174 kidney transplant recipients with data spread over different decades from all over the world shows favorable outcomes with live birth rates exceeding that in the recent national population. Majority of patients preserve their graft. However, pregnancy after renal transplant confers significant risk in terms of maternal and fetal adverse events, including increased rates of preeclampsia, gestational diabetes, cesarean section rates, and pregnancy induced hypertension. The risk of prematurity and low birth rate are also high. Areas which need to be studied in the future include type of immunosuppression and its correlation with specific pregnancy outcomes; and evaluation of risk factors associated with specific maternal and fetal adverse events. The definitions used in evaluating these outcomes also need to be standardized. The results of this study can help the health care providers with appropriate counseling and individualized management of this high risk population.Additional file 1:Reproducible search strategy. (DOCX 149 kb)Additional file 2:Subgroup analysis of various pregnancy outcomes in kidney transplant recipients for studies published from 2000 to 2017. (DOCX 16 kb)"}
+{"text": "Background: For medical students seeking additional specialty experience in Med-Peds, in-person electives have often been a source of mentorship and guidance.\u00a0The COVID-19 pandemic has impacted the ability for the completion of in-person clerkships for medical students across the nation.\u00a0Virtual opportunities to increase exposure to Med-Peds programs and didactics are lacking at this time.\u00a0Objective: To develop a virtual Med-Peds student elective that serves to increase awareness of the Med-Peds specialty, exposure to Med-Peds topics and relevant didactics, and exposure to Med-Peds specific mentorship when on-site clerkships are not available due to the COVID-19 pandemic.\u00a0Methods:\u00a0Fifteen medical students participated in a virtual Med-Peds student elective utilizing Zoom .\u00a0Three separate cohorts of five students each completed two-week elective experiences.\u00a0The virtual elective curriculum was created using asynchronous and synchronous learning modalities.\u00a0Sessions were composed of self-directed learning topics, peer-to-peer interactive case discussions, resident-led didactics, and attending physician-led didactics and mentorship sessions.\u00a0A pre-survey was administered at the beginning of the elective and a post-survey was administered at the end of the elective to assess the effectiveness of the elective, student experiences with Med-Peds mentors, and students\u2019 general perceptions of Med-Peds as a residency application choice.\u00a0Results:\u00a0All students (100%), rated the Med-Peds elective to have exceeded their expectations.\u00a0All students indicated this elective had been extremely (100%) valuable to increase their understanding and interest in Med-Peds . Compared to prior to the elective, most were very likely (87%) or likely (7%) to apply to Med-Peds as their top (preferred) specialty. Similar to pre-survey data, one-third (33%)\u00a0of the students were still likely to apply to an alternate specialty in addition to Med-Peds.\u00a0Hundred percent of students indicated that the mentorship component of the elective exceeded their original expectations.\u00a0While most students indicated that they are much more strongly considering applying to Med-Peds as a top (preferred) specialty, the number of students who continue to consider dual-application to include either categorical Internal Medicine, categorical Pediatrics, or Family Medicine did not differ before and after completion of the virtual elective.\u00a0Conclusions: Implementation of a virtual medical student elective focusing on exposure to Med-Peds can strengthen medical students\u2019 interest in the combined specialty despite a paucity of previous experiences or an affiliated Med-Peds program. This new type of rotation can positively impact a student\u2019s view of a hospital system and a residency program when in-person clinical rotations are not available. In light of the COVID-19 pandemic, medical students\u2019 exposure to direct patient care has been significantly limited.\u00a0As guided by the Association of American Medical Colleges (AAMC), clinical rotations for medical students were suspended amidst the pandemic first surge .\u00a0Thus, mFor medical students interested in applying to combined Internal Medicine-Pediatrics (Med-Peds), rotation changes are thought to have detrimental impacts on student interest in the field. Med-Peds requires an interest in two specialties, Internal Medicine and Pediatrics, and many medical students have not completed both third-year clerkships by the time of the beginning of their official fourth year. Many students still experience difficulty completing third-year clerkships and obtaining important sub-internship (sub-I) rotations early in their fourth-year schedule to close educational gaps, decide on residency specialty choices, and obtain letters of recommendation. Up to 200 students per year rely on specific Med-Peds fourth-year rotations to provide a deeper understanding and experiential knowledge of the Med-Peds specialty .\u00a0FurtherWe developed a series of two-week virtual Med-Peds student electives, not for credit, to help close the gap for third-year students not on any clinical rotation and without much exposure or mentorship to Med-Peds. The objectives of the virtual elective were to enable students to understand the combined specialty of Med-Peds in general, to explore the kinds of career\u00a0paths Med-Peds physicians pursue, and to learn topics (adult and pediatric) germane to the practicing Med-Peds physician. At the time of this publication, this is the only known virtual Med-Peds student elective amongst the 77 Med-Peds residency programs.\u00a0This study will describe the structure and outcomes of this rotation.Participant selectionMedical students were invited electronically based on their application already received to our in-person Med-Peds fourth-year elective at ChristianaCare.\u00a0These students were unable to be offered an in-person rotation due to pandemic restrictions\u00a0and the inability for student schedules\u2019 to coincide with in-person elective opportunities later in the academic year.\u00a0A total of twenty medical students were offered with five students that declined or did not respond to our offer.\u00a0A total of fifteen medical students agreed to participate in the virtual elective.Participant characteristicsAll students were not on any clinical rotation.\u00a0Fourteen students were from medical schools not affiliated with any Med-Peds residency program. Four students had not completed their Internal Medicine clerkship and two students had not completed their Pediatrics core clerkship prior to the virtual elective.\u00a0Five students had prior interactions with Med-Peds residents for specialty advice, three students had experiences with Med-Peds faculty, four students had received mentorship from a Med-Peds program leader, and three students had no Med-Peds trained mentors.\u00a0All fifteen students had varied experiences with mentors trained in a specialty other than Med-Peds but did not have a common \u201cgo to\u201d person type to learn about Med-Peds. Table MaterialsZoom  was utilized as the platform for the virtual Med-Peds student elective.\u00a0A daily Zoom \u201croom\u201d was created for participants and presenters to log into utilizing a meeting ID and passcode.\u00a0Students were expected to use a webcam, computer audio, and the chat feature. A separate google drive was created to serve as a repository for all documents relevant to the elective.Curriculum designThe ChristianaCare Virtual Med-Peds student elective was designed as a two-week interactive experience utilizing the virtual platform Zoom for delivery of didactic content as well as interactive experiences.\u00a0Three separate cohorts, each for two weeks, consisting of five medical students per cohort participated during May and June 2020.\u00a0The curriculum had six different components, emphasizing concepts of adult learning theory. The content was available for both synchronous and asynchronous learning.\u00a0Core components of the curriculum included (1)\u00a0daily self-directed learning times encouraged to increase exposure to a variety of evidence-based guidelines and relevant literature; (2)\u00a0peer to peer (student) interactive case-based sessions as well as podcast shares to promote motivation for learning and team building amongst the cohort; (3)\u00a0Med-Peds resident-driven sessions focused on case discussions, NEJM Knowledge+ interactive patient cases, resident scholarly activity, and discussions on residency planning, wellness, and mentorship; (4)\u00a0Med-Peds attending sessions, with a mixture of ChristianaCare and non-ChristianaCare trained attendings. Topics were based on their own specialized clinical and professional interests and expertise; (5)\u00a0cases, didactics, and readings for content; and (6)\u00a0reflective writing assignment at the end of the elective that focused on their experience, understanding of Med-Peds, and whether or not their goals prior to starting the elective had been met.The schedule was generally from 9 am to 5 pm, and most individual sessions were up to one hour in duration.\u00a0All students had the lunch hour as protected time off and breaks were scattered throughout the day. Topics were chosen based on medicine and pediatric core curriculum highlighted by the American Board of Internal Medicine (ABIM) and American Board of Pediatrics (ABP) blueprints .\u00a0All stuOn average, each cohort received seven sessions of self-directed learning, six sessions of peer group learning, 10 sessions of resident-led learning, and 20 sessions of attending physician-led learning.\u00a0A sample schedule is shown below in Table StudyAn anonymous 11-question pre-survey and 16-questions post-survey was administered prior to and at the completion of the elective, respectively, via SurveyMonkey.\u00a0This study was deemed exempt by the ChristianaCare institutional IRB as it was\u00a0conducted in an educational setting using normal educational practices.\u00a0All students (100%) from all three cohorts completed both pre and post-surveys.Pre-surveyStudents indicated that gaining mentorship within Med-Peds, experiences with different Med-Peds people, program-specific information, and exposure to different job-niches of Med-Peds were extremely important for interest in the virtual elective.OverallAll students (100%), rated the Med-Peds elective to have exceeded their expectations. All would recommend the ChristianaCare Virtual\u00a0Med-Peds\u00a0student elective to another student in the future and all think more positively about the residency program.Elective constructThe students were surveyed about aspects of the elective and are listed in the table below.\u00a0All participants were overall very satisfied or satisfied with the virtual platform. Fourteen of 15 students submitted their most memorable takeaway and are categorized below in Table Fourteen of 15 students submitted their least favorite aspect of the elective, of which four responded that they had no least favorite aspect.\u00a0The other 10 students\u2019 comments are categorized below in Table Specialty and student impactAll students indicated this elective had been extremely (100%) valuable to increase their understanding and interest in Med-Peds . Students rated the impact of the elective on their decision to apply to Med-Peds and to other residency program types, as depicted in Table Compared to prior to the elective, most were very likely (87%) or likely (7%) to apply to Med-Peds as their top (preferred) specialty. However, compared to prior to the elective, two-thirds of students are very likely (33%) or likely (33%%) to apply to Med-Peds as their only specialty which is similar to pre-survey.Alternate specialties  considered by students are in Table All students (100%)\u00a0rated the Med-Peds elective and mentorship to have exceeded their expectations. Direct feedback on the degree of mentorship they received, including the following comments:\u201cI did not expect to get so much useful advice about the application process in this elective.\u00a0It also helped solidify the real focus that exists in Med-Peds for mentoring trainees at all levels.\u201d\u201cTo this point, I have not had a strong relationship with a mentor, but this rotation showed me how important and impactful it can be to have a good mentor.\u201d\u201cOverwhelming support from the APD and PD who provided listening ears and advice that was specific to my concerns and needs.\u201d\u201cI wasn\u2019t sure what to expect beforehand, but it was an excellent experience that showed me the program feel.\u00a0There were so many mentors that would give open and honest answers, open to all questions.\u201d\u00a0In the midst of the COVID-19\u00a0pandemic where the cessation of clinical experiences was required, the ChristianaCare Virtual Med-Peds student elective rotation provided a direct insight into the combined specialty through a virtual platform.\u00a0The overall feedback from the rotation was highly positive, and students found the experience to be extremely valuable.\u00a0The virtual platform was viewed favorably, and overall students felt that they had a strong sense of connection with the peers in their respective cohorts, as well as the program faculty and residents they encountered, with all students indicating that they would strongly recommend this elective rotation to future applicants.While most students indicated that they are much more strongly considering applying to Med-Peds as a top (preferred) specialty, the number of students who continue to consider dual-application to include either categorical Internal Medicine, categorical Pediatrics, or Family Medicine did not differ before and after completion of the virtual elective.\u00a0The barriers students indicated were external to what a virtual elective was intended to overcome, and included personal reasons such as geographic limitations, concerns about competitiveness to include being an osteopathic candidate, and advice to dual-apply.\u00a0Although the virtual elective did not decrease the number of individuals considering dual-application, it is plausible that without an experience as such, fewer students would consider applying to Med-Peds as a top specialty and/or consider applying to a lower ratio of Med-Peds programs to categorical programs. Further follow-up after the residency match will be valuable in identifying the number of students from this elective who applied and matched into med-peds and what impacted their final rank lists.\u00a0A highly valuable part of the elective which exceeded expectations for participants was career mentorship in Med-Peds.\u00a0While all students had exposure to Med-Peds mentors to varying degrees, the majority of the students were receiving most of their career advice from non-Med-Peds trained physicians.\u00a0The elective provided an opportunity to receive individualized mentorship in addition to general career advising from various Med-Peds physicians at all levels of training and practice.\u00a0All students indicated that this was among the most important takeaway from the elective, and felt this highlighted the general culture and camaraderie that exists within Med-Peds trained providers.\u00a0Further work to be explored includes connecting the local Med-Peds mentors with medical school deans and career counselors in order to increase general awareness of Med-Peds and the diverse careers possible within this unique specialty.Overall, 100% of students strongly agreed or agreed that the elective rotation strengthened their decision to apply to Med-Peds.\u00a0At the same time, 93% (14/15) of students disagreed or strongly disagreed that the elective strengthened their decision to apply to another residency program type.Limitations of this virtual Med-Peds student elective includes a small sample size of fifteen students in addition to a single program experience.\u00a0There was no comparison to a control group or to an in-person med-peds elective. All presenters were currently affiliated with the same institution which can limit students\u2019 generalizability to the specialty as a whole. We also did not assess the relative competitiveness of students in the elective to confirm or refute the common concern about a students\u2019 competitiveness.\u00a0Implementation\u00a0of a virtual medical student elective rotation focusing on exposure to Med-Peds can strengthen medical students\u2019 interest in the combined specialty despite a paucity of previous experiences or an affiliated Med-Peds program. This new type of rotation can positively impact a student\u2019s view of a hospital system and a residency program when in-person clinical rotations are not available. More specialty-specific mentorship directed to students and to those that generally advise medical students about the competitiveness of the field is needed. Expansion of this elective to other times of the year or even when in-person rotations resume should be considered. A national Med-Peds virtual elective can be developed using the framework provided from this small study specifically targeting students without a nearby Med-Peds program."}
+{"text": "Proteinuria is a hallmark of kidney disease. Therefore, measurement of urine protein content plays a central role in any diagnostic work-up for kidney disease. In many cases, proteinuria analysis is restricted to the measurement of total protein content knowing that very high levels of proteinuria (nephrotic proteinuria) are characteristic of glomerular disease. Still, proteinuria can also be a manifestation of impaired tubular protein reabsorption or even be physiological. This review will discuss the physiology of renal protein handling and give guidance on a more sophisticated analysis of proteinuria differentiating albumin, low-molecular weight proteins and immunoglobulins. These non-invasive tests are available in most routine clinical laboratories and may guide the clinician in the diagnostic process before ordering far more expensive (molecular genetic testing) and/or invasive (kidney biopsy) diagnostics. Pediatric Nephrology. The paper by Beara-Lasic et al. [Besides serum creatinine, blood pressure, and urinalysis, the measurement of urinary protein excretion plays a central role in the recognition and classification of renal disease. Even small amounts of proteinuria, i.e., microalbuminuria, are associated with dismal outcomes and are therefore included in the staging of chronic kidney disease according to the KIDGO guidelines . This isc et al.  also pubc et al. \u201311.2/h or protein-creatinine ratio of < 180 mg/g (20 mg/mmol)). Still, there are three situations when proteinuria may be physiological: (i) orthostatic proteinuria [Under normal circumstances, urine is almost free of protein  feteinuria , 14. In Water and small solutes up to the size of inulin (5 kDa) can pass the glomerular filter freely. For larger molecules, permeability is inversely related to molecular size. Therefore, LMW proteins with a molecular mass between 10 and 20 kDa such as \u03b11-microglobulin, \u03b2-2 microglobulin, cystatin C, retinol-binding protein (RBP), and many other macromolecules including hormones and cytokines also pass the glomerular filter in considerable amounts  rather than being degraded in lysosomes.By contrast, the intact glomerular membrane is almost impermeable to albumin due its larger size and negative charge causing reflection of this anionic molecule \u201323. In rBoth the classical view and Dickson\u2019s findings imply that substantial amounts of albumin will be detected in the urine of individuals with defective tubular protein reabsorption but normal glomeruli, and does not necessarily imply a glomerular origin of albuminuria. This is illustrated by an increased urine albumin-creatinine ratio of 38 mg/mmol in patients with Dent disease, who have impaired proximal tubular protein absorption , 26, repPathological proteinuria may result from two principal mechanisms (or a combination of the two): (i) excessive permeability of the glomerular barrier for protein or (ii) impaired reabsorption of protein in the proximal tubule. While there is an association between nephrotic range proteinuria and glomerular disease, there is considerable overlap with non-glomerular disease which can also cause large proteinuria and albuminuria .anionic proteins, i.e., albumin and transferrin, while most other proteins have much less affinity for protons. Therefore, the limits of detection vary considerably between different proteins: 150 mg/l for albumin, 200 mg/l for transferrin, 500 mg/l for IgG, 600 mg/l for \u00df2-microglobulin, and > 1000 mg/l for immunoglobulin light chains [concentration and (anti-) diuresis therefore strongly influences sensitivity and specificity of this test.The first screening for proteinuria is by urine dipstick. This colorimetric method is based on a change in pH in the presence of t chains . Urine dt chains . It shouIn most clinical laboratories, total protein is measured using the colorimetric biuret  or a turIn order to achieve high sensitivity , urine albumin concentrations are measured by immunoturbidimetry or nephelometry using anti-albumin antibodies . This meAs stated above, urine protein concentration is strongly influenced by (anti-)diuresis. Therefore, proteinuria is quantified either in timed urine samples or by normalizing for urine creatinine concentration as a surrogate marker of (anti-)diuresis . The forStudies comparing both methods for albuminuria and total proteinuria suggest that analysis of spot urine is sufficiently accurate in clinical practice , 51, 52.The commonly used unit to express the protein-creatinine ratio is gram/gram, and this can be transformed to SI units (g/mmol) by dividing by 9.Alb) and IgG (FEIgG) [Alb (hazard ratio 35.2 using a cutoff 0.0325%) and FEIgG (hazard ratio 37.1 using a cutoff 0.043%) were strong predictors of end-stage kidney disease with a median follow-up of 7 years.In heavy glomerular proteinuria, the selectivity index (SI) describes if urine protein is largely composed of albumin\u00a0and transferrin (\u201cselective\u201d) or if significant amounts of very large proteins, such as IgG, are present too. The SI is calculated as the relation of IgG in blood and urine related to transferrin (uIgG \u00d7 sTf / sIgG \u00d7 uTf) . An SI \u2264 (FEIgG) . In theiSeveral authors have reported LMW proteinuria in patients with documented glomerular disease , 58\u201362. Van den Brand et al. studied LMW-protein markers \u00df2-microglobulin and \u03b11-microglobulin as predictors of disease progression in idiopathic membranous glomerulopathy , 63. Ther = 0.54, p = 0.19)Several papers have addressed LMW proteinuria as a potential predictor of steroid resistance in childhood nephrotic syndrome , 60, 61.In recent years, a number of urine markers have been identified for the prediction of imminent acute renal failure. These include neutrophil gelatinase-associated lipocalin (NGAL)  and kidnCarter et al. found high inter- and intra-individual variability of serum and urine markers of AKI in patients with chronic kidney disease. However, the changes during AKI were high, indicating that these markers are still clinically useful if baseline values are available . For uriA recent meta-analysis of four studies in children showed an AUROC of 0.85 (95% CI 0.81\u20130.88) for urinary cystatin C , while tPediatric Nephrology argues against FSGS as primary diagnosis in their patient and points towards the diagnosis of Dent disease [As outlined above, the presence and amount of various proteins in the urine varies considerably across the spectrum of renal disease, in particular in distinguishing patients with an isolated tubulopathy from patients with chronic kidney disease involving the glomeruli and chronic kidney disease of non-glomerular origin . Here, a limited strategy measuring albumin, \u03b11-microglobulin, and creatinine is able to separate these entities with high sensitivity and specificity . Beara-L disease .Beara-Lasic et al. did not present cutoffs to distinguish chronic kidney disease of tubulointerstitial origin from glomerular disease. These two entities can be separated using urinary albumin and total protein concentrations (AUROC 0.82), whereas the albumin-total protein ratio (AUROC 0.61) and the \u03b11-microglobulin-creatinine ratio (AUROC 0.53) performed poorly. In this setting, \u03b11-microglobulin should be related to total protein (AUROC 0.82) or albumin concentration (AUROC 0.82).Smith et al. used the albumin-total protein ratio to distinguish tubular from glomerular proteinuria defined by urine protein electrophoresis and immunofixation in some 1000 urine samples . In theiThese findings can also be used in the diagnostics of macroscopic hematuria. Serum albumin constitutes about 55% of total protein in healthy persons . In postA detailed analysis of proteinuria can provide important diagnostic and prognostic information. These tests are cheap, non-invasive, and rapidly available in most clinical laboratories and an important adjunct to renal biopsy and modern molecular genetic techniques. From a historical perspective, taking a close look at urine was the starting point of laboratory medicine more than 2000 years ago ."}
+{"text": "Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium with high immunostimulatory and pathogenic potential involved in the onset and progression of periodontitis, a chronic disease characterized by aberrant immune responses followed by tooth-supporting bone resorption, which eventually leads to tooth loss. While several studies have provided evidence related to the virulence factors of A. actinomycetemcomitans involved in the host cell death and immune evasion, such as its most studied primate-specific virulence factor, leukotoxin, the role of specific lipopolysaccharide (LPS) domains remain poorly understood. Here, we analyzed the role of the immunodominant domain of the LPS of A. actinomycetemcomitans termed O-polysaccharide (O-PS), which differentiates the distinct bacterial serotypes based on its antigenicity. To determine the role of the O-PS in the immunogenicity and virulence of A. actinomycetemcomitans during periodontitis, we analyzed the in vivo and in vitro effect of an O-PS-defective transposon mutant serotype b strain, characterized by the deletion of the rmlC gene encoding the \u03b1-L-rhamnose sugar biosynthetic enzyme. Induction of experimental periodontitis using the O-PS-defective rmlC mutant strain resulted in lower tooth-supporting bone resorption, infiltration of Th1, Th17, and Th22 lymphocytes, and expression of Ahr, Il1b, Il17, Il23, Tlr4, and RANKL (Tnfsf11) in the periodontal lesions as compared with the wild-type A. actinomycetemcomitans strain. In addition, the O-PS-defective rmlC mutant strain led to impaired activation of antigen-presenting cells, with less expression of the co-stimulatory molecules CD40 and CD80 in B lymphocytes and dendritic cells, and downregulated expression of Tnfa and Il1b in splenocytes. In conclusion, these data demonstrate that the O-PS from the serotype b of A. actinomycetemcomitans plays a key role in the capacity of the bacterium to prime oral innate and adaptive immune responses, by triggering the Th1 and Th17-driven tooth-supporting bone resorption during periodontitis. Aggregatibacter actinomycetemcomitans is a small, non-motile, Gram-negative coccobacillus, resident in the oral cavity of humans and non-human primates, that preferentially colonizes the tissues that surround teeth , its specific receptor (RANK), and its soluble decoy osteoprotegerin (OPG) , also known as tumor necrosis factor ligand superfamily member 11 (in (OPG) , 5. Indein (OPG) , 7. Initone loss \u20138.A. actinomycetemcomitans (A. actinomycetemcomitans serotypes are recognized based on the antigenicity of the O-polysaccharide (O-PS) immunodominant component of their lipopolysaccharide (LPS), being the serotypes a, b, and c the most frequently detected in humans  and \u03b1-L-rhamnose (L-Rha), linked by a non-reducing \u03b2-D-N-acetyl-galactosamine  residue, and the O-PS from serotypes a and c consists of 6-deoxy- \u03b1-D-talose  and 6-deoxy-\u03b1-L-talose , respectively  is required for the L-Rha synthesis and its assembling to the O-PS structure, and the genetic deletion of the rmlC gene encoding this enzyme completely abolish the production of the O-PS moiety, leading to a generation of an O-PS-defective A. actinomycetemcomitans strain . In order to analyze the role of O-PS of A. actinomycetemcomitans, two mutant strains were used: the rmlC mutant strain, characterized by the absence of detectable O-PS, and the waaL mutant strain, characterized by impaired production of O-PS compared with the VT1169 wild-type strain, which was used for comparison , supplemented with or without specific antibiotics, at 37\u02daC and under capnophilic conditions. The rmlC and waaL mutant strains were grown in the presence of 50 \u00b5g/ml spectinomycin, and the +rmlC/rmlC and +waaL/waaL complemented strains were grown in the presence of 1 \u00b5g/ml chloramphenicol. To obtain a reliable number of live bacteria with their whole antigenic potentiality for the periodontitis induction and cell stimulation, growth curves were made and bacteria were obtained at the exponential growth phase as previously described  and the Stockholm Regional Ethics Committee.For periodontitis induction, eight-week-old wild-type BALB/c female mice were obtained at the estrus stage of the estrous cycle, to avoid the potential influence of sex hormones on bone metabolism. For 9 CFU/ml of each A. actinomycetemcomitans strain were microinjected using a 26s-gauge syringe . A total of three microinjections were performed every 48\u00a0h into the palatal interproximal gingiva between the first and second molar of the right and left side of the maxilla  +rmlC/rmlC complemented strain-infected group, (d) waaL mutant strain-infected group, (e) +waaL/waaL complemented strain-infected group, (f) sham-infected mice, which received PBS without bacteria, and (g) untreated animals. The mice were euthanized after 30 days by a single overdose of ketamine/xylazine anesthesia, and samples of maxillae, palatal periodontal tissues, and cervical lymph nodes were collected for further analysis. No changes were detected in the body-weight of mice throughout the study  equipment , as previously described , 32. 3D-Ahr, Ifng, Il6, Il10, Il17, Il22, Il23, Il1b, Tlr2, Tlr4, and RANKL (Tnfsf11), 10 ng of cDNA were amplified using the appropriate primers . For the draining cervical lymph nodes, the cell suspensions were obtained through mechanic disruption of the three bilateral cervical lymph nodes that drain the periodontal tissues , using a 70-\u03bcm cell-strainer in the presence of cold PBS.In order to obtain cells to perform the flow cytometric analysis, the palatal periodontal tissues and cervical lymph nodes that drain the periodontal tissues were processed, as previously described . For the+ (Th1), ROR\u03b3t+ (Th17), and AhR+ (Th22) lymphocytes within the periodontal lesions and cervical lymph nodes were analyzed by identifying the expression of their specific transcription factors using flow cytometry, as previously described  analysis of 11-parameter flow cytometry data obtained from untreated mice was performed using the FlowJo software . Samples were randomly downsampled to 10,000 events per sample, and analysis was run on equivalent numbers of events per sample. Following downsampling, 11-parameter samples were concatenated and visualized with tSNE (Barnes-Hut implementation) in FlowJo. The following parameters were tuned in preliminary experiments and then used as default values: iterations, 550; perplexity, 40; eta, 200; theta, 0.5. All parameters, except for lineage marker CD45 and DAPI live/dead marker, were included in the analysis.6 cells/well) in 24-well plates were challenged with the rmlC or waaL mutant strains, the +rmlC/rmlC or +waaL/waaL complemented strains, or the VT1169 wild-type strain of A. actinomycetemcomitans, at a multiplicity of infection (MOI) 3 for 20\u00a0h for flow cytometric assays, or 8\u00a0h for qRT-PCR assays. Untreated cells or cells treated with 1 \u03bcg/ml of commercial Escherichia coli-derived LPS  were used as controls. The selected MOI was based on dose-response assays (Splenocytes were harvested from C57BL/6 background mice and maintained in IMDM medium (Gibco), supplemented with heat-inactivated 10% fetal calf serum , 100 U/ml penicillin, 100 \u03bcg/ml streptomycin, and 2% beta-mercaptoethanol . Splenocytes , and the first-strand cDNA was synthesized using the iScript RT Supermix , according to the manufacturer\u2019s instructions. In order to quantify the mRNA expression levels for In order to analyze the surface expression of co-stimulatory molecules CD40 and CD80 in B lymphocytes, dendritic cells, and macrophages obtained from total splenocytes, the following antibodies were used: CD19 (6D5), MHC-II (M5/114.15.1), CD11c (N418), CD11b (M1/70), Ly6C (HK1.4), CD80 (16-10A1), and CD40 (HM40-3) . Live/Dead Fixable viability dyes  were used to exclude dead cells. All the experiments were acquired using a FACS LSR Fortessa X20 flow cytometer  and analyzed with FlowJo software .Statistical analysis was performed using the GraphPad Prism v.4.01 software . Heatmaps from qRT-PCR fold-change data were done in Excel v.16.39 software . The normality of data distribution was established using the Kolmogorov-Smirnov test. When a parametric analysis was carried out, the statistical differences were determined using the ANOVA and the Tukey or Holm-Sidak post-hoc tests. When a nonparametric analysis was carried out, the statistical differences were determined using the Kruskal-Wallis and Dunn tests. A difference was considered significant when p < 0.05.A. actinomycetemcomitans mutant strains used in this study were characterized by changes in the O-PS expression, we carried out the electrophoretic assays of their purified LPS. The silver-stained profile of the LPS obtained from both the rmlC and waaL mutant strains revealed the absence of the O-PS domain , Th17 , Th22 (Il22), and T regulatory (Il10) lymphocyte responses in the palatal periodontal tissues by qRT-PCR , the key factor involved the osteoclast differentiation and activation. In periodontal lesions induced with the A. actinomycetemcomitans rmlC mutant strain, the expression levels for RANKL (Tnfsf11) were significantly lower as compared with those induced with the VT1169 wild-type strain  (+CD3+CD4+ROR\u03b3t+) (+CD3+CD4+AhR+) , T lymphocytes (CD45+CD3+CD90+), innate lymphoid cells (CD45+CD90+CD3-), macrophages (CD45+CD64+CD11b+), DCs (CD45+CD11c+MHC-IIhi), neutrophils (CD45+Ly6G+Ly6Cmid), and monocytes (CD45+Ly6C+Ly6G-) , including dendritic cells (DCs), macrophages, and B lymphocytes , we examA. actinomycetemcomitans serotype b strain alters the CD40 and CD80 surface expression on bacteria-challenged splenocytes-derived APCs. Splenocytes were used based on the similarities in APC proportions between the spleen and periodontal tissues in mice  in experimental periodontal lesions, as compared with the wild-type strain. These variations were focalized in the periodontal tissues, without significant disturbance in the T helper subsets within the cervical lymph nodes. During the characterization of the gingival immune compartment, we also determined that the most frequent periodontal APCs were B lymphocytes and DCs. In these cells, O-PS-defective A. actinomycetemcomitans strain challenge triggered a lower expression of the co-stimulatory molecules CD40 and CD80, as well as lower expression of Tnfa and Il1b, as compared with the wild-type strain, suggesting that the lack of this bacterial moiety could affect their capacity to prime T lymphocytes towards the pro-osteolytic phenotypes.b VT1169 wild-type strain was capable of inducing bone loss. Conversely, when the periodontal lesions were induced with the rmlC mutant strain lacking the O-PS moiety, the mice developed less severe alveolar bone loss. Based on this observation, we propose that the O-PS domain exerts a critical role in the virulence of the serotype b cluster. This may be due to changes in the periodontal immune cell network mediating the activation of tooth-supporting bone resorption in response to structural variations in bacterial LPS, as it was previously reported with the purified LPS of A. actinomycetemcomitans serotype b , and the CD40/CD40L interactions, in turn, \u2018license\u2019 DCs for T-cell priming by overexpressing CD80 and CD86  signaling to determine the pathways involved in the recognition of O-PS by periodontal APCs. In this context, the immune response against P. gingivalis LPS is unusual due to its unique and heterogenous lipid A structure, being considered an agonist for TLR-2 as well as an agonist or antagonist for TLR-4   , and in m (T1SS) . Hence, poptosis ; thus, icomitans  and we uA. actinomycetemcomitans strains belonging to the serotype b were used in all experiments due to their higher immunogenic potential as compared with the other serotypes. Indeed, A. actinomycetemcomitans strains belonging to the serotype b have demonstrated a higher capacity to trigger Ifng, Tnfa, Il1b, Il6, Il12, and Il23 expression in human monocyte-derived DCs as compared with A. actinomycetemcomitans strains belonging to the other serotypes  and the Stockholm Regional Ethics Committee.GM conceived the study, designed and performed most experiments, analyzed the data, and was involved in drafting the article. FC, EC, JA, and CT-A performed experiments. AH and EV designed experiments, analyzed the data, and critically evaluated and supplemented the article. RV conceived the study, designed experiments, analyzed the data, and prepared the article for submission. All authors contributed to the article and approved the submitted version.This study was financially supported by grant FONDECYT 1181780 from the Chilean Governmental, Agencia Nacional de Investigaci\u00f3n y Desarrollo (ANID). GM is a recipient of a Ph.D. Scholarship CONICYT 21170297 from ANID.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": "Ogresuchus furatus gen. et sp. nov., based on a semi-articulate specimen located in a titanosaurian sauropod nesting ground. The new taxon challenges current biogeographical models about the early dispersal and radiation of sebecid crocodylomorphs, and suggests an origin of the group much earlier than previously expected. Moreover, the new taxon suggests a potential convergent evolution between linages geographically isolated. Taphonomic evidences suggest that Ogresuchus died almost in the same place where fossilized, in a dinosaur nesting area. Biometric and morphologic observations lead to speculate that Ogresuchus could easily predate on sauropod hatchlings.Sebecosuchia was a group of highly specialized cursorial crocodyliforms that diversified during the Cretaceous and persist until the end of the Miocene. Their unique combination of cranial and post-cranial features indicates that they were active terrestrial predators that occupied the apex of the Late Cretaceous terrestrial ecosystems, even competing with theropod dinosaurs. Here, we report the discovery of the earliest sebecid worldwide, and the first from Eurasia,  During that time, the crocodomorph assemblage was characterized by the predominance of Laurasian eusuchian lineages, despite other minority groups like Gondwanan notosuchians were also present3. The enigmatic genus Doratodon is considered the only representative of the clade Notosuchia in the Late Cretaceous of Europe so far5, although the taxon is poorly known due to its scattered and fragmentary record3. On the other hand, the presence of isolated ziphodont teeth in several localities from Iberian Peninsula6 seems to indicate the presence of a more diverse notosuchian fauna than previously suspected.Late Cretaceous continental faunas from Europe are famed for showing a unique mosaic of taxa with both Gondwanan and Laurasian affinities10 . The combination of these characters leads speculates that sebecosuchians were active terrestrial predators that could even compete with medium-size theropod dinosaurs as top predators during the Mesozoic9. Currently, two families can be recognised within Sebecosuchia: Baurusuchidae and Sebecidae12. While baurusuchids become especially diverse, abundant, and nearly exclusive from the Late Cretaceous of South America12, albeit putative remains of baurusuchids are mentioned in Asia13 and Africa14; sebecids diversified during the Palaeocene until the Miocene, also in South America12.Among notosuchians, the clade Sebecosuchia represents a group of highly specialized crocodyliforms characterized for their unique anatomical treats, showing triangular-shaped and laterally compressed skull, a significant reduction of the number of mandibular teeth, zhiphodont dentition, hypertrophied caniniform teeth, and cursorial limb morphologyHere, we describe the first sebecid sebecosuchian of Eurasia on the based of a semi-articulate specimen, including cranial and post-cranial elements, which was discovered in a dinosaur nesting ground at the Southern Pyrenees (south-western Europe). This finding represents the oldest record of Sebecidae worldwide and sheds light on the early evolution of the group, and provides clues on the feeding behaviour of these terrestrial predators.15).Crocodylomorpha Walker, 1970 (sensu Clark15).Crocodyliformes Hay, 1930 , in reference to the inferred feeding behaviour that included infant individuals, like the mythological creature from European folk tales; and \u2013suchus, from the Greek Souchos meaning crocodile. Species name after furatus, from the Latin furari meaning to be stolen, in reference to the unfortunate event that took place during the fieldworks (see Supplementary Information Genus name after MCD-7149 (Museu de la Conca Dell\u00e0), a semi-articulate skeleton preserving the anterior part of the rostrum and several axial and appendicular elements .El Mirador site, . High cemented grey marl level from the \u201clower grey unit\u201d of the Tremp Formation; early Maastrichtian  carinae; presence of apicobasal ridges on the enamel of the incisiviform and caniniform teeth; presence of apicobasal ridges on the enamel of posterior teeth; large and aligned neurovascular foramina on lateral surface of the maxilla; foramen in perinarial depression of the premaxilla; very large incisive foramen; absence of a large nutrient foramen on palatal surface of the premaxilla-maxilla contact; palatal surface of the maxilla without rugose surface; nasal-maxilary contacts remain parallel to each other (do not converge anteriorly or posteriorly); postzygapophyses located dorsally to the transverse processes in dorsal vertebrae.The right premaxilla, the left maxilla, some teeth, the palatine and the palpebral are the best-preserved cranial remains, although a fragmentary right prefrontal could be also present. Complete descriptions for these bones were possible after a micro CT-scanning Fig.\u00a0.Figure 2The right premaxilla is exposed on the rock in lateral view. It is a medio-laterally thin bone, and dorso-ventrally higher than rostro-caudally wide. The caudal margin is sinuous, making a dorso-caudal projection of the premaxilla for the contact with the nasals, and articulating with the lost right maxilla in a sigmoid suture  cannot be assessed. The prezygapophyses seem to fuse with the transversal processes from the 7th dorsal vertebra on, as described in other related taxa as Campinasuchus20. At middle length the shaft makes torsion, being antero-posteriorly flattened at proximal half and medio-laterally fattened at distal half.In addition, some dorsal ribs are also identified. These elements are flattened. The proximal end shows the capitulum and the tuberculum for articulating with the associated vertebrae. Capitulum and tuberculum are separated by a well-marked U-shaped depression. The shaft is ventrally curved and shows a median longitudinal depression, unlike Only the right ulna, and the metacarpals I, II, III and IV are well identified. The proximal epiphysis of the right radius is also probably preserved Fig. .20. It is exposed in lateral side. In lateral view, the bone is arquated, displaying a concave anterior margin and a concave posterior one. The bone becomes shaper on its distal portion. The distal condyles are lost. The proximal end is cranio-caudally expanded. The proximal articular surface is concave, with the caudal olecranon process more developed than the cranio-lateral one. The lateral face bears a shallow longitudinal groove for the insertion of M. extensor carpi radialis brevis pars ulnaris, delimited caudally by a ridge for the insertion of M. flexor ulnaris21.The ulna is an elongated and latero-medially flattened bone, as in other sebecids, baurusuchids, and notosuchiansThe proximal epiphysis of the radius is not well preserved. In proximal view it is a sub-squared bone with wide condyles, but it is strongly damaged hampering the assessment of detailed morphology.M. interossei is observed21. These bones are similar to those referred to other baurusuchids19.Metacarpals were identified based on it general outline. The metecarpals I and II are almost complete, but the II, IV and the probable V are distally broken. Metacarpals decrease in width and robustness from the I to the V, being the first the largest. Each of them has an expanded proximal portion for articulating with the next metacarpal. The width of this expansion also decreases in size accordingly. In MI and III, the distal condyles bear a circular central depression for the attachment of A partial left femur, both tibiae and an indeterminate metatarsal were identified.The femur is broken in two parts. The shaft seems almost cylindrical, but both proximal and distal ends are lost hindering any accurate morphological description.B. albertoi16, Sebecus22, Stratiotosuchus23 and Mariliasuchus8, differing from the straight condition in Crocodylia. Left tibia is preserved only in its distal portion, but the right tibia is almost complete. The tibial shaft is bowed posteriorly and medially, as in Sebecus22. This tibia is expanded at both ends, although the proximal articular surface is not well preserved. The distal end of the tibia is divided into lateral and medial portions. The medial portion is mesio-distally projected, forming an oblique distal margin. The lateral portion is well developed. This condition is present in other notosuchians as Stratiotosuchus, Notosuchus, Araripesuchus, Yacarerani, Pissarrachampsa and Sebecus25.Both tibiae are exposed in posterior view. They are long and medially curved bones, as in M. interossei, as in the metacarpals. Based on the moderate expansion of the proximal end, this bone is tentatively considered as the metatarsal I.A left metatarsal is well preserved. It is a long and slender bone, compressed cranio-caudally. The shaft is almost straight with expanded proximal and distal ends. The proximal end shows well-marked lateral and medial condyles separated by a shallow concavity. The distal condyles are rounded, making a squared epiphysis. A lateral circular concavity is observed in both sides for the attachment of the 26. The maxilla of Ogresuchus specially resembles that of Gondwanasuchus27, but although both taxa show apicobasal sulci on their teeth, Gondwanasuchus bears serrated dentition. The ornamentation pattern of Ogresuchus is also similar to Caipirasuchus teeth, although Caipirasuchus shows a highly specialized dentition composed by three serrated morphotypes in a continuous tooth row, not separated by a premaxillary-maxillary notch12. The anterior palpebral of Ogresuchus is unusually elongated. This bone differs from the morphology observed in most basal notosuchians, baurusuchids and sebecids; although comparisons are hindered because of the palpebral is not preserved in several species. On the other hand, the shape of the anterior palpebral is reminiscent to Gondwanasuchus27 and Araripesuchus tsangtsangana24. Finally, The absence of antorbital fenestra in Ogresuchus differs from many basal notosuchians and some baurusuchids32. This condition is similar to those observed in some basal notosuchians, a few baurusichids, and sebecids33.Based on the reconstructed 3D model, the general outline of the skull Fig.\u00a0, especiaOgresuchus furatus was assessed based on the character-taxa matrices and methodology of Pol et al.12. The tree search strategy resulted in 1,150 most parsimonious trees of 1613 steps, before the second round of TBR; and exceeded the maximum number of trees retained in memory  after that. Therefore, the analysis was rerun excluding 3 unstable taxa pruned in the analysis reported by Pol et al.12\u2014Pabwheshi, Pehuenchesuchus and Coringasuchus. The analysis resulted in 3,456 most parsimonious trees of 1604 steps, after the final round of TBR . As a result, Ogresuchus was recovered as a member of Sebecidae, within Sebecosuchia  until the Early Cretaceousian Fig.\u00a0, it is lOgresushus\u2009+\u2009Zulmasuzhus\u2009+\u2009Sebecus spp. might, indeed, to be considered common synapomorphic characters shared by both South American and African linages, remaining invariable for several millions of years. Most of these characters are related to the contact area between maxilla and premaxilla (see Supplementary Information), and it might suggest that the basic configuration of the anterior part of the snout that defines the group was already present in the early members of the family.Although being a preliminary speculation, two potential scenarios might explain such results. On one hand, the phylogenetic features that define the clade grouping Ogresuchus with Palaeogene South American sebecids could result as a convergent evolution phenomenon. In this case, the alleged allopatric speciation of the South American and African linages could drive some taxa to acquire similar morphological features. In fact, convergent evolution among crocodyliforms is well known, and several evidences suggest that ecomorphological convergent adaptation (i.e. occupation of similar dietary niche) can contribute more signal than phylogenetic relatedness39. If so, this could imply that the real phylogenetic position of Ogresuchus could be obscured by its ecomorphological convergence with other taxa.On the other hand, the phylogenetic treats that group 42. Based on recent allometric equations20, the body length of the Ogresuchus was estimated in 1.09\u00a0m and its body mass in only 9.04\u00a0kg of weight  and the Palaegene (i.e. Sebecus) doubled the size of Ogresuchus (2\u20133\u00a0m in length) but notably exceeded its weight (c. 70\u00a0kg)22.Body size is one of the most important biological features because its intimate relationship to the ecology of any organism, the skeleton of Ogresuchus is incomplete and not fully articulated although the bones are preserved in good condition and show certain anatomic-like arrangement . Although it has been stated that the jaw of sebecosuchians could produce orthal movements with a wide opening46, the small cranial size of Ogresuchus probably could not load an efficient bite force upon the thick megaloolithid eggshell. In addition, none of the 30 eggshells surrounding the skeleton nor any of the 1,000 fragments discovered in the El Mirador site16 shown evidences of the characteristics hole marks and cracking associated to predation51. These observations are consistent with previous studies pointing out that sebecosuchians had no specific adaptations for opening eggs47.As a first approach, it can be stated that the small body size of Ogresuchus could produce some kind of predation pressure on a 3-kg-weigh, 40-cm-long neonate titanosaur53. One of the most highlighting features of Ogresuchus is its labio-lingual compressed dentition without serration and marked apicobasal ridges on the tooth crown, a dental condition shared with some theropod dinosaurs like Buitretraptor and Compsognathus54. Although non-serrated grooved teeth are commonly referred to purported piscivorous tetrapods55, the blade-like morphology of caniniform teeth of Ogresuchus could likely pierce the soft flesh of a baby titanosaur that, in addition, hatched with no defensive elements (i.e. osteoderms56). Thus, a feeding behaviour that incorporates the ingestion of hatchling dinosaurs should be in the scope of further palaeobiological analyses of Ogresuchus.On the contrary, it is likely that Ogresuchus furatus in Late Cretaceous deposits from southern Europe \u201cpushes back\u201d the origination time of the once top terrestrial predators sebecid sebecosuchians. According to our interpretations, sebecids would have originated the previous to the full break of Gondwana (middle Albian). If so, a remainder group of sebecids could evolve in Africa for several millions of years until some taxa could reached the Late Cretaceous European archipelago. As occurs with many other taxonomic Cretaceous groups from Europe, a cornerstone in this hypothesis relies in future discoveries of terrestrial fossils from the Early Cretaceous of the African continent. Anyway, whatever the factor drifting the evolution of the group, phylogenetic and morphological evidences might suggest certain degree of ecomorphological convergence between early members of the family and more derived sebecid taxa. Furthermore, taphonomic and biometric evidences provide evidences that lead speculating that Ogresuchus furatus could stalk neonate titanosaure sauropods, incorporating them as a part of diet.In conclusion, the discovery of 12. The new taxon was coded for this study and included in the dataset, resulting in a final matrix of 412 characters and 110 OTUs. According to methods described by Pol et al.9, the character 5 was excluded from the analysis and the characters 1, 3, 6, 10, 23, 37, 43, 44, 45, 49, 65, 67, 69, 71, 73, 77, 79, 86, 90, 91, 96, 97, 105, 116, 126, 140, 142, 143, 149, 167, 182, 187, 193, 197, 226, 228, 279, 339, 356, 357, 364, 368 were set as additive because represent nested sets of homologies and/or entail present and absence information. The character matrix was analysed using a maximum parsimony approach  in TNT 1.558. An heuristic tree search of 10,000 replicates of Wagner trees with random addition sequences was performed followed by TBR branch-swapping, collapsing zero-length branches. According to Pol et al.12, all most parsimonious trees recovered in this search were subjected to a final round of TBR branch-swapping.The data matrix and the phylogenetic methodology were preformed following Pol et al.O. furatus were performed based on the holotype MCD-7149  cranial and appendicular reconstructions of 49 Figs.\u00a0, Video 1Supplementary information 1Supplementary video 1Supplementary video 2Supplementary information 2"}
+{"text": "Asthma is a chronic respiratory disease which is not curable, yet some patients experience spontaneous remission. We hypothesized that epigenetic mechanisms may be involved in asthma remission.Clinical remission (ClinR) was defined as the absence of asthma symptoms and medication for at least 12\u00a0months, and complete remission (ComR) was defined as ClinR with normal lung function and absence of airway hyperresponsiveness. We analyzed differential DNA methylation of ClinR and ComR comparing to persistent asthma (PersA) in whole blood samples (n\u2009=\u200972) and nasal brushing samples (n\u2009=\u200997) in a longitudinal cohort of well characterized asthma patients. Significant findings of whole blood DNA methylation were tested for replication in two independent cohorts, Lifelines and Epidemiological study on the Genetics and Environment of Asthma (EGEA).PEX11 . The whole blood DNA methylation levels of this CpG were also different between ClinR and healthy subjects. One ComR-associated CpG  that annotated to TCF7L2 (Transcription Factor 7 Like 2) was replicated and associated with expression of TCF7L2 gene. One out of seven ClinR-associated CpG sites and 8 out of 129 ComR-associated CpG sites identified from whole blood samples showed nominal significance (P\u2009<\u20090.05) and the same direction of effect in nasal brushes.We identified differentially methylated CpG sites associated with ClinR (7 CpG sites) and ComR (129 CpG sites) in whole blood. One CpG  associated with ClinR was replicated and annotated to We identified DNA methylation markers possibly associated with clinical and complete asthma remission in nasal brushes and whole blood, and two CpG sites identified from whole blood can be replicated in independent cohorts and may play a role in peroxisome proliferation and Wnt signaling pathway. Asthma is a chronic airway disorder that affects more than 300 million people around the world. Asthma cannot be completely cured with the available treatment up to now. However, some asthma patients may grow out of this disease, which is called asthma remission.Asthma remission is more common in patients with childhood onset asthma, and the proportions of remission differ in different age groups at follow-up  [ILRL1 and IL13 in lung tissue [Asthma remission is associated with both genetic and environmental factors. Environmental factors including breast feeding and having pets in childhood were reported to be positively associated with asthma remission . A receng tissue .DICER1, STX3, and LIPIN1 in blood cells, and CDHR3, FBXL7 and NTRK1 in nasal epithelial cells [. [ACKR2 and DGKQ by gene expression.Epigenetic mechanisms such as DNA methylation may help to build a link between genetic factors and the environment. DNA methylation can be regulated by SNPs, and can reflect environmental exposures, ageing, cell type constitution and activation . DNA metal cells , 9. Vermcells [.  identifiAlthough this latter paper provided a proof of concept of the relation between asthma remission and DNA methylation, bronchial biopsies are not easy to obtain for further studies. DNA methylation in whole blood and nasal epithelium is a good proxy for bronchial epithelium and can therefore also help to understand the mechanism of asthma remission. Here, we hypothesize that epigenetic mechanisms may be involved in asthma remission, reflected in different DNA methylation patterns in whole blood and nasal epithelium. To test this hypothesis, we performed an EWAS of whole blood and nasal DNA in subjects with PersA, ClinR and ComR. We investigated a longitudinal cohort in which asthma was initially carefully defined and the remission status was assessed during follow-up (median 39\u00a0years). We subsequently replicated the whole blood DNA results in two independent cohorts, and also verified the top results from whole blood DNA in cells obtained by nasal brushing.A full description of methods is provided in the online supplement.Subjects included in this study were from long-term follow-up studies in the University Medical Center Groningen (UMCG). This study consists of two sources of subjects, (1) subjects from the third visit 2013\u20132014) of a longitudinal study described previously by Carpaij et al  of a lon and wereWe replicated findings in whole blood in two independent cohorts: the Lifelines population-based cohort in The Netherlands (where we could replicate our results on ClinR using Illumina 450\u00a0K array) and the Epidemiological study on the Genetics and Environment of Asthma (EGEA) cohort in France, a case control and family study on asthma, where we could replicate our results on ClinR and ComR with methylC-capture sequencing . No coho20 ) methacholine \u2a7d 39.3\u00a0mg\u00a0mL\u22121)), and (4) FEV1% predicted pre-bronchodilator\u2009>\u200980%. PersA was defined as the presence of asthma symptom and/or the use of asthma medication. Detailed phenotype definitions of the replication cohorts are described in the Additional file The presence of PersA, ClinR and ComR was determined at the most recent visit. ClinR was defined according to the following criteria: 1) No use of any asthma medication, and (2) no symptoms (asthma attacks and/ or wheezing) in the past year. ComR was defined as ClinR combined with (3) no AHR  with 436,824 CpG sites remained for following steps.We used robust linear regression to determine the differential methylation between persistent asthma and asthma remission: (1) PersA versus ClinR, and (2) PersA versus ComR, in whole blood and nasal brushing samples, with adjustment for covariates that are known to affect DNA methylation . For whole blood samples, we performed adjustment on the percentage of monocytes, B cells, NK cells, CD4\u2009+\u2009T cells, CD8\u2009+\u2009T cells, and granulocytes, which were predicted by the Houseman  algorith\u20137, which is 0.05 / 436,824) in whole blood DNA were selected for replication in two independent cohorts, Lifelines and EGEA. Lifelines did not include an assessment of AHR, so only included a ClinR phenotype available for replication. The weighted Z-score method was used to meta-analyze the results of discovery and replication cohorts, considering EGEA used methylC-capture sequencing method, which was different from discovery and Lifelines (450\u00a0K array), to investigate DNA methylation. CpG sites that passed the epigenome-wide significance threshold of P\u2009<\u20091.14\u2009\u00d7\u200910\u20137 (Bonferroni correction) in the meta-analysis of all studies were considered to be replicated. Finally, top sites identified in whole blood were verified in nasal brushes.Genome-wide significant CpG sites that passed Bonferroni correction (P\u2009<\u20091.14\u2009\u00d7\u200910cis expression quantitative trait DNA methylation (cis-eQTM) analysis. Blood eQTM was assessed in the BIOS consortium dataset [Significant CpG sites were annotated by GREAT v3.0.0 . We corr dataset . Matched dataset . The gen dataset . To chec1 and FVC level compared to PersA, but no difference regarding FEV1%predicted and FVC %predicted values was identified among the groups , resulting in four CpG-gene pairs that showed negative correlation  were significantly associated with ClinR  in nasal brushes, with one pair ) also being nominal significant and in the same direction in the PIAMA dataset . The Q-Q plot  in nasal brushes, and four pairs also showed nominal significant and in the same direction in PIAMA dataset . The ClinR-associated CpG (cg13378519) was located on Chr 1 and close to gene PEX11B . The CpG site was not available in the EGEA study and was only replicated in Lifelines. This CpG was lower methylated in ClinR subjects compared to PersA  and TCF7L2 (Transcription Factor 7 Like 2). This CpG was lower methylated in ComR subjects compared to PersA in whole blood, but showed no obvious difference in nasal brushes .We replicated the top findings in whole blood in two independent cohorts. In replication cohort EGEA, four ClinR-associated CpG sites and 93 ComR-associated CpG sites were available, with one CpG site for ClinR and one for ComR showing nominal significance (P\u2009<\u20090.05) and the same direction as the discovery cohort. In the replication cohort Lifelines, which only had the ClinR phenotype, seven ClinR-associated CpG sites were all available with one CpG with P\u2009<\u20090.05 and the same direction. After meta-analysis, one CpG associated with ClinR and another one CpG associated with ComR were replicated , known to affect DNA methylation at multiple sites in the genome  gene and TLX1 (T Cell Leukemia Homeobox 1) gene, in their top list of remission vs persistent asthma reached nominal significance (P\u2009<\u20090.05) and showed the same direction of effect in our results of ComR in whole blood  and LOC100507389, were also present in our results of DMRs associated with ClinR in nasal brushes . One CpIn this study, we identified one CpG site associated with ClinR and a different CpG site associated with ComR from whole blood, that were replicated in independent cohorts. We also revealed that 25 CpG sites were associated with asthma remission phenotypes in nasal brushes. In the nasal dataset, two differentially methylated regions were previously observed in another DNA methylation study of asthma remission using bronchial biopsies. This is the first epigenome-wide association study of asthma remission in both whole blood and nasal epithelial cells. This study is important since understanding asthma remission may provide new leads for future asthma treatments, and epigenetic studies may help to reveal these cellular mechanisms.PEX11B gene. PEX11B encodes a protein of the PEX11 family [PEX11B gene in human cells induces peroxisome proliferation [TCF7L2 gene and also correlated with the expression level of this gene. The TCF7L2 gene product is a transcription factor that plays a role in the Wnt signaling pathway. SNPs in TCF7L2 were previously reported to be strongly associated with type-2 diabetes and this gene plays a role in the pancreatic insulin secretory response to incretins [The two replicated CpG sites were annotated to genes which were not known to be involved in asthma (remission) before. ClinR-associated CpG cg13378519 was located in the promoter region of 1 family . Overexpferation , 24. Ourncretins , 26. Prencretins  and it hncretins . However. [Although remission subjects do not have asthma symptoms any more, they still may be different from healthy people. Airway abnormalities such as basement membrane thickening still exist in clinical and complete remission subjects . Vermeul.  reportedDNA methylation is cell-type specific. Both of the studied cells or tissues are highly heterogeneous and this may confound the association between DNA methylation and disease. Thus, we applied the widely used Houseman\u2019s cell type correction method in blood samples, and for nasal brushes, we used SVA which showed good performance in cell type correction when reference data is lacking . DNA metPRKCH (Protein Kinase C Eta) plays a key role in epithelial tight junction regulation which is important in maintaining the integrity and function of the airway epithelial barrier [PDE1B (Phosphodiesterase 1B) encoded a protein belonging to phosphodiesterases (PDEs) family, and various PDE inhibitors showed anti-inflammatory, anti-remodelling and bronchodilator effect and are potential treatment of asthma [DNA methylation might be related to the regulation of gene expression, and eQTM analysis may help to understand the function of CpG sites. Among the eQTM genes that correlated with ClinR-associated CpG sites in whole blood, the protein encoded by  barrier ; PDE1B  and DMRcate (FDR) methods. Table S3 Correlation analysis of DNA methylation and gene expression levels  for asthma remission in blood. Table S4. Pathway analysis of eQTM genes correlated with ClinR and ComR in blood. Table S5. Genomewide significant  CpGs associated with ClinR in nasal brushes. Table S6. Differentially methylated regions associated with ClinR in nasal brushes identified by both comb-p  and DMRcate (FDR) methods. Table S7. Look up of ClinR-assocaited CpGs (indentified from blood) in the results of nasal brushes. Table S8. Correlation analysis of DNA methylation and gene expression levels  for asthma remission in nasal brushes. Table S9. 129 genome-wide significant  ComR-associated CpGs in whole blood in discovery cohort. Table S10. Differentially methylated regions associated with ComR in whole blood identified by both comb-p  and DMRcate (FDR) methods. Table S11. Genome-wide significant  CpGs associated with ComR in nasal brushes. Table S12. Differentially methylated regions associated with ComR in nasal brushes identified by both comb-p  and DMRcate (FDR) methods. Table S13. Look up of ComR-associated CpG sites (indentified from blood) in the results of nasal brushes. Table S14. Summary statistics of replicated CpGs (identified from blood) before and after adjusting for BMI and rhinitis. Table S15. Summary statistics of significant CpGs  before and after adjusting for BMI and rhinitis. Table S16. Look up of CpGs associated with ClinR identified by Vermeulen et al. in this study.Additional file 3: Figure S1. Lung function of different groups at baseline and last visit. In this figure, t-test was used in comparing means of any two groups . Figure S2. Estimated cell proportions among different groups. In this figure, t-test was used in comparing means of any two groups (ns: P>0.05). Figure S3. Quantile\u2013quantile plot for epigenome-wide meta-analysis of the association between asthma remission and blood DNA methylation (n = 72). (a) ClinR, (b) ComR. Figure S4. Quantile\u2013quantile plot for epigenome-wide meta-analysis of the association between asthma remission and nasal DNA methylation (n = 97). (a) ClinR, (b) ComR. Figure S5. Boxplot illustrating DNA methylation levels of cg24788483 in persistent asthma, clinical remission and complete remission subjects in discovery cohort. (a) DNA methylation levels in blood, (b) DNA methylation in nasal brushes.  Figure S6. Density distributions of DNA methylation levels of two replicated CpGs in 72 blood samples from discovery cohorts. Figure S7. Regional association plot of two replicated CpGs. For each plot from top to bottom the tracks included are: 1) Log10 from the discovery 4 years model with CpGs indicated by dots. 2) Annotation tracks for the plotted genomic region taken from UCSC Genome Browser. 3) Pairwise correlation matrix across the displayed CpGs. Figure S8. DNA methylation levels of two replicated CpGs in remission subjects and asthma patients stratified by ICS usage. In this figure, t-test was used in comparing means of any two groups ."}
+{"text": "To provide insight into the nuclear function of \u03b2-DG, we characterized the interaction between \u03b2-DG and emerin at the molecular level. Emerin is a major NE protein that regulates multiple nuclear processes and whose deficiency results in Emery\u2013Dreifuss muscular dystrophy (EDMD). Using truncated variants of \u03b2-DG and emerin, via a series of in vitro and in vivo binding experiments and a tailored computational analysis, we determined that the \u03b2-DG\u2013emerin interaction is mediated at least in part by their respective transmembrane domains (TM). Using surface plasmon resonance assays we showed that emerin binds to \u03b2-DG with high affinity (KD in the nanomolar range). Remarkably, the analysis of cells in which DG was knocked out demonstrated that loss of \u03b2-DG resulted in a decreased emerin stability and impairment of emerin-mediated processes. \u03b2-DG and emerin are reciprocally required for their optimal targeting within the NE, as shown by immunofluorescence, western blotting and immunoprecipitation assays using emerin variants with mutations in the TM domain and B-lymphocytes of a patient with EDMD. In summary, we demonstrated that \u03b2-DG plays a role as an emerin interacting partner modulating its stability and function. The nuclear envelope (NE) of animal cells is composed of three distinct compartments: a double nuclear membrane (the inner and outer nuclear membranes), the nuclear pore complex (NPC) and the nuclear lamina. The NE acts as a hub for a variety of nuclear processes, including gene expression, DNA repair and chromatin organization . It contDespite the critical importance of the NE, its protein composition is not yet completely characterized, and it is likely that additional protein components remain to be unveiled. In this regard, we recently described that \u03b2-dystroglycan  is a new NE protein in myoblasts . DG is aWithin the nucleus \u03b2-DG forms a complex with the NE proteins emerin and lamins A/C and B1, to preserve nuclear structure and function ,10 and pE. coli as GST fusion proteins  ; thus, wproteins A, while n vector  and a coST alone B. These led \u03b2-DG  and the led \u03b2-DG D. CollecTo demonstrate the implication of the TM domains for \u03b2-DG\u2013emerin interaction in a membrane environment in cells, vectors expressing GFP fused to the TM of \u03b2-DG (GFP\u2013TM\u03b2-DG) or to the TM of emerin (GFP\u2013TM\u2013Eme) were prepared to carry out localization and GFP-based immunoprecipitation (IP) assays. C2C12 myoblasts were transiently transfected to express GFP alone, GFP\u2013\u03b2-DG, GFP\u2013TM\u03b2-DG, GFP\u2013emerin or GFP\u2013TM\u2013Eme A and theC. elegans (14 nM) -methionine . Likewise, GST and GST-tagged emerin proteins were incubated with [S35]-\u03b2-DG. Thereafter, glutathione beads were recovered by centrifugation at 1000\u00d7 g at 4 \u00b0C and washed three times in 1 mL of ice-cold washing buffer , and then, bound radioactive proteins were mixed (1:1) with 2X Lemmli loading buffer  and further separated by 10% SDS\u2013polyacrylamide gels. Gels were fixed 1 h with fixing buffer , dried, exposed on a phosphor screen and imaged using the Typhoon trio .In vitro binding of \u03b2-DG with emerin was analyzed by GST-based binding assays. Emerin and \u03b2-DG proteins were synthetized in vitro using pSG5\u2013emerin and pSG5\u2013\u03b2-DG plasmids and the TNTreported . AffinitCells cultured on coverslips were fixed for 10 min with 4% paraformaldehyde, permeabilized for 10 min by exposure to 0.2% Triton X-100 in PBS and blocked for 20 min with 0.5% gelatin in PBS at room temperature, prior to incubation overnight at 4 \u00b0C with appropriate primary antibodies and then with the appropriate secondary fluorochrome-conjugated antibodies . Double immunostaining was carried out by incubation overnight at 4 \u00b0C with the second primary antibody and the next day with the corresponding secondary antibody. Cells were incubated for 10 min at room temperature in PBS with 1-mg/mL diamidino-2-phenylindole (DAPI)  to stain nuclei. After washing, coverslips were mounted on microscope slides with VectaShield  and examined on a confocal laser scanning microscope  employing a Plan Neo Fluor 63\u00d7 (NA = 1.4) oil-immersion objective. Manders overlap coefficients were calculated for double labeling immunofluorescences, using red (Alexa 594 nm) and green (FITC 488 nm) channels and the ImageJ plugin JACoB. Quantitative analysis to determine the nuclear to cytoplasmic ratio of fluorescence (Fn/c) was carried out using Image J software.v/v] Tween-20) and 6\u201315% [w/v] low-fat dried milk) and further incubated overnight at 4 \u00b0C in agitation with the appropriate primary antibody. After three washes in TBS-T, the membranes were incubated with the appropriate horseradish peroxidase\u2013conjugate secondary antibody  and developed in X-ray films , using ECL western blotting analysis system , according to the manufacturer\u2019s instructions. Blot images were acquired with the Gel Doc EZ System  and subjected to densitometric analysis using Image Lab 6.0.1 software . To normalize protein expression, the band intensity of the target protein was divided by the band intensity of the loading protein (actin). To calculate emerin half-life, the emerin protein level was assessed by densitometry analysis of autoradiograms, during a time course of cycloheximide treatment. The band pixel intensity of emerin was divided by the band pixel intensity of actin (loading control) for each lane and the normalized emerin level present in WT C2C12 myoblasts at t0 was set at 1.0. Normalized emerin values were fit to exponential decay curves to calculate protein half-life and the linear regression plot was obtained using Graphpad Prism 6 software (www.graphpad.com).Cells were centrifuged, washed with PBS pH 7.4 and resuspended in 150 \u03bcL lysis buffer , homogenates were then clarified by centrifugation at 4 \u00b0C and protein concentration determined by the Bradford method. The suspension was then sonicated at 3.5 microns on Soniprep 150, applying 3 bursts of 15 s each, with 30 s interval between each sonication. Lysate aliquots (40\u201380 \u03bcg of protein) were mixed (1:1) with 2X Laemmli buffer , prior to be electrophoresed on 10% SDS\u2013polyacrylamide gels, using the electrophoresis running buffer . Proteins were then transferred to nitrocellulose membranes  on Transblot apparatus , using the transfer buffer . Membranes were then blocked for 1 h at room temperature in agitation with TBS-T ), 1X complete protease inhibitor tablet, 0.5% (v/v) Triton X-100 and 0.5-mM PMSF and precipitated proteins separated by SDS-PAGE for western blotting analysis. Immunoprecipitation using the GFP-Trap\u00ae bead system  was performed in accordance with the manufacturer\u2019s instructions.Recombinant protein G-agarose beads  were equilibrated with gentle agitation for 4 h in lysis buffer and then C2C12 whole cell extracts (1 mg) incubated with the beads for 2 h at 4 \u00b0C, followed by incubation of the cleared extracts overnight at 4 \u00b0C with 5 \u03bcg of anti-hemagglutinin (HA) immunoprecipitating antibodies. As a negative control, parallel incubations with an irrelevant IgG antibody were performed. After that, equilibrated protein G-agarose beads blocked previously with 4% BSA were added to the protein extracts and incubated overnight at 4 \u00b0C. Immune complexes were collected by centrifugation at 1250\u00d7 www.graphpad.com) using the values from 30 different injections. p-values of <0.05 were considered statistically significant. The data were expressed as means \u00b1 standard errors of the means.Kinetics analyses were performed in a Reichert  SR7500DC dual-channel SPR instrument. For analysis of the kinetics of emerin\u2013\u03b2-DG and emerin\u2013TM of \u03b2-DG interactions, planar polyethylene glycol/carboxyl sensor slides were used. The flow rate for all steps was determined with phosphate-buffered saline with Tween-20 (PBST) at 40 \u00b5L/min. GST\u2013emerin was coupled to the sensor by passing N-ethyl-N\u2032-(3-dimethylaminopropyl) carbodiimide\u2013N-hydroxy-succinamide (EDC\u2013NHS) for 5 min, immobilizing GST\u2013emerin in 20-mM sodium acetate buffer (pH 4.0) at 12.5 \u00b5g/mL just over the left channel for 8 min and blocking with 1-M ethanolamine (ETN) for 10 min. The kinetic analysis with the ligands  were done by injecting five increasing concentrations of ligand in PBST in triplicates for 150 s to see association phase, after that, PBST was load for 150 s to see the dissociation phase. Between each one of triplicate experiments, PBST was perfused for 15 min to achieve a new baseline before the next injection, using several injections from lower to higher concentration of the analyte without regeneration ,39. Datahttp://zhanglab.ccmb.med.umich.edu/I-TASSER/) [http://molprobity.biochem.duke.edu/). Further, the structure models of helix-rich TM domains (residues 225\u2013247 in emerin and 551\u2013773 in \u03b2-DG) were raised from homology modeling with I-Tasser server. The resultant structures were subjected to protein\u2013protein docking using the ClusPro 2.0 server (https://cluspro.bu.edu/signup.php) [In initial analyses, protein structure models of emerin (259 amino acids long) and \u03b2-DG (893 amino acids long) were carried out by open modeling using I-Tasser server (TASSER/)  and its nup.php) , which rIn summary, we showed that binding of \u03b2-DG with emerin takes place at least in part, through the association of their corresponding TM domains. This interaction is biologically relevant because \u03b2-DG confers stability on emerin and positively regulates its function; reciprocally, emerin is required for \u03b2-DG to be targeted to the NE. The potential involvement of \u03b2-DG in the phenotypes observed in patients affected by EDMD deserves further investigation."}
+{"text": "IL-36\u03b3-OV had dramatic therapeutic efficacies in multiple murine tumor models, frequently leading to complete cancer eradication in large fractions of mice. Mechanistically, IL-36-\u03b3-armed OV induced infiltration of lymphocytes and dendritic cells, decreased myeloid-derived suppressor cells and M2-like tumor-associated macrophages, and T cell differentiation into effector cells. Further study showed that IL-36\u03b3-OV increased the number of tumor antigen-specific CD4+ and CD8+ T cells and the therapeutic efficacy depended on both CD8+ and CD4+ T cells. These results demonstrate that these IL36\u03b3-armed OVs exert potent therapeutic efficacy mainly though antitumor immunity and they may hold great potential to advance treatment in human cancer patients.In this study, we aimed to apply the cytokine IL-36The online version contains supplementary material available at(10.1007/s00262-021-02860-4)  Recently, immune checkpoint inhibitors (ICIs) have been approved to treat a variety of cancers, such as melanoma, non-small cell lung cancer, renal cell carcinoma, and MSI-high colorectal carcinoma, resulting in great advances in cancer immunotherapy . However\u03b3 (IL-36\u03b3), formerly IL1F9, is a member of the IL-1 family , anti-mouse CD8 Ab at 250\u00a0\u03bcg/injection , and anti-mouse CD4 Ab at 150\u00a0\u03bcg/injection  for depletion of NK, CD8Long-term surviving mice bearing intra-peritoneal MC38 tumor treated with OVs were used for tumor cell re-challenge . In those cured mice and na\u00efve mice (control), 5.0e5 MC38-luc cancer cells were injected subcutaneously into the right flank, and 5.0e5 Lewis lung cancer cells into the left frank. Tumor appearance was recorded up to day 40 post re-challenge with cancer cells.tumor (mm3)\u2009=\u2009(L\u2009\u00d7\u2009W2)/2.Tumor growth was monitored via digital caliper volume measurement and compared to na\u00efve C57BL/6 mice inoculated with MC38-luc tumor implants at the same time. Tumor volume was calculated as: VBUV395 conjugated anti-mouse CD45 (clone: 30-F11), BUV737 conjugated anti-mouse CD4 (clone: GK1.5), Pacific Blue conjugated anti-mouse CD8a (clone: 53\u20136.7), PE-CF594 conjugated anti-mouse Foxp3 (MF23), PE conjugated anti-mouse Tim-3 (clone: 5D12), and Alexa Fluor 647 conjugated anti-mouse CD206 (clone: MR5D3) were purchased from BD Bioscience. PE-Cy7 conjugated IFN-\u03b3 (clone: XMG1.2) and FITC conjugated CD11b (M1/70) were purchased from eBioscience. Pacific Blue conjugated anti-mouse MHC II (clone: M5/114.15.2), PE conjugated anti-mouse Gr-1 (clone: RB6-8C5), BV510 conjugated anti-mouse CD24 (clone: M1/69), and APC-Cy7 conjugated anti-mouse F4/80 (clone: BM8) were purchased from Biolegend.\u03b3 staining, cells were stimulated for four hours with 50\u00a0ng/ml phorbol 12-myristate 13-acetate  and 1\u00a0\u00b5g/ml ionomycin (Sigma) in the presence of 10\u00a0\u00b5g/ml Brefeldin A. After stimulation, cells were stained for antibodies to surface markers, followed by fixation permeabilization with Fixation and Permeabilization buffer (eBioscience) according to the manufacturer\u2019s instructions. Then cells were stained with antibodies to intracellular markers. All the samples were applied to LSRII or Fortessa FACS (BD Biosciences) and analyzed by using Flowjo software (Tree star).At indicated time points, lavaged cells were collected and analyzed using flow cytometry as previously described . For IFNMC38 colon cancer and panc02 pancreatic cancer were previously transduced with a lentivirus expressing firefly luciferase (MC38-luc and panc02-luc), thus allowing bioluminescence imaging. The growth of transplanted cancers was monitored by in vivo bioluminescent live animal imaging with the Xenogen IVIS 200 Optical In Vivo Imaging System . Live animal bioluminescence imaging was performed for two purposes. One was to ensure that tumor implants were present and that groups had comparable tumor burdens, and the other was to monitor tumor progression, and was thus performed periodically after treatments.Animal health status and survival was monitored closely. Abdominal girth of mice bearing intraperitoneal tumor implants was monitored with caliper measurement and mice were sacrificed when girth exceeded 1.5\u2009\u00d7\u2009original measurements. Mice either succumbed to their disease or were sacrificed when abdominal girth exceeded allowable measurements as described above. Mice with subcutaneous tumors were sacrificed when tumors reached a maximum diameter of 2\u00a0cm, became ulcerated, and/or interfered with murine activity.+ T cells, CD3+CD4+, and CD3+CD8+ T cells per area was calculated using the number of cells positive for the antibody versus the total number of cells. Student\u2019s t-test was used to analyze statistical significance.Resected tumors were fixed for 2\u00a0h in 2% paraformaldehyde and incubated in 30% sucrose overnight. Sections were cut (5\u00a0\u00b5m) and stained with combined primary antibodies CD3 Alexa 488 , CD4 Alexa 594 , and CD8 Alexa 647  and nuclei were labeled with Hoechst dye . Images were acquired digitally from nine fields under each condition. Density of positive cells was evaluated by automated image analysis using Nikon Elements . Percentage of CD3Once subcutaneous tumors reached 5\u00a0mm in dia, mice were treated intravenously with 1.0e8 pfu of OVs or PBS administered via tail vein injection. Tumor tissues were recovered two, four, and six days after viral or mock treatment and then homogenized using Precellys\u00ae 24 Tissue Homogenizer . Single cells were collected for various assays.RNA was isolated from tumor homogenates of subcutaneous MC38-luc tumor implants using the RNeasy kit . The synthesis of cDNA was then performed using from 2\u00a0\u03bcg of RNA using qScript\u2122 cDNA SuperMix  and Dyad\u00ae Peltier Thermal Cycler . Quantitative PCR was then performed using TaqMan analysis with PerfeCTa\u00ae qPCR SuperMix  on the StepOnePlus System . All PCR primers were purchased from Thermo Fisher Scientific .\u2212\u0394CT), where \u0394CT\u2009=\u2009CT (Target gene)\u2009\u2212\u2009CT (HPRT1 or GAPDH).Relative gene expression was compared to a housekeeping gene, either hypoxanthine\u2013guanine phosphoribosyltransferase (HPRT1) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and then expressed as fold increase . Once isolated, 2.0e4 CD8+ T cells were stimulated with 4,000-rad-irradiated MC38 cells or control cancer cells (at 2.0e4) in RPMI 1640 media supplemented with 10% FBS at 37\u00a0\u00b0C, 5% CO2 for 24\u00a0h. Following incubation, the plates were appropriately washed and then incubated with biotinylated \u03b1-mouse IFN-\u03b3 antibody . The plates were developed using Vectastain Elite ABC and AEC Peroxidase substrate (SK-4200) kits according to vendor protocols . Finally, the plates were read and analyzed using an ImmunoSpot\u2122 analyzer and software .Briefly, at day 7 or at an indicated time after i.p. inoculation of 5.0e5 of MC38-luc colon tumor cells, tumor-bearing mice were treated i.p. with 1.0e8 pfu of OVs or PBS. On the specific time as indicated, intraperitoneal lavage was performed, during which 5\u00a0mL of 2% FBS-containing PBS was injected into the peritoneal cavity using an 18-gauge needle, and then the cavity was gently agitated before the volume was aspirated and repeated up to two times. Lavage fluid was collected and strained over a 100\u00a0\u03bcM cell strainer, and red blood cells were lysed using ACK Lysing Buffer and then strained over a 40\u00a0\u03bcM cell strainer. The CD8t test. Animal survival was assessed using Kaplan\u2013Meier survival curves and analyzed using log rank (Mantel-Cox) test. A p value of\u2009<\u20090.05 was considered statistically significant. Standardized symbols are used in the figures, as follows: * p\u2009<\u20090.05; ** p\u2009<\u20090.01; *** p\u2009<\u20090.001; **** p\u2009<\u20090.0001; and ns: not significant.GraphPad Prism version 7  was used to analyze the experimental data. Analysis was performed using one-way ANOVA test and nonparametric Student\u2019s \u03b3, we constructed three IL-36\u03b3-armed VVs, namely vvTK-IL-36\u03b3, vvDD-IL-36\u03b3, and vvTD-IL-36\u03b3, by inserting an active form of IL-36\u03b3 into three VV backbones with different tumor selectivity and oncolytic activities  and vvTK-IL-36\u03b3, the secreted IL-36\u03b3 in the media was determined by Western blot analysis . Finally, we examined their oncolytic potency of two pairs of OVs in MDA-MB-468 human breast cancer cells. In this case, the vvTD pairs displayed slight better activity of oncolysis than vvTK- pair (p\u2009<\u20090.01)  at 1.0e8 pfu per mouse. They were monitored for toxicity and efficacy through appearance, tumor size, and survival. The parental virus vvTK- treatment prolonged survival of tumor-bearing mice . To determine whether memory antitumor T cells were generated in the cured mice, we re-challenged the tumor-free mice with the same tumor cells . These results suggested that the adaptive antitumor immunity against MC38 tumor might have cross-reacted with Lewis lung cancer. As a control, both MC38 and Lewis lung cancer grew in 100% na\u00efve mice. These results indicated that an IL-36\u03b3-armed OV was a strong antitumoral agent with the ability to induce memory antitumor immune responses.We first examined the antitumoral activities of these OVs in an intraperitoneal MC38 tumor model as described previously . On day ice Fig.\u00a0b. Treatmval Fig.\u00a0b compare\u03b3 were also confirmed at the mRNA level using RT-qPCR . mRNA of the viral marker gene A34R was detected at 60\u00a0h but reduced to basal levels by day 6 . These data demonstrated that the viruses were replicated for a few days, and by day 6, they were reduced to low levels. In contrast, IL-36\u03b3 protein was sustained at a high level in the infected tumor tissues six days after OV administration.We also examined the kinetics of viral replication and IL-36\u03b3 expression in tumor tissues . IL-36\u03b3 was significantly increased in tumor homogenates on days 2 and 6 after administration of OVs as measured by ELISA . The elevated levels of IL-36\u03b3-armed OVs. In MC38-luc s.q. tumor model, vvTD inhibited tumor growth, and vvTD-IL-36\u03b3 showed improved antitumor efficacy  in prolonging mouse survival. In B16 tumor model  and vvTD (p\u2009=\u20090.0016), respectively. These data collectively demonstrated that IL-36\u03b3-armed OVs were potent antitumoral agents in multiple syngeneic murine tumor models.We also established subcutaneous MC38-luc and B16 models, and peritoneal Panc02-luc tumor models to test the activity of IL-36\u03b3 at 1.0e8 pfu per mouse. On day 4 post-treatment, tumor tissue sections were analyzed using immunofluorescence microscopy for immune cell markers such as CD3, CD4, and CD8 for the assessment of the level of infiltrating T cells in the tumor tissues . We also found a trend\u2014that the therapeutic effect was dependent on NK cells under these conditions (p\u2009=\u20090.06).Since T cells were increased by IL-3636\u03b3 Fig.\u00a0. The groi.p.) grown MC38-luc tumors. We collected cells lavaged from the peritoneal cavities of tumor-bearing mice on day 6 post-virotherapy and analyzed the immune cells using multi-color flow cytometry  in total lymphocytes , but not monocytic MDSCs (m-MDSCs) was reduced when treated with vvTK-IL-36\u03b3 compared to control OV and non-treatment  were significantly reduced by IL-36\u03b3-OV  at the abdominal site, and IL-36\u03b3-expression further enhanced this effect. Together, these data indicated that IL-36\u03b3 helped shape a more immunogenic TME and enhanced adaptive antitumoral immunity.To gain further insight into the underlying mechanisms, we comprehensively studied immune cells lavaged from the abdominal cavity of intraperitoneally  . The reason is not clear, even though we could speculate that IL-36\u03b3 probably promoted tumor-specific T cell traffic to tumor tissue area.We also examined the numbers of tumor antigen-specific T cells in the MC38 tumor-bearing mice treated with PBS, vvTD, or vvTD-IL-36+ T cells undergo a highly orchestrated activation process [+ (or CD137+) has accurately correlated with naturally occurring tumor-reactive T cells in cancer patients [+ T cells [+CD44+CD8+ T cells in the TME . Next, we examined both tumor-antigen- and viral antigen-specific CD8+ T cells , tumor antigen peptide from p15E (p15E604\u2013611), and viral antigen peptide from B8R (B8R20\u201327). When unloaded or stimulated with OVA, 2\u20134% of T cells were 4-1BB+CD8+ effector T cells from mice treated with PBS, vvTD, or vvTD-IL-36\u03b3. When re-stimulated with p15E peptide, 4-1BB+CD8+ T cells were up to 2% from mice treated with PBS,\u2009~\u20097.5% with vvTD, and up to 10.5% in vvTD-IL-36\u03b3"}
+{"text": "The aim of this study was to assess the reliability and validity of Turkish version of the Xerostomia Inventory XI in patients with primary Sj\u00f6gren\u2019s syndrome (pSS). A cross-sectional survey study design and analysis were used to assess the reliability and validity of the Xerostomia Inventory XI. A total of 69 patients with pSS  were included. The Xerostomia Inventory XI (TR) was applied twice at an interval of 15 days. The test-retest reliability was assessed with the intraclass correlation coefficient (ICC), and the internal consistency of multiitem subscales by calculating Cronbach\u2019s alpha values. The correlations between ESSPRI, basal and stimulated salivary flow (BSF-SSF), Oral Health Impact Profile-14 (OHIP-14) and Oral Health-Related Quality of Life-UK (OHRQoL-UK) Questionnaire were evaluated to determine the construct validity. The ICC value for test/retest reliability of the Xerostomia Inventory XI (TR) was 0.993. The internal consistency was 0.869. There were low to high correlations between Xerostomia Inventory XI (TR) and ESSPRI, BSF, SSF, OHIR-14 total and OHRQoL-UK total. The Turkish version of the Xerostomia Inventory XI was found to be clinically valid and reliable to be used in clinical evaluations and rehabilitation interventions in patients with pSS. Primary Sj\u00f6gren\u2019s yndrome (pSS) is a chronic, multisystemic, autoimmune and idiopathic disease that involves all exocrine glands, mainly the salivary and tear glands. Lymphocytic infiltration of the salivary and tear glands causes complaints of xerophthalmia and xerostomia. In addition to this, there may be complaints such as dry nose, dry cough, or dry skin [1] . These dryness complaints greatly affect daily quality of life [2]. The pSS prevalence in Turkey has been found to be 0.72% according to the American-European classification criteria and to be 1.56% according to the European criteria [3].SSaliva, which plays a critical role in oral health and maintaining comfort, has antibacterial, lubricant, remineralising, digestive, buffering and cleansing properties . Dry mouth (xerostomia), which is one of the main symptoms in patients with pSS, reduces the quality of life. Patients complain about a reduced amount of saliva, which causes problems in speaking, swallowing and chewing of food, thereby significantly reducing quality of life. Patients feel the need to drink water, especially when they eat dry food, wake up, or talk for a long time [6]. The amount of saliva can be increased with the use of chewing gum, sugar-free desserts, lemon or artificial saliva products [7].A decrease in the amount of saliva negatively affects the intraoral flora, and changes also occur in the oral flora of patients with pSS. The effect of the oral mucosa in these patients is to reduce Ig-A secretion and weaken the antibacterial defence system. It is extremely important that the pH of the oral cavity is continuously protected. A reduction in oral cavity pH is common in these patients, but if the pH can be kept stable, the impairment in mineralisation is reduced. A change in pH and loss of the antibacterial effect of saliva cause tooth decay. Therefore, patients should pay attention to oral hygiene [8]. - Patients with pSS are diagnosed according to the American-European Consensus Group\u00a0 or the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria . Although these criteria provide an advantage for diagnosis, xerostomia is not correlated with disease activity [11]. Xerostomia is a subjective feeling. Therefore, instead of a tool to evaluate this symptom with a single question, a measurement tool should be used which can evaluate it from multiple angles and includes the complexity of the symptom [3]. The relationship between the subjective data of dry mouth of pSS patients and saliva flow rate has been reported to be weak [12]. Therefore, it has been suggested that it would be more appropriate to evaluate the symptoms of xerostomia using scales such as isual nalogue cale, Xerostomia Inventory and Sicca Symptom Inventory [13]. Although the correlation is weak, saliva flow rate was used as objective data in this study as in other version studies, but in addition, questionnaries were applied to assess the validity of the Turkish version in the evaluation of oral symptoms. These questionnaires were the EULAR Sj\u00f6gren yndrome atient VASSPeporting ndex (ESSPRI) and quality of life  Oral Health-Related Quality of Life-United Kingdom (QHRQOL-UK). The Xerostomia Inventory XI (XI) is a widely-used questionnaire in disease-specific assessments and research. It examines the symptoms of xerostomia multidimensionally and consists of 11 items. The original English version of the survey was developed in 1999 by the Australian dentist, Murray Thompson et al [14]. The survey items show that both experimental and behavioral features have excellent reliability, validity, responsiveness [11]. XI enables patients to express their complaints clearly, as the questionnaire consists of yes/no binary answers, and multiple responses ranging from 1 (never) to 5 (RI) \u201c\u201dvery often), thereby allowing patients to express the severity of symptoms without limitations.\u201c\u201dThe XI has been translated and culturally adapted in many countries such as Spain [13], Greece [15], South Korea [11], Portugal [14] among others . No validity and reliability studies of the XI have been conducted previously in Turkish. The aim of this study was to assess the reliability and validity of the Turkish version of the XI in patients with pSS.The study sample was formed of patients who presented at the heumatology linic. The study was completed with a total of 69 patients, considering that the number of samples should be at least 5 times the number of items in the questionnaire [16] and the incidence of the disease is low [18].RCInclusion criteria were a diagnosis of pSS according to the revised ACR/EULAR classification criteria [10], medical treatment stable for 6 months before the study, age 2065 years, ability to speak and understand Turkish fluently and voluntary participaton in the study. Exclusion criteria were defined as any treatment between the first test and the retest, having received radiation to the head and neck region, other causes of xerostomia, cancer patients, salivary gland inflammation, a history of another rheumatic disease, any psychiatric or cognitive impairment, acquired immunodeficiency syndrome, pre - existing lymphoma, graft versus disease, or a history of anticholinergic drug use. The data of patients for whom any changes in drug treatment were made during the study were not included and the participation of the patient was terminated.A cross-sectional survey study design was used to assess the reliability and validity of the Turkish version of XI.Demographic data were collected in face-to-face interviews. All participants were evaluated under the same conditions by the same rheumatologist and physiotherapist experienced in the field of rheumatological rehabilitation. The xerostomia symptom of all the patients was evaluated with XI, disease activity with the ESSPRI, oral health with the OHIP-14 and oral health-related quality of life with the QHRQOL-UK scale. In addition, after a 5-min rest in a quiet laboratory environment, basal and stimulated (citric acid 2%) saliva flow rate was measured with the saliva flow rate test. The evaluations were made in a single session lasting 4560 min.ute - utesFor the retest, the patients completed the XI for a second time after an interval of one week.The recommended procedures of XI for Turkish validity and reliability were followed  and were carried out in 5 stages. First, the XI was translated from English into Turkish by 2 independent translators whose native language was Turkish and had a good level of English. A synthesis of the translations was produced. This was then back-translated by 2 other independent translators with English as a foreign language and good knowledge of both languages. No translator was able to access the original version and they were not informed about the concept of the survey. Later, all versions of the questionnaire were combined by 4 physiotherapists who had not been involved in the translation but were experienced in the treatment of individuals with pSS and by translators who made bidirectional translations. Then, the prefinal version was prepared and applied to 10 patients with pSS to evaluate each item. The final Turkish version of XI (XI-TR) was produced when there was no problem about the clarity and comprehensibility of the statements of each item (Table 1). The XI consists of 11 items. The patients were asked to select the best response for each item describing their symptoms during the previous two weeks. The responses are scored between 1 and 51: ever, 2: , as :Nardly ever, 3: ccasionally, 4: airly often, and 5: ery often. The total of the item scores provides the total score ranging between 11 and 55, with a higher score indicating more severe symptoms [7].The OHIP-14 is a scale used to evaluate oral health comprehensively, questioning physical and mental conditions. It consists of 14 items, evaluating 7 areas of functional limitations, physical pain, mental distress, physical disability, social disability, mental disability and disability. Responses are given with a 5-point Likerttype rating, with a total score ranging from 0 to 56. A high score indicates that the patient has more difficulties and a decreased quality of life. Turkish validity and reliability studies have been conducted for the OHIP-14 . This consists of 16 items to evaluate how oral and dental health affect the general health and quality of life of individuals. It is divided into 4 main groups of physical state, symptom, social state and mental state. Items are scored with a 5-point Likert-type rating to give a total score of between 16 and 80. The responses are scored as 1 =very badly, 2= badly affected, 3= no effect, 4= good effects, and 5 = very good effects. A higher total score indicates a better quality of life . RIESSPRI scoring is a symptom severity assessment scale completed by the patient and used in the evaluation of SS. Items referring to patient complaints of fatigue, pain and dryness are scored from one to ten. The arithmetic average of the scores is then calculated to give a final value. ESSPRI score <5 is considered as an acceptable disease condition, and a score of \u22655 as a sign of high activity [24]. For this measurement, the patients with pSS were asked to attend the clinic at 09:00 after overnight fasting. To determine the amount of basal saliva, the individual was rested for 5 min before starting the measurement. Then, the tare of the measuring cup was determined and the individual was asked to collect his or her saliva into the measuring cup for 10 min in a quiet environment. The amount of saliva collected was measured on a sensitive scale and recorded in grams. The method for measuring the amount of stimulated saliva was the same following the application of two drops of citric acid (2%) onto the tongue to stimulate saliva production [25]. All statistical analyses were performed using SPSS\u00a0v. 25.0 software\u00a0.\u00a0\u00a0Continuous variables were expressed as\u00a0mean\u00a0\u00b1 standard deviation (SD), median (minimum-maximum)values\u00a0and\u00a0categorical variables as number (n) and\u00a0percentage (%).\u00a0Construct validity was investigated using the Spearman rank correlation coefficient. Correlations were categorized as low (r:0.100.49), moderate (r:0.500.69) or high (r:0.701.00). The scale reliability was measured using the Cronbach\u2019s alpha (a) coefficient.\u00a0\u00a0n---The testretest method was used to assess the scale\u2019s stability over time. The correlation between the two measurements was calculated using the intra class correlation coefficient (ICC). Internal consistency reliability was evaluated with Cronbach\u2019s alpha coefficients.\u00a0The study group of pSS patients comprised 92.8% females with a mean age of 54.81 \u00b1 8.77 years. The demographic and clinical characteristics of the patients included in the study are given in Table 2. The descriptive statistics of the XI-TR test and retest, ESSPRI, salivary flow rate test, OHIP-14 and OHRQoL-UK questionnaires are summarized in Table 3. Descriptive analyses of the XI-TR test/retest are given in Table 4.In all items, the test-retest examinations of the XI-TR scale were found to have a very high ICC. The obtained ICCs and confidence intervals are given in Table 4. The scale items werre detemined to be reliable.The internal consistency coefficient  of the XI-TR scale was found to be 0.869 indicating that the scale was quite reliable. When the \u201cCronbach\u2019s Alpha if tem eleted\u201d values were examined, it was seen that there was no need to remove any item from the scale and all items were very reliable (Table 5). AID3.3. Constructand external validityThe relationships between XI-TR and ESSPRI, salivary flow rate test, OHIP-14 and OHRQoL-UK scale scores are given in Table 6. Correlations were categorized as low (r 0.10\u20130.49), moderate (r0.50\u20130.69), or high (r 0.70\u20131.00) [26]. There was determined to be a positive moderate to high correlation between XI-TR score and ESSPRI, OHIP-14 total and subscale scores (p< 0.05). A significant low to moderate correlation was determined between XI-TR score and salivary flow rate test, OHRQoL-UK total and subscale scores (p<0.05) (Table 6). These results showed that XI-TR is valid.The XI-TR questionnaire was found to be clinically valid and reliable for use in clinical evaluations, and rehabilitation interventions for patients with pSS. This questionnaire was originally developed to assess xerostomy in Australian elderly people [7]. Then it was applied to head and neck cancer patients who received radiotherapy [27] and to diabetic patients [28]. Later, it was applied to patients with pSS to evaluate the symptoms of sicca [3].Since the basal and stimulated salivary flow rate test is an objective evaluation method, the OHIP-14, OHRQoL-UK and ESSPRI questionnaires, which examine the effectiveness of oral health on quality of life and disease activity, are widely accepted because of their reliability and validity, and they have been determined as the gold standard for determining construct validity . Therefore, these assessment methods were used to evaluate the construct validity of XI-TR. There was determined to be a moderate to high positive correlation between the XI-TR score and ESSPRI, OHIP-14 total and subscale scores. A low to moderate negative correlation was determined between the XI-TR score and salivary flow rate test, OHRQoL-UK total and subscale scores. These results demonstrated that XI-TR is valid. In other version studies, only the salivary flow rate test has been used for validity . Although it was reported that the salivary flow rate test has a low level of correlation with xerostomia findings, that no other outcome measures were used constituted a limited aspect of those studies. In the current study, the results were strengthened by the use of many valid Turkish questionnaires including xerostomia findings and quality of life, and the significant correlations between them.Test/retest reliability measures the stability of answers to questions of a scale over time. Reliability indicates the accuracy and repeatability of the measurement performed. The more similar the scores of the test-retest measurements, the more reliable the questionnaire. [31]. The Cronbach alpha coefficient and the internal consistency determine the relationship between the items that make up the scale. The closer that the value is to one indicates high internal consistency and that it is reliable [32]. Cronbach alpha values in different language versions of the scale have been reported to be 0.89 and 0.87 for Spain, 0.86 for Korea and 0.90 for Portugal with pSS patients. In Germany, the Cronbach alpha value was 0.92 in patients with head and neck cancer. The internal consistency coefficient of XI-TR was calculated as 0.869 in this study, which was high and similar to the literature. It is recommended that the Cronbach alpha coefficient be above 0.8 in health-related studies [32]. Therefore, the score obtained from this study shows that the scale is quite reliable. The ICC varies between 0.0 and 1.0. The more similar the answers given by all individuals to the questions, the less variability in scoring and the ICC value will increase. In literature, ICC ranges have been reported as 0.590.91 for Spanish, 0.480.812 for Korean and 0.790.94 for Portuguese versions. In the current study, the ICC value was 0.853\u20130.994, demonstrating that XI-TR has test-retest reliability in patients with pSS. According to Fleiss (1986), if the ICCs is <0.40 it shows poor reliability, and if >0.75, it shows perfect reliability [33]. A strength of the current study was that not only the salivary flow rate test was used to evaluate xerostomia findings, but also Turkish questionnaires with validity and reliability in this subject. Another strength was that all of the pSS population evaluated participated in the retest. In studies applied in other countries, it can be seen that the initial number was not reached in the retest [11]. Xerostomia is not only seen in individuals with pSS. It can be recommended that in future studies, validity and reliability Turkish version studies are applied to other groups, such as the elderly [34], Parkinson\u2019s disease patients [35], and those undergoing head and neck radiotherapy [36], in order to be used in other possible conditions that may cause xerostomia. In the light of all the results obtained in this study, XI- - -\u2018\u2019 \u2018\u2019-TR scale is valid and reliable in Turkish for patients with pSS.This study was conducted in accordance with the Declaration of Helsinki. Approval for the study was granted by the Local Ethics Committee of Pamukkale University. All individuals were informed verbally and informed consent forms were signed. The code is 60116787-020/54466 (meeting dated 06.08.2019 and numbered 14)."}
+{"text": "CliniciOver the past several decades, there have been dramatic changes in health care with the evolution of technology, increased regulatory burden, and the exponential growth of clerical burden, partly brought about by the widespread introduction of electronic health records. These developments come at a cost to the well-being and work-life balance of clinicians. There are several key drivers Figure  that can3Unfortunately, there are serious personal and professional costs associated with clinician burnout Figure . Persona78935111213151617Assessing burnout and its drivers by conducting surveys is a critical step to understanding the factors that need to be addressed to cultivate an engaged and efficient workforce and to improve clinician burnout. Hence, burnout is a metric to measure and monitor, whereas clinician well-being is the goal in cardiology and medicine. With this joint statement, the American College of Cardiology (ACC), American Heart Association (AHA), European Society of Cardiology (ESC), and World Heart Federation call for global action in health care reform, research, and policy development to address clinician well-being. Further, we aim to generate awareness about the impact of burnout and potential solutions to improve clinician well-being in cardiovascular medicine.Survey data among 2,274 U.S. cardiologists and fellows-in-training reported that more than one-quarter reported being burned out and almost 50% were stressed; only 23.7% reported that they were enjoying their work. Women reported burnout more frequently compared with men. Also, midcareer cardiologists more frequently reported burnout compared with fellows-in-training and early- or late-career cardiologists. Neither type of practice setting nor type of cardiovascular subspeciality had an impact on burnout, but burned-out respondents did report more time spent in direct clinical practice. Overall, most were satisfied with their career; however, burned-out respondents were less satisfied with achieving their professional goals or their desired financial compensation, and were less likely to recommend cardiology as a career. Compared with their peers, burned-out physicians were less likely to report feeling valued or being treated fairly at work. Drivers associated with burnout among cardiologists include lack of control over workload, a hectic work environment, misalignment of values, and insufficient documentation time . Data am2021Health care organizations have predominately focused on the concept of \u201cfixing the employee\u201d with individual-focused programs (self-resiliency and stress management training) as the solution to improving well-being; however, much more effort needs to be tailored to systemic issues that affect the work environment. Intentional refinement of practice environments with highly functioning teams within which clinicians can optimally care for patients enables all team members to find value and purpose in their work and can result in improved outcomes for the health care organization and patients. It is imperative for health care organizations to support the psychosocial health of their employees and be accountable for a holistic approach. One such method is the Stanford WellMD Professional Fulfillment Model, which incorporates culture of wellness, practice efficiency, and personal resiliency domains , while aOrganizational infrastructure is necessary to provide the foundational means to maintain work environments within which clinicians may thrive. The burnout and wellness hierarchy model, adapted from Maslow\u2019s hierarchy of needs, provides a prioritized stepwise approach for health care leaders to address clinicians\u2019 professional fulfillment: basic physiological and mental health needs first; safety and security second; respect from administrators, colleagues, and patients third; appreciation and interpersonal connection fourth; and finally, resources and time to treat patients and practice medicine fully . For insIn addition, health care organizations need to provide employees with a structure to allow for confidential reporting of mistreatment and also to destigmatize clinicians\u2019 access to mental health resources. Hospital credentialing committees need to refine their inquiries into mental health conditions so that it does not perpetuate a culture of either under-reporting of mental health conditions or undertreatment due to the fear of the loss of hospital privileges among clinicians.Regular assessment of clinician burnout is essential to understand the effectiveness of implemented strategic pilot projects. Similar to the concepts of prevention of cardiovascular disease, secondary prevention is achieved with targeted approaches to prevent recurrent burnout; however, investment in primary and primordial prevention is equally crucial to reduce or potentially avoid burnout all together.Clinician wellness is a broad entity that requires medical specialty societies to develop a multipronged approach to make meaningful progress. To support their members, medical societies will need to continue to provide recommendations to health care organizations and advocate for meaningful health policy changes. Furthermore, development of cardiology-specific tools that may improve practice efficiency or clinician knowledge base in a timely and convenient fashion is imperative. Medical specialty societies are also key in developing a sense of community for their members. Engaged members of medical specialty societies often cite global social networks as a recurring theme for ongoing involvement in societal committees and attendance at national conferences. However, medical societies will need to continue to expand their initiatives in diversity and inclusion so that all members feel valued and have a sense of belonging. In addition, the medical specialty societies will need to consider innovative ways to deliver educational content and offer networking opportunities, especially in light of resource constraints and increased need for virtual platforms.In March 2020, the ACC\u2019s Board of Trustees commissioned the Task Force on Clinician Well-Being to help focus its strategy to address clinician well-being. Several surveys have been launched to assess the prevalence of and drivers of burnout amongst ACC members. Member sections are integrating well-being themes in their various offerings to members, including toolkits, webinars, and conferences. Recent webinars share cardiologists\u2019 perspectives of mental health conditions as well as inform the viewers regarding signs and symptoms of anxiety and depression, encourage treatment, and provide members the contact information for confidential counseling resources. Additionally, clinician well-being was incorporated into the 2020 ACC/AHA Professionalism and Ethics conference, and the formal recommendations were recently published .Also, in Europe, nearly 4,000 medical faculty and researchers from 17 ESC member countries in 2018 completed the ESC-sponsored \u201cCulture and Leadership in Cardiology in Europe\u201d C-Change Faculty Survey. Overall, cardiologists and research professionals have high leadership aspirations and a high degree of personal satisfaction in their work. However, approximately one-quarter of individuals did not view their institutions as providing the best opportunities for meaningful work, and one-third did not feel they were supportive of career advancement. The ESC has been using these findings to guide future strategic initiatives that aim to provide appropriate support for cardiologists in Europe and enable satisfaction within their work environment.The ACC, AHA, ESC, and World Heart Federation acknowledge that clinician well-being is paramount to providing high-quality patient care and are committed to improving the well-being of the cardiovascular workforce.Medical specialty societies will need to continue to identify and promote for the reduction of administrative complexities and practice inefficiencies, which are burdensome and hinder professional fulfillment and patient care. Advocating for appropriate initiatives to improve clinician well-being and mental health resources, while also influencing the elimination of non-essential regulation and document requirements, is key. Furthermore, assisting in combatting the stigma of mental health conditions and the negative ramifications by licensing boards of a history of treated medical health conditions are crucial endeavors for medical specialty societies. Collaboration with other specialties is necessary, including leveraging the expertise of psychiatrists and clinical psychologists. The House of Cardiology will continue to work together and strategize to maintain the well-being of our profession and keep the joy in medicine."}
+{"text": "In recent decades the conservation of cultural heritage has been attracting increasing research interest from many different scientific disciplines. Lately, the co-integration of chemistry and physics with biological techniques has shaped our understanding of the living microbial communities on sites of cultural heritage . This concept has been applied to the study of some of Leonardo da Vinci's most emblematic drawings. This includes the famous Leonardo autoritratto (self-portrait). Results were achieved via Oxford Nanopore sequencing technology. The drawings' microbial bio-archive showed a relatively high contamination with human DNA and a surprising dominance of bacteria over fungi. The study also showed the presence of cellulose degraders, such as several members of the phyla Proteobacteria, Actinobacteria, and Firmicutes among bacteria, and fungi belonging to the classes Sordariomycetes and Eurotiomycetes. Interestingly, the microbial profile correlated with that of Rome or Turin were also identified, indicating the importance of environmental and storage conditions on the specific microbiota .This Research Topic discusses the very interesting concept of the definition of \u201cbio-archive\u201d for the determination of a core microbiome with the scope of monitoring, cataloging, and providing added-value to artworks . Illumina MiSeq technology showed that after treatment an enrichment of bacteria able to foster calcium carbonate biomineralization was achieved, and this supported stone consolidation. This is an excellent example of how bio-conservation treatment can safely and effectively be applied to temples, sculptures, and stuccos, and how metagenomics is an effective tool to assess the quality of the intervention .The application of -omics techniques has also found great applications in archaeology with a specific focus on stone materials. Two studies dealt with stone conservation and degradation. The first study evaluated the changes in bacterial diversity of severely degraded tuff stone and lime plaster at the archaeological Maya site of Copan (Honduras) after treatment with the patented sterile M-3P nutritional solution . In addition, the study reported a meta-taxonomical overview (via Illumina sequencing) of the bacterial and fungal microbial community .Another example on the protection of stone showed the biodegradation potential by microorganisms against the protective epoxy resin in the archaeological Ruins of Liangzhu City (Cina) . The main picture arising from the integration of metagenomic with recent chemical (Raman spectroscopy) and physical  approaches, provided a complete picture of ecological connections, which includes different microbial degraders penetrating the parchment at different stages and triggering microbial successions according to collagen availability. The mini-review showed the parchment's degradation stages, including hydrothermal degradation of the two main collagen populations, the less stable collagen, classified as native, and the more stable, the so called stabilized collagen .A valuable approach of integration among -omics approaches has been proposed by studying the biodeterioration dynamics of parchment. Such dynamics have been explored by adopting a multidisciplinary approach combining standard microbiological methods with high-throughput molecular, chemical, and physical techniques (The main overview from the papers published in this Research Topic is that the multi-omics revolution for microbial cultural heritage conservation is only just beginning. More exciting discoveries will occur in the future through the integration of genomics, transcriptomics, proteomics, metabolomics specialties with microbial cultivable techniques and restoration/conservation applications. Multidisciplinary approaches are therefore the key for this bright future and essential to unraveling the role that microorganisms can provide in the conservation of cultural heritage, from the perspectives of both biodeterioration and bio-restoration.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "An important number of breast and ovarian cancer cases is due to a strong genetic predisposition. The main tool for identifying individuals at risk is recognizing a suggestive family history of cancer. We present a prospective study on applying three selected clinical guidelines to a cohort of 1000 Slovenian women to determine the prevalence of at-risk women according to each of the guidelines and analyze the differences amongst the guidelines.Personal and family history of cancer was collected for 1000 Slovenian women. Guidelines by three organizations: National Comprehensive Cancer Network (NCCN), American College of Medical Genetics in cooperation with National Society of Genetic Counselors (ACMG/NSGC), and Society of Gynecologic Oncology (SGO) were applied to the cohort. The number of women identified, the characteristics of the high-risk population, and the agreement between the guidelines were explored.NCCN guidelines identify 13.2% of women, ACMG/NSGC guidelines identify 7.1% of women, and SGO guidelines identify 7.0% of women from the Slovenian population, while 6.2% of women are identified by all three guidelines as having high-risk for hereditary breast and ovarian cancer.We identified 13.7% of women from the Slovenian population as being at an increased risk for breast and ovarian cancer based on their personal and family history of cancer using all of the guidelines. There are important differences between the guidelines. NCCN guidelines are the most inclusive, identifying nearly twice the amount of women as high-risk for hereditary breast and ovarian cancer as compared to the AGMG/NSCG and SGO guidelines in the Slovenian population.The online version contains supplementary material available at 10.1186/s12885-021-08400-8. Family history of cancer is the most important risk factor for breast and gynecological cancer development after sex and age . The preHowever, it is less common to refer unaffected women to genetic counseling for gynecological cancers, even when they harbor a family history of cancer and genetic counseling and testing would provide important information for their cancer risk evaluation . IdentifSince general population screening for BRCA pathogenic variants is currently not recommended due to low general population prevalence (1 in 300 to 500) , it is iClinical guideline\u2019s specificity and sensitivity for selection of pathogenic variant carriers from women with a confirmed diagnosis of breast or gynecological cancers have been estimated in several studies \u201321. TherWe aimed at comparing clinical guidelines for identification of women at risk and referral to genetic counseling for hereditary breast and ovarian cancer issued by three professional organizations \u201315 that An interview was conducted amongst a thousand women from the general population, patients of the Outpatient Clinic of the Division of Gynecology and Obstetrics, University Medical Centre Ljubljana, Slovenia, between January 2018 and September 2019. Inclusion criteria for our study was female sex, age above 18\u2009years, and patient status of any of the outpatient specialist gynecologic clinics of the Division of Gynecology and Obstetrics, UMC Ljubljana for various gynecologic diseases . Exclusion criteria was the inability to communicate in the Slovenian language.The women completed an interview that included: contact information, personal history of gynecologic and other cancers, previous genetic testing/pathogenic variant found in the family, and family history of gynecologic and other cancers. Information was collected on all known cancerous diseases within a family, age at diagnosis of cancer, family relation to the interviewee, type of cancer, and the bloodline of the relative. The questionnaire used to conduct these interviews was developed for this study  .Our study population consisted of a group of 1000 women, aged 18 to 88\u2009years old. The median age is 36\u2009years old. Women presented both personal and family history of cancer. Namely, 3% of women had a personal history of breast and/or ovarian cancer. Family history of breast and/or ovarian cancer ) was reported in 21.1% of women in our cohort, and of those, 6.3% of women had a FDR with breast cancer and 1.7% of women had a FDR with ovarian cancer. No personal or family history of any cancer was reported by 27.8% of interviewees , and the number and percentage of women from our study population qualifying for each criterion with each guideline are shown in Table\u00a0There are differences in the criteria definition and description in the NCCN, ACMG/NSGC, and SGO guidelines. Consequently, different guidelines identify a varied number of women as high-risk for hereditary breast and ovarian cancer Table . NCCN guOur analysis showed that 23.0% of women from our cohort had a personal or family history of breast or ovarian cancer up to second-degree relatives. Out of these women, 60%, representing 13.7% of all women, were identified as high-risk for hereditary breast and ovarian cancer by at least one guideline; 6.2% of all women were identified as high risk for hereditary breast and ovarian cancer by all of the guidelines analyzed , where there is no age limit defined and based on the criterion of early onset of breast cancer . NCCN identifies an additional 3.5% of women as high-risk, based on the criterion of pancreatic/ prostate cancer in the family, however, more than half (2.1%) of those women do not meet any other criteria and/or are not identified as high-risk by the other two guidelines.p\u00a0<\u20090.001, and only a moderate agreement between NCCN guidelines and the remaining two guidelines ; ; p\u00a0<\u20090.0A comparison of the three referral guidelines has revealed differences among the criteria with each of the guidelines. The criteria an individual at any age with a known pathogenic/ likely pathogenic variant in a cancer susceptibility gene within the family and male breast cancer in family identify the same number women from our study group as high risk for breast and ovarian cancer with all three analyzed guidelines. Using other criteria, different guidelines identify a varied number of women as high risk for HBOC.NCCN guidelines are the most inclusive amongst the guidelines, identifying nearly double the number of women compared to the other two guidelines. A group of 58 women have been identified as high-risk only by NCCN, with 21 of those women identified due to having a FDR/SDR with pancreatic or prostate cancer in the family. NCCN guidelines included this criterion in a 2019 update  based onACMG/NSGC and SGO guidelines identify 7.1 and 7.0% of women as high-risk, respectively, with some differences amongst the guidelines. The criterion that uniquely identifies women as high-risk with ACMG/NSGC guidelines is having a FDR with breast cancer at ages between 45 and 50\u2009years present in the family. This criterion results in the identification of six additional women. Testing first-degree relatives of women with the diagnosis of breast cancer between ages 45 and 50\u2009years might be reasonable, since a recent study showed that the peak incidence of breast cancer in BRCA1 pathogenic variant carriers occurred between the ages 41 and 50 . SGO guiApplication of various combinations of criteria to our study group reveals that 6.2% of women from our cohort were concordantly identified as high-risk by all guidelines. The criteria that selected these women as high-risk group were a BRCA1/2 pathogenic variant present in the family, patient or a FDR with two or more primary breast cancers, ovarian cancer, breast cancer before age 45 or male breast cancer, or had a combination of three or more specific cancers present in the family. These shared criteria may represent the core criteria of the guidelines, identifying the highest risk women, since it has already been suggested that agreement of multiple guidelines might be considered for the selection of women with the highest risk for the pathogenic variant, viewing each guideline as an \u2018expert\u2019 and all guidelines as an \u2018expert panel\u2019 , 23.It has recently been observed that the prevalence of BRCA pathogenic variant carriers is higher than previously estimated  and thatThe aim of the identification of high-risk women in the population is to reduce morbidity and mortality in those women by referring them to appropriate screening and prevention . Our stuThe limitation of our study is that we cannot disclose the specificity and sensitivity of each of the guidelines for the identification of pathogenic variant carriers in high-penetrance cancer genes due to several reasons. Most importantly, the 1000 women from our study population have not been molecularly tested and their BRCA status has not been determined. Because of that, we cannot assess the sensitivity of the guidelines, since we are unable to determine whether some of our average risk population might be asymptomatic carriers of a pathogenic variant in a cancer predisposition gene without a family history of cancer and, therefore, part of the high-risk population. This can be partly explained by the fact that pathogenic variant carriers sometimes lack indicative family of cancer because of different reasons: imperfect reporting of cancer disease in the family, presence of adoption or risk-reducing surgeries in a family, families with few female relatives or a small family size . SecondlIn conclusion, the analysis of cancer family history of 1000 women from the general population shows that NCCN, ACMG/NSGC, and SGO guidelines identify 13.7%, an important proportion of women as high-risk for hereditary breast and ovarian cancer. NCCN guidelines identify nearly double the number of women compared to the remaining two guidelines as having an increased risk for HBOC in the Slovenian general population.Additional file 1. Questionnaire used to conduct the interviews."}
+{"text": "The triglyceride-glucose (TyG) index has been proposed as a convincing indicator of insulin resistance and has been found to be associated with atherosclerosis among diabetic patients. However, the relationship between the TyG index and arteriosclerosis in subjects with prediabetes and new-onset type 2 diabetes (T2D) remains uncertain. The purpose of this study was to assess the degree of carotid plaque burden in patients with prediabetes and new-onset T2D and to investigate the association between the TyG index and the degree of carotid plaque burden in this population.This was a cross-sectional observational study that included 716 subjects aged 40\u201370 years old with prediabetes or new-onset T2D. Demographic, anthropometric, and laboratory measurements were collected. Participants underwent carotid arteriosclerosis evaluation by ultrasonography, and the degree of atherosclerosis was evaluated according to the carotid plaque burden. The TyG index was calculated.P < 0.001). The high TyG index group had a higher atherosclerotic plaque burden than the low TyG index group (P < 0.001). Multiclassification logistic regression analysis showed that the TyG index was positively associated with a high plaque burden , while no association was found between the TyG index and a low/moderate plaque burden. This association remained consistent in the subgroup analysis. In multiple linear regression analysis, sex, age, and the TyG index were found to be independently associated with carotid plaque burden. For each unit increase in the TyG index, the risk of a high carotid plaque burden increased 1.595-fold.The population was stratified into high or low TyG index groups according to the median TyG index value. Higher values were associated with a higher BMI and waist circumference as well as higher total cholesterol, triglyceride, low-density lipoprotein cholesterol, plasma glucose, glycated hemoglobin, fasting C-peptide, and C-reactive protein levels (A high TyG index was positively associated with a high carotid plaque burden in subjects with prediabetes and new-onset T2D. Clinicians should pay close attention to the TyG index to help these patients receive the greatest benefit from early intervention. Diabetes mellitus, a leading cause of disability worldwide, is a well-known risk factor for atherosclerotic cardiovascular disease (ASCVD) . A previIndeed, in the prediabetic state, the risk of cardiovascular events is persistent, and atherosclerosis might appear prior to a diagnosis of diabetes mellitus , 4. AccoCarotid plaque burden plays an essential role in the progression of ASCVD. For the early diagnosis of arteriosclerosis, imaging modalities such as ultrasound and coronary angiography are usually needed, and performing such procedures during routine monitoring of an asymptomatic population on a large scale may be difficult as they are expensive and time-consuming. Therefore, early recognition of simple and accurate indicators of carotid plaque burden that can be applied in daily clinical practice could aid in the early identification of patients at high risk of ASCVDs . InsulinBased on the above background, the current study evaluated whether and how the simple calculated TyG index was associated with the carotid plaque burden in subjects with prediabetes and new-onset T2D without any ASCVDs. The results of this work can contribute to the early recognition of patients at high risk of cerebrovascular accidents.Between January 2018 and January 2021, a total of 4,394 hospitalized patients with abnormal blood glucose were screened. After a review of clinical information consisting of medication usage data, self-reported medical history, glycated hemoglobin (HbA1c), fasting blood glucose, and oral glucose tolerance test (OGTT) results, 992 patients with a diagnosis of prediabetes or new-onset T2D at our hospital were included. The inclusion criteria were as follows: (1) diagnosis of prediabetes or new-onset T2D; (2) age between 40 and 70 years old; (3) no history of atherosclerotic cardiovascular disease; (4) no severe renal dysfunction; (5) no previous lipid-lowering treatment; and (6) carotid ultrasonography.The criteria for prediabetes included a fasting plasma glucose (FPG) level between 100 mg/dL (5.6 mmol/l) and 125 mg/dL (6.9 mmol/l) and/or an HbA1c ranging between 5.7 and 6.4%, without a history of diabetes or the use of any antidiabetic drugs. Criteria for new-onset T2D included an incidental finding of FPG \u2265 126 mg/dL (7.0 mmol/l) and/or HbA1c \u2265 6.5% for no more than 6 months, without a history of diabetes or the use of any antidiabetic drugs , 16. AthBased on the inclusion criteria, 276 patients were excluded, and 716 patients were ultimately included in the analysis . The stu2).Demographic information (including sex and age) and anthropometric measures  were collected. Body mass index (BMI) was calculated using the equation weight (kg)/squared value of height  \u2265 140 mmHg or diastolic blood pressure \u2265 90 mmHg on two different occasions or a history of antihypertensive therapy.Venous blood samples were collected from the subjects after an overnight fasting for the analysis of total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), FPG, HbA1c, and high-sensitivity C-reactive protein (hs-CRP). We calculated the TyG index using the equation log [(fasting TG (mg/dl) \u00d7 FPG (mg/dl)/2] .Carotid ultrasonography was performed by trained and experienced sonographers using a color ultrasound diagnostic apparatus equipped with a linear array transducer. The common, bifurcation, external, and internal carotid arteries were examined bilaterally from the transverse and longitudinal orientations to evaluate the carotid intima-media thickness (IMT), the presence and morphology of atherosclerotic plaque, and the presence of carotid stenosis.1: low plaque burden ; PS2: moderate plaque burden ; and PS3: high plaque burden . The carotid ultrasonography results were reviewed by two independent experienced sonographers blinded to the clinical data. Discrepancies were resolved by consensus.Carotid intimal thickening was defined as 1.0 \u2264 IMT \u2264 1.5 mm, and atherosclerotic plaque was defined as a localized IMT \u2265 1.5 mm or a relative focal thickening of more than 50% of the IMT of the surrounding tissue. A finding of two or more plaques was defined as multiple plaques. According to morphology and echo characteristics, we divided the plaques into stable plaques or unstable plaques. Plaques with hyperechoic, homogeneous echoes and a smooth surface were defined as stable plaques. Hypoechoics plaques and those with mixed echoes or ulcerations were defined as unstable plaques . CarotidP \u2264 0.05 was considered indicative of statistical significance. All statistical analyses were performed by using SPSS version 21.0 .The subjects were divided into two groups based on their TyG index values (high or low), and the baseline clinical data and ultrasound findings of the two groups were compared. The Kolmogorov\u2013Smirnov test was used to evaluate the normality of the distribution of continuous variables. Data are presented as medians (interquartile ranges) for continuous variables with a non-normal distribution and as percentages for categorical variables. Group differences were analyzed using the Wilcoxon rank sum test for continuous variables and the chi-square test for categorical variables. A chi-square test for trend was used to assess the trend of carotid plaque burden prevalence with a high TyG index. To assess the possible influencing factors of carotid plaque burden of the subjects, multiclassification logistic regression analysis was performed by calculating odds ratios (ORs) and 95% confidence intervals (CIs). Finally, we developed a linear regression model to explore the correlation between the change in the TyG index and the carotid plaque burden after adjusting for sex, age, tobacco use, TGs, HDL-C, FPG, and HbA1c. A The demographic and baseline data are presented in M = 7.84) was used to divide the participants into high and low TyG index groups, and subgroup analyses of the data were performed. Subjects with a higher TyG index value were more likely to have higher BMI and waist circumference (25.30 [22.95\u201327.40] vs. 24.29 [22.05\u201326.43)] kg/m2, P < 0.001 and 90.00 [84.00\u201395.88] vs. 85.00 [80.00\u201392.75] cm, P < 0.001, respectively) than subjects with a lower TyG index value. TC and TG values were higher in the high TyG index group than in the low TyG index group (5.21 [4.38\u20136.10] vs. 4.31 [3.69\u20134.93)] mmol/L, P < 0.001 and 2.20 [1.68\u20132.97] vs. 1.14 [0.94\u20131.44] mmol/L, P < 0.001, respectively). Accordingly, the high TyG index group exhibited a higher level of LDL-C (3.44 [2.74\u20134.01] vs. 2.78 [2.28\u20133.25)] mmol/L, P < 0.001) and a lower level of HDL-C  than the low TyG index group. Moreover, FPG, HbA1c, and fasting C-peptide levels were higher in the high TyG index group than in the low TyG index group . Finally, subjects in the high TyG index group had higher levels of hs-CRP than those in the low TyG index group .The median value of the TyG index (P < 0.001), more unstable plaques , and a higher PS . On the other hand, the proportions of subjects with a high TyG index were similar between PS1 and PS2 , while no association between the TyG index and a low/moderate plaque burden was found in the study population.Univariate logistic regression analysis was conducted with the possible influencing factors of carotid plaque burden as independent variables and PS as the dependent variable. Factors that were statistically significant in the univariate analysis were introduced into the unordered multiclassification logistic regression model, and the analysis results are shown in 2), and hypertension (no or yes), the association between the TyG index and incident high plaque burden remained consistent , sex , BMI . TyG index showed the strongest association with the carotid plaque burden. For each unit increase in the TyG index value during this period, the plaque burden score increased 1.595-fold.To further evaluate the extent to which the TyG index was associated with carotid plaque burden in our population, multiple linear regression analysis was performed. First, possible risk or protective parameters were included in the univariate analysis, and the results showed that sex, age, smoking, triglycerides, HDL cholesterol, fasting blood glucose, HbA1c, and the TyG index were meaningful. These parameters were then used to construct a regression model by using a multiple linear regression equation, and the results are displayed in This cross-sectional study shows for the first time that a high TyG index value in subjects with prediabetes and new-onset T2D is significantly associated with an increased risk of high carotid plaque burden as assessed by ultrasound. The association remained consistent even when the subjects were stratified by age, sex, BMI, and hypertension. No association between the TyG index and a low/moderate plaque burden was found. Furthermore, we found that compared with other factors, the TyG index showed the strongest association with the carotid plaque burden. For each unit increase in the TyG index, the risk of a high carotid plaque burden increased 1.595-fold.Prediabetes is an intermediate stage in which glucose tolerance progresses from normal to uncontrolled intolerance. As a multifactorial metabolic disorder, prediabetes has been confirmed to have a causal relationship with ASCVD , 21. In In previous studies, one of the difficulties in identifying the risk factors involved in arteriosclerosis progression was the quantification of arteriosclerotic severity. In the present study, carotid plaque burden was used as a marker of atherosclerosis, which manifested as the change in the plaque score obtained from different carotid districts. As reported in the literature, the prevalence of carotid artery plaque shows a good correlation with the presence of atherosclerosis and can well reflect the overall severity of atherosclerosis in the vasculature , 26. TheP = 0.05). This could be explained by the possibility that plaque erosion, but not plaque burden, is the intermediate between smoking and ASCVD , the National Key R&D Program of China (2018YFC0114900), Development Project of National Major Scientific Research Instrument (82027803), National Natural Science Foundation of China (81971623), Key Project of Natural Science Foundation of Zhejiang Province (LZ20H180001), Zhejiang Medicine and Health Science and Technology Project (2021KY1147), and Shaoxing Medical Key Discipline (2019SZD05).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Familial hypercholesterolemia (FH) is an inherited metabolic disorder with a high level of low-density lipoprotein cholesterol and the worse prognosis. The triglyceride-glucose (TyG) index, an emerging tool to reflect insulin resistance (IR), is positively associated with a higher risk of atherosclerotic cardiovascular disease (ASCVD) in healthy individuals, but the value of TyG index has never been evaluated in FH patients. This study aimed to determine the association between the TyG index and glucose metabolic indicators, insulin resistance (IR) status, the risk of ASCVD and mortality among FH patients.Data from National Health and Nutrition Examination Survey (NHANES) 1999\u20132018 were utilized. 941 FH individuals with TyG index information were included and categorized into three groups:\u2009<\u20098.5, 8.5\u20139.0, and\u2009>\u20099.0. Spearman correlation analysis was used to test the association of TyG index and various established glucose metabolism-related indicators. Logistic and Cox regression analysis were used to assess the association of TyG index with ASCVD and mortality. The possible nonlinear relationships between TyG index and the all-cause or cardiovascular death were further evaluated on a continuous scale with restricted cubic spline (RCS) curves.p\u2009<\u20090.001). The risk of ASCVD increased by 74% with every 1 unit increase of TyG index . During the median 114-month follow-up, 151 all-cause death and 57 cardiovascular death were recorded. Strong U/J-shaped relations were observed according to the RCS results . A higher TyG index was independently associated with both all-cause death and cardiovascular death. Results remained similar among FH patients with IR (HOMA-IR\u2009\u2265\u20092.69). Moreover, addition of TyG index showed helpful discrimination of both survival from all-cause death and cardiovascular death (p\u2009<\u20090.05).TyG index was positively associated with fasting glucose, HbA1c, fasting insulin and the homeostatic model assessment of insulin resistance (HOMA-IR) index (all TyG index was applicable to reflect glucose metabolism status in FH adults, and a high TyG index was an independent risk factor of both ASCVD and mortality. Atherosclerosis cardiovascular disease (ASCVD) is the leading cause of death in both developed and developing countries . AtherosThe triglyceride-glucose (TyG) index, calculated as Ln , is an emerging tool to reflect insulin resistance (IR) . IR is thttps://www.cdc.gov/nchs/nhanes/irba98.htm). Informed consent from all the participants was obtained before participating.NHANES is a two-year-cycle cross-sectional survey conducted by the Centers for Disease Control and Prevention (CDC) of America, involving a home interview and a medical examination, offering demographics, socioeconomic status, dietary and health information as well as physical and physiological measurements of U.S. population . NHANES There were 1456 out of 116876 participants in the survey diagnosed as FH in accordance with a Dutch Lipid Clinical Network (DLCN) index that was higher than or equal to 3 points, as described previously . Among tInformation on socioeconomic conditions, behavior and history of diseases was obtained through questionnaires by experienced interviewers. Drinking was defined as having at least 12 alcohol drinks per year; smoking was defined as having smoked\u2009\u2265\u2009100 cigarettes in life . HistoryThe period of follow-up lasted from the date of the interview through the last follow-up time, Dec 31 2019, or the date of death, whichever came first. Records from the NDI provided information on these including participants' causes of death. The endpoints for this study included all-cause mortality and cardiovascular death, which encompassed cardiac death  and vascular death  [Data were analyzed using SPSS complex sample module version 22.0  and R (version 4.2.2). The Kolmogorov\u2013Smirnov normality test was adopted to test the normality of continuous variables. Normally distributed variables were described with mean\u2009\u00b1\u2009standard deviation (SD). Variance analysis was adopted to compare the mean levels while Chi-square tests were chosen to compare the percentages of categorical variables across the different groups.Spearman correlation analysis was used to test the association of TyG index and various established glucose metabolism-related indicators  with 95% confidence interval (CI) for the relationship between TyG index and ASCVD. The possible nonlinear relationships between TyG index and the all-cause or cardiovascular death were further evaluated on a continuous scale with restricted cubic spline (RCS) curves based on the multivariable Cox proportional hazards models, with four nodes at the fixed percentiles of 5%, 35%, 65% and 95% of the distribution of TyG index. The event-free survival rates among the groups were estimated by the Kaplan\u2013Meier method and compared by the log-rank test. Cox regression analysis was used to assess the association between TyG index and mortality. The following variables were utilized as covariates in the study population: age, gender, BMI, smoke, drink, HOMA-IR, low-density lipoprotein cholesterol (LDL-C), creatinine and hypertension. To assess the added prognostic value of TyG index beyond the original model, C-index was calculated, using predict.coxph function to predict Cox model with predict value type\u2009=\u2009\"survival\". Furthermore, a sensitivity analysis was applied to further investigate the association of TyG index with ASCVD and mortality in FH patients with IR (HOMA-IR\u2009\u2265\u20092.69) . A two-sp\u2009<\u20090.001), in addition to higher occurrence rate of diabetes and impaired fasting blood glucose  than those with lower TyG index. Besides, individuals with higher TyG index were also more likely to be older, white and had higher BMI, systolic blood pressure (SBP), fasting total cholesterol (TC) and LDL-C as well as lower high-density lipoprotein cholesterol  compared with those with relatively lower TyG index. There was no significant difference among the four groups in DLCN score, poverty-income ratio, drink, smoke, hypertension history, diastolic blood pressure (DBP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) as well as serum creatinine (p\u2009>\u20090.05).Among the 941 FH participants in this study, 204 (21.68%) had TyG index\u2009<\u20098.5 (low TyG index), 311 (33.05%) had TyG index\u2009\u2265\u20098.5 and\u2009\u2264\u20099.0 (moderate TyG index), and 426 (45.27%) had TyG index\u2009>\u20099.0  among FH patients, indicating that TyG index was applicable to FH individuals to reflect the glucose metabolism status.As a novel indicator of IR, the diagnostic value of TyG index in FH population was examined by assessing the association with those well-recognized indicators reflecting glucose metabolism status including fasting blood glucose, HbA1c, fasting insulin, HOMA-IR and HOMA-IS. As shown in Table p\u2009=\u20090.01) in the group with high TyG index compared with the group with low TyG index; while no significant difference was observed between the group with moderate and low TyG index . For every 1 unit increase of TyG index, the risk of ASCVD increased by 74% after adjustment . In the sensitivity analysis, TyG index remained significantly associated with ASCVD in FH patients with IR . These results indicated that increase of TyG index was independently associated with the elevation of ASCVD risk among FH adults.Given the idea that TyG index is associated with the development and prognosis of cardio-cerebrovascular diseases , 27, we p for non-linearity\u2009=\u20090.0083 and 0.0046, respectively). The study population was divided into three groups  due to the strong U/J-shaped relation between TyG index and mortality. Figure\u00a0p\u2009=\u20090.035). No significant difference was observed in the incidence of cardiovascular mortality (log-rank p\u2009=\u20090.093). Cox analysis was utilized to further assess the association of TyG index with all-cause death and cardiovascular death (Table p\u2009=\u20090.01 for every 1 unit increase of TyG index) and cardiovascular death . In sensitivity analysis . The adjusted HR was 1.61  for all-cause death and 2.09  for cardiovascular death in the group with high TyG index comparing with the group with moderate TyG index. However, no significant difference was observed when comparing the two groups reported low and moderate TyG index . In Cox prediction models, C-statistic values were 0.489 (95%CI 0.437\u20130.542) and 0.408 (95%CI 0.324\u20130.491) for survival from all-cause death and cardiovascular death with traditional risk factors, respectively (Table p\u2009=\u20090.042) and cardiovascular death . These results demonstrated that TyG index was an independent risk factor for all-cause death and cardiovascular death among FH adults.In this study, the median follow-up duration is 114\u00a0months . In Fig.\u00a0Herein, we combined NHANES data from 1999 to 2018, and a total of 941 FH participants with TyG index and follow-up data accessible were finally included. The capabilities of TyG index to reflect glucose metabolism status (hyperglycemia and IR) and predict the risks of ASCVD and mortality were preliminarily verified. As a cost-effective tool, TyG index integrates fasting glucose and triglycerides levels and could provide an early relevant clinical evaluation of glucolipid metabolic disorder such as IR, and potential prediction value of ASCVD and mortality risks. To the best of our knowledge, this is the first study to examine the value of TyG index among adults with FH.vs. the bottom quartile [r, 0.462; p\u2009<\u20090.001), HbA1c , fasting insulin  and HOMA-IR  in FH population. Therefore, TyG index seems to be applicable to FH patients.As a hallmark of T2D, IR is a state of decreased sensitivity and responsiveness to the action of insulin . Arguablquartile . Howeverquartile . Whereasquartile . FH is aquartile \u20138. Furthquartile . Given tquartile . Resultsp\u2009=\u20090.01), suggesting T2D and IR led to additional ASCVD risk [p for trend\u2009=\u20090.02) for each SD increase in the TyG index. Herein, we found that high TyG index acted as an independent risk factor of ASCVD and mortality in FH adults. To be specific, the risk of ASCVD, all-cause death and cardiovascular death increased by 74% after adjustment , 55%  and 79%  for every 1 unit increase of TyG index after multivariable adjustment. These results indicated that IR plays an essential, detrimental role in FH patients, which could be another explanation for the residual risks of FH. Evaluation and treatment of IR should also be emphasized since most of current therapies focus on the management of LDL-C [vs. 0.489, p\u2009=\u20090.042) and cardiovascular mortality .Despite major advances in understanding of the disease and effective therapies such as lipid-lowering drugs and dietary interventions, FH is still underdiagnosed and undertreated . As a reCVD risk . As a reCVD risk , 27. In CVD risk . In anotof LDL-C . Besidesp\u2009=\u20090.0083 for all-cause death and 0.0046 for cardiovascular death, respectively) and the moderate TyG index group had the lowest risk of mortality. When compared with the moderate TyG index group, the low TyG index group had a trend toward an increased risk of all-cause and cardiovascular mortality , despite no significant differences. A potential cause of the phenomenon is the effect of certain parameters which could not be adjusted, such as hypoglycemia. TyG index was significantly correlated with blood glucose . The low TyG index group had a trend of worse prognosis, which may be caused by lower blood glucose. Nevertheless, we failed to provide robust statistical evidence on the elevated risk of mortality in the low TyG index group compared with the moderate TyG index group mainly due to the limited sample size.Surprisingly, a strong U/J-shaped relation was observed according to the RCS results (The current study has several limitations to be noted. Firstly, the small sample size may have limited the statistical power to detect some associations as significant when comparing different groups, as mentioned above. However, up to 114\u00a0months of median follow-up duration helps to improve statistical efficiency. Secondly, the data of TyG index was obtained only at baseline and it\u2019s hard to control for possible changes in blood glucose and TG during the follow-up in theory. However, it\u2019s still considered a valid method to evaluate the long-term effects of TyG index according to a large number of reports , 39, 40.Conclusively, results in this pilot study suggested that TyG index was applicable to reflect glucose metabolism status in FH adults, and a high TyG index was an independent risk factor of ASCVD and all-cause mortality in the same population."}
+{"text": "Multiple sclerosis (MS) is the most frequent non-traumatic neurological debilitating disease among young adults with no cure. Over recent decades, efforts to treat neurodegenerative diseases have shifted to regenerative cell therapy. Adipose tissue-derived stromal vascular fraction (SVF) comprises a heterogeneous cell population, considered an easily accessible source of MSCs with therapeutic potential in autoimmune diseases. This study aimed to assess the regenerative capacity of low-level laser-activated SVF in an MS cat model.Fifteen adult Persian cats were used in this study: Group I , Group II  intrathecal injection), and Group III . The SVF was obtained after digesting the adipose tissue with collagenase type I and injecting it intrathecal through the foramen magnum.P \u2264 0.05) recovered in Group III, and the Basso, Beattie, and Bresnahan (BBB) scores of hindlimb locomotion were significantly higher in Group III (14 \u00b1 0.44) than Group II (4 \u00b1 0.31). The lesion\u2019s extent and intensity were reduced in the magnetic resonance imaging (MRI) of Group III. Besides, the same group showed a significant increase in the expression of neurotrophic factors: BDNF, SDF and NGF  compared with Group II . Furthermore, SVF co-treated group revealed a significant (P \u2264 0.05) increase in oligodendrocyte transcription factor (Olig2) and myelin basic protein  that was decreased in group II . Moreover, group III showed a significant (P \u2264 0.05) reduction in Bax and glial fibrillary acidic protein  as compared with group II . The transmission electron microscopy demonstrated regular more compact, and markedly (P \u2264 0.05) thicker myelin sheaths (mm) in Group III (0.3 \u00b1 0.006) as compared with group II (0.1 \u00b1 0.004). Based on our results, the SVF co-treated group revealed remyelination and regeneration capacity with a reduction in apoptosis and axonal degeneration. The results showed that the pelvic limb\u2019s weight-bearing locomotion activity was significantly (SVF is considered an easy, valuable, and promising therapeutic approach for treating spinal cord injuries, particularly MS.The online version contains supplementary material available at 10.1186/s13287-022-03222-2. Spinal cord injuries in mammals cause degenerative neurons and axons, resulting in significant sensorimotor dysfunction, paraplegia, or tetraplegia , 2. MultThe stromal vascular fraction (SVF) is a by-product of adipose tissue harvesting and digestion. It consists of various non-expanded cells, including adipose mesenchymal stem cells (ASCs), adipocytes, T regulatory cells, macrophages, endothelial precursor cells, and numerous leukocytes , 21. TheWhile considerable effort in treating spinal cord injury focuses on small laboratory animals, large animal models are needed to properly evaluate stem cell therapy\u2019s safety and clinical efficacy after spinal cord injury .Therefore, our study is designed to evaluate the therapeutic potential of autogenic Low laser-activated SVF transplantation in the functional recovery and structural remodeling in the ethidium bromide-induced MS in cats by magnetic resonance imaging (MRI), histopathological and immunohistochemical examination, transmission electron microscopy, and gene expression analysis by RT-PCR.The Veterinary Medicine Cairo University Institutional Animal Care and Use Committee approved all experimental animal protocols (Vet-CU-IACUC) with approval number Vet Cu12/10/2021/392, as well as shelters approval sheets were applicable for this study.ad libitum at ideal room temperature (20\u201323\u00a0\u00b0C). All cats were evaluated before the study to exclude any animals suffering from nervous manifestations. The animals were grouped into three (n\u2009=\u20095). Group I , Group II , and Group III . Sample collection was applied 28\u00a0days post-SVF injection in all groups.Fifteen male adult Persian cats (2\u20133\u00a0years) were collected from different shelters around Giza and housed in the Faculty of Veterinary Medicine, Anatomy Department with a minimum acclimation period of one week before major surgery. Food and water were available The spinal cord demyelination was performed in groups (Gp II and Gp III) using ethidium bromide  , 24. TheThe wound was sutured with a simple continuous pattern for muscles and subcutaneous tissues using Vicryl size 3\u20130 and silk size 2\u20130 for the skin in a single interrupted pattern. Animals were injected with a systemic course of antibiotics for 5 days: analgesics  and daily dressing to the wound.Fourteen days after induction, animals were administered either a single dose of normal saline (Group II) or laser-activated SVF intrathecally through the foramen magnum (Gp III).On day 14 post-induction, under general anesthesia, a small skin incision in the inguinal region was aseptically operated, and adipose tissue (~\u200920\u00a0g) was collected from S/C tissue in a 50-ml sterile falcon tube.The tissue was processed following a sterile protocol in the laminar airflow. The collected fat was chopped into small pieces, washed thrice with phosphate-buffered saline, and then added an equal volume of 0.1% collagenase type I into it. The tissue was incubated in a rotary incubator at 37\u00a0\u00b0C, with constant agitation for one hour. After digestion, an equal volume of DMEM containing 10% fetal bovine serum was added to neutralize the collagenase. The SVF was centrifuged at 800\u00a0g for 10 minutes to separate the collagenase . Then, t2. After 24\u00a0h, the medium was replaced to remove non-adherent cells; half of the medium was replaced every 2 days until the cells reached a confluency of 80%\u201390%. The offspring produced in this step was referred to as the first passage (P1) cells after 7\u201312\u00a0days. Then, 1\u2009\u00d7\u2009106 cells were inoculated for 4 days in the culture medium (second passage) [DMEM was placed ten times the cell sedimentation volume in a centrifuge tube. The cells were then inoculated at a density of 30%\u201350% into a culture flask, followed by adding a complete medium to a final volume of 10\u00a0ml; the flasks were placed in an incubator at 37\u00a0\u00b0C with 5% COpassage) .Following the second passage, stem cells were harvested. Cells were treated with a 10% trypsin EDTA solution for 5\u201310\u00a0min in the incubator, followed by a wash. The cell pellet was then incubated for one hour with 1% bovine serum albumin containing primary antibodies against the following cell surface markers: CD 34, CD73, CD45, CD44, and CD105. The cells were then incubated for 30\u00a0min with the secondary antibody before immunophenotyping using a fluorescence-activated cell sorting cell analyzer .7 cells per plate. Then, they were differentiated into chondrogenic and adipogenic lineages to demonstrate the isolated cell\u2019s mesenchymal phenotypes.After the third passage, the cells were trypsinized and placed at 10The adipogenic medium containing 100 nM dexamethasone, 50 mg/ml indomethacin, and 100 ML ascorbic acid, was added to each well and changed every 3 days to induce adipogenesis. After 21\u00a0d, the culture medium was removed, and the cells were fixed with 4% formalin at room temperature for one hour before being stained with Oil Red O solution in 10% isopropanol for 15\u00a0min. The adipose droplets were visualized using a light microscope .To induce chondrogenesis, the media that contained Dulbecco\u2019s modified Eagle\u2019s medium with 1% fetal bovine serum, 6.25 lg/ml insulin (Sigma), 10 ng/ml transforming growth factor-b1 (Sigma), and 6.25 lg/ml transferrin (Sigma) was added to each flask. The media was changed every 3 days. Chondrogenic differentiation was assessed via Safranin-O staining. .6 nucleated cells) was activated using a red laser diode (635\u00a0nm) for 10 minutes at 7\u00a0cm, then injected directly into the foramen magnum on day 14 post-induction of the MS as declared by [The stem cell preparation , and QT-PCR. The timeline of the study was provided  under general anesthesia. The technique for spinal imaging protocol was performed in T11 to L3  and included transverse T2-weighted (TR/TE 3290/99\u00a0ms) and T1-weighted (TR/TE 651/12\u00a0ms), sagittal STIR (TR/TE/TI 3310/ 61/140\u00a0ms) sequences sagittal, and dorsal T2-weighted (TR/TE 2880/111\u00a0ms) and T1-weighted (TR/TE 623/1\u00a0ms).Then, all animals were anaesthetized with xylazine 1\u00a0mg/Kg/IM  and ketamine 5% (Ketamar\u00ae 5% Sol. Amoun Co. A.R.E) I/M 10\u00a0mg/kg. After muscular relaxation, they were euthanized humanely by injecting sodium thiopental at 2.5%  in the lethal doses of 67\u00a0mg/Kg/IV,  and the For further morphological assessments, the fascia and back muscles were dissected until the vertebral column's spinous process and the vertebral body was reached. Then, the spinous process was carefully severed using a bone cutter, and the spinal cord was excluded outside the vertebrae.The spinal cord specimens were fixed in 10% Neutral Buffered Formalin for 24\u00a0h. The specimens were regularly processed for paraffin blocks, dehydrated in a graded sequence of ethyl alcohol, cleared in xylene, and embedded in paraffin. Sections of 4\u20135\u00a0\u03bcm thickness were obtained and stained by H&E  for ligh5-\u00b5m tissue sections were fixed into adhesive slides, rehydrated to water, and subjected to heat-induced epitope retrieval in a microwave for 15\u00a0min. After washing, tissue sections were incubated with primary antibodies  at a dilution of 1:150 for 12\u00a0h in a refrigerator. Furthermore, sections from the SVF co-treated group were incubated with primary antibody against CD44 at a dilution of 1:1000. Then additional washing steps. Later, HRP-labeled secondary antibodies  were added at a dilution of 1:1000 for two hours at room temperature. To develop the color, DAB-Substrate Kit was used. Control negative slides were performed by deleting the primary antibody. Positive immunostaining was quantified as area percentage  using cellSens dimensions (Olympus software).Small, 1-mm spinal cord specimens from all groups were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer for a few hours before being post-fixed in 1% osmium tetroxide for one hour. Then, 1-\u00b5m semi-thin sections were cut and stained with toluidine blue. Selective ultrathin sections were cut and stained with uranyl acetate and lead citrate. Finally, the obtained sections were analyzed via TEM et al.-Azhar University\u2019s Regional Center for Mycology and Biotechnology (RCMP) (JEOL 1010). Furthermore, the myelin sheath thickness was assessed in at least 30 myelinated axons/samples using the ImageJ program.\u2212 \u0394\u0394CT [Total RNA was extracted using the easy-spin Total RNA Extraction Kit  as directed by the manufacturer. A Nanodrop ND-1000 spectrophotometer was used to assess the quality and quantity of RNA (Nanodrop Technologies). The cDNA was created using M-MuLV Reverse Transcriptase (NEB#M0253) as per the provided protocol. Real-time reverse transcription (RT)-PCR was used to examine the target gene expression, and mRNA levels were determined using qRT-PCR with the HERAPLUS SYBR Green qPCR kit (#: WF10308002). The primer sets are presented in Table \u2212 \u0394\u0394CT .Table 1PP\u2009\u2264\u20090.05. The statistical software package Origin Pro, version 2016, was used to calculate Pearson\u2019s correlation coefficient. The gene expression analysis, immunohistochemistry, and transmission electron microscopy (TEM) results were analyzed using GraphPad Prism version 8.4.3 (686) in a one-way ANOVA, and the values are presented as mean\u2009\u00b1\u2009SEM (n\u2009=\u20095 cats/group). Different superscript letters indicate a significant difference at P\u2009\u2264\u20090.05.A two-way ANOVA was applied to the data from completely random samples to examine the gait scores. The Fisher\u2019s post hoc test was used to compare the effects of the various treatments. The significant differences between the groups and experiment days were indicated in the columns by letters. Values are presented as mean\u2009\u00b1\u2009SEM (n\u2009=\u20095 cats/group). Different superscript letters indicate a significant difference at In the control negative group, the spinal cord segments T11\u2013L3) measured 6\u20136.5\u00a0cm. The cord appeared like a whitish-long cylindrical tube enclosed by the dura mater with no gross lesion. However, in the EB-treated group, the spinal cord was defined by the existence of a noticeable reddish-brown hemorrhagic sizable region (2\u20133\u00a0cm). The spinal cord of the SVF co-treated group seemed to be a whitish tube with a small localized brown lesion (<\u20091\u00a0cm)  and very low expression for CD34 and CD45, indicating the criteria of ADMSCS  decrease in the mean myelin thickness of spinal cord nerve fibers in the EB treatment group compared with the control group. Conversely, the SVF co-treated group revealed a substantial  increase in the mean myelin thickness compared with the EB treatment group  difference in the myelin thickness between the control and SVF co-treated groups.As presented in Fig.\u00a0oup Fig.\u00a0. FurtherP value\u2009<\u20090.05) decreased levels of NGF, SDF, and BDNF. The SVF co-treated group significantly  increased the transcript levels of the studied genes. These results confirmed the protective role of SVF against EB-induced damage  was used as an internal control. The EB-treated group showed significantly  . FurtherNGF is vital for the neurons during the differentiation of the nervous system. Significant increases in NGF synthesis in inflammatory tissues in patients with an inflammatory disease have been reported . ChangesBased on our results, SVF can improve the MS cat model through different mechanisms. Firstly, the ability of MSCs to increase migration of oligodendrocyte progenitors and differentiation into oligodendrocytes that were evidenced by a strong positive immunoreactivity against CD44 antibodies in the SVF co-treated sections. CD44 is a cell surface receptor that plays a key role in mediating cell migration and consider one of MSCs important markers.  recordedOur study demonstrated that the transplantation of SVF intrathecally could alleviate the adverse effects of ethidium bromide-induced demyelination in a cat model of MS. The therapeutic effects of SVF improve the motor function of the hindlimb, ameliorate the lesion severity, alleviate the induced histopathological alterations, enhance the remyelination capacity, significantly increase the gene expression of neurotrophic factors and the immunoreactivity of olig2 and MBP, and reduce Bax, and GFPA immunoreactivity. To date, this is the first investigation elucidating the global effects of SVF treatment in the cat MS model.The fast and excellent response, besides easily application of SVF injections in acute cases, gave attention to becoming a routine application in spinal cord injuries like accidents, MS, etc.Further studies are required to evaluate the long-term and multiple dose effects of SVF in the treatment of spinal cord injuries.Additional file 1. Supplementary figures."}
+{"text": "The crystallinity and the growth rate of crystalline structures of polyethylene glycol and polyethylene blocks in polyethylene-b-polyethylene glycol diblock copolymers (PE-b-PEG) were evaluated and compared to polyethylene and polyethylene glycol homopolymers. Melting and crystallization behaviours of PE-b-PEG copolymers with different molecular weights and compositions are investigated by differential scanning calorimetry (DSC). The polyethylene/polyethylene glycol block ratio of the copolymers varies from 17/83 to 77/23 (weight/weight). The influence of the composition of PE-b-PEG copolymer on the ability of each block to crystallize has been determined. Thermal transition data are correlated with optical polarized microscopy, used to investigate the morphology and growth rate of crystals. The results show that the crystallization of the polyethylene block is closer to the polyethylene homopolymer when the copolymer contains more than 50 wt. % of polyethylene in the copolymer. For PE-b-PEG copolymers containing more than 50 wt. % of polyethylene glycol, the polyethylene glycol block morphology is almost similar to the PEG homopolymer. An important hindrance of each block on the crystallization growth rate of the other block has been revealed. Amphiphilic diblock copolymers are macromolecules with a hydrophobic block and a hydrophilic block. These copolymers are able to self-organize in bulk or in solution. These specific properties have attracted a great deal of research interest in the past decade due to their potential in applications as industrial surfactants . The majCrystallization of diblock copolymers composed of one amorphous block and one semi-crystalline block has been studied in the literature ,7 as PEOCommonly used as surfactants, polyethylene-block-polyethylene glycol copolymers (PE-b-PEG) are amphiphilic, and both blocks, hydrophilic PEG and hydrophobic PE, might be expected to crystallize into two distinctive crystalline phases.The objective of this work is to study the effect of the block ratio on the PE-b-PEG crystallinity. The effects of the molecular weight and the weight ratio of the two polymer blocks on the PE-b-PEG crystallinity will be evidenced by the characterization of various PE-b-PEG copolymers of different compositions. The thermal properties and specifically the crystallinity degree of each block will be characterized by differential scanning calorimetry. The morphology and the growth rate of the crystalline phase of each block are investigated by optical microscopy under polarized light in combination with a hot-stage. In order to evidence the influence of the blocks ratio, the results obtained for PE-b-PEG copolymers are compared with the thermal and morphological properties of the PE and PEG homopolymers.Different PE-b-PEG copolymers have been used. The general formula of these copolymers is given in nM = 2050 g\u00b7mol\u22121), and polyethylene (PE) conditioned as powder . These PE and PEG homopolymers have low molecular weights, closed to the molecular weights of PE blocks and PEG blocks in the studied copolymers. However, molecular weights of PE and PEG blocks of the copolymers are smaller than those of the corresponding homopolymers. Shorter chains lengths are not provided by the supplier, homopolymers with the closest chains lengths have been thus chosen for the study. The following results obtained can be influenced by the non-equal chain lengths between copolymer blocks and the homopolymers, but the main interest of this study is to compare diblock copolymers. The results will be consequently discussed by referring to the properties of PE and PEG blocks of close molecular weight to the blocks of the copolymer existing in the literature.PE-b-PEG copolymers of various molecular weights were supplied by Sigma Aldrich . PE-b-PEG copolymers are conditioned as pellets. Two homopolymers were also studied: polyethylene glycol (PEG) conditioned as flakes . The average values of these data are presented in The ethylene/ethylene oxide ratio and the number average molecular weight of the copolymers indicated by the supplier have been verified by proton nuclear magnetic resonance  was used. Samples of ca. 10 mg were placed in sealed aluminum pans; an empty aluminum pan was used as a reference. Two temperature ramps were applied in order to cancel the thermal history. A heating rate of 10 \u00b0C/min and a cooling rate of 10 \u00b0C/min were applied between \u221280 \u00b0C and 160 \u00b0C.The morphology of PE, PEG and PE-b-PEG copolymers was observed by the use of an Olympus BX51  optical microscope equipped with polarized light. The polarized light is particularly interesting to differentiate the crystalline and the amorphous phases. Magnifications of \u00d7500 and \u00d7100 were used. The microscope is also equipped with a CCD camera connected to a computer in order to record each step of the melting and the crystallization. The temperature of the sample was controlled by the use of a Linkam LTS350 hot-stage coupled to the microscope.In order to be observed by optical microscopy in transmission mode, thick films of PE, PEG and PE-b-PEG were obtained by casting a polymer layer between two glass slides. A glass/pellet/glass sandwich is placed in oven and heated up to a molten state at 120 \u00b0C, and kept at this temperature during 15 min. This temperature is above the melting point of PE and PEG, ensuring a complete flowing of both blocks. After 15 min in the oven, the glass slides were separated in such a way that about 200 \u00b5m of melted polymer was deposited on each of them. Both glass slides were cooled at ambient temperature.The melting of polymers was recorded during a heating ramp from the ambient temperature to 120 \u00b0C at a rate of 10 \u00b0C/min. The crystallization of polymers has been recorded during a cooling ramp running from 120 \u00b0C to the ambient temperature (20 \u00b0C) at a rate of 2 \u00b0C/min.The spherulites and lamellas sizes were directly measured on the images recorded by the CCD camera. The initial and final crystallization temperatures were determined from the video recording the crystallization. The growth rate of these crystalline structures was obtained from the evolution of the size as a function of time, directly determined from the videos recorded by the CCD camera.m max (maximum of the endothermic peak) is equal to 52 \u00b0C. The crystallization occurs during the cooling ramp between 39 \u00b0C and 24 \u00b0C. The maximal crystallization temperature Tc max (maximum of the exothermic peak) is equal to 33 \u00b0C.cX was calculated using the formula below, where \u0394mH0 is the melting enthalpy of 100% crystalline PEG .The degree of crystallinity 96.8 J/g ,15), andnM = 8000 g\u00b7mol\u22121) [nM = 3400 g\u00b7mol\u22121) [nM = 1000 g\u00b7mol\u22121 to nM = 20,000 g\u00b7mol\u22121, the degree of crystallinity varies from 85% to 99% [Very close heat of fusion of 191 J/g (g\u00b7mol\u22121)  and 183 g\u00b7mol\u22121)  have bee% to 99% ,16,17. TThe morphology of bulk PEG was observed by optical microscopy under polarized light at ambient temperature. PEG film is obtained by deposition at 120 \u00b0C (melted state) on a glass slide and is cooled rapidly at ambient air see . Large sm initial and the final melting temperature Tm final correspond to the first disappearance of spherulites and to the complete disappearance of spherulites, respectively. The initial crystallization temperature Tc initial and the final crystallization temperature Tc final correspond to the first appearance of spherulites and to the complete covering of the glass slide with PEG spherulites, respectively.Melting and crystallization temperatures of PEG have been also determined by optical microscopy thanks to the coupled CCD camera recording the spherulites melting and crystallization during the heating and the cooling ramps, respectively see . The iniThe melting and crystallization temperatures of PEG observed by optical microscopy are very close to those observed by DSC see . Some diThe characteristic temperatures observed by DSC were not influenced by the different rates applied. An explanation can be given to the more important difference between crystallization temperatures. As the heating stage is cooled by ambient air during crystallization, the cooling step at 2 \u00b0C/min induces a less precise control of the temperature below 35 \u00b0C. Moreover, if primary crystallization is easily visible on optical images, the secondary crystallization is less visible, and complete covering of the image by spherulites does not mean that secondary crystallization is ended. The secondary crystallization is particularly visible in The melting temperatures observed by DSC and optical microscopy are different even if the heating rates applied are the same for both techniques. Using optical microscopy, the area of interest is a circle of a few millimeters in diameter. The melting event might have thus begun for lower melting temperatures elsewhere. From The evolution of the spherulites size and growth rate as a function of the crystallization temperature is presented in \u22121. The value of 17 \u00b5m/s is very slow compared to 0.14 cm/s, but might be explained by the different cooling ramp used in the quoted reference, and also by the different conditioning of PEG 2,000,000 g\u00b7mol\u22121 as powder.The average growth rate of spherulites in PEG homopolymer is equal to 17 \u00b5m/s. This represents a reference to reveal the influence of the PE block in PE-b-PEG diblock copolymer. It is also important to point out that the growth rate decreases as the crystallization temperature is increased. The spherulitic morphology of crystallized PEG has been observed in many studies ,16,17, rBulk thermal properties of PE homopolymer were also investigated. cX of PE homopolymer was calculated using \u0394mH0, the melting enthalpy of 100% crystalline PE . The degree of crystallinity of this PE is equal to 44%.Melting and crystallization peaks are very wide, and the limits for peaks integration are therefore difficult to define. The degree of crystallinity (285 J/g ) and \u0394HmcX = 30%) and 222 J/g (cX = 77%), respectively [nM = 1700 g\u00b7mol\u22121) and the crystallinity degree (44%) related to the melting enthalpy (126 J\u00b7g\u22121), the experimental PE homopolymer studied belongs to the low density polymers, which is useful in the comprehension of the PE block in the PE-b-PEG copolymer.The very wide melting peak indicates that the crystalline organisation is less perfect compared with PE with a higher molecular weight. Peacock  and Mandectively . ConsideThe morphology of polyethylene was observed by optical microscopy under polarized light at ambient temperature after being deposited at the melting state (120 \u00b0C) on a glass slide and rapidly cooled at ambient temperature . SpherulThe melting of PE was recorded between 30 \u00b0C and 110 \u00b0C by optical microscopy equipped with a CCD camera and coupled with a hot-stage applying a heating ramp from the ambient temperature to 120 \u00b0C at 10 \u00b0C/min. Between 86 \u00b0C and 89 \u00b0C see , one canMelting and crystallization temperatures of bulk PE were determined by optical microscopy during a cooling and a heating ramp applied to a hot-stage. These temperatures were compared to the characteristic temperatures obtained by DSC and gathered in c final. When PE crystallizes, spherulite growth until 50 \u00b0C is observed. Below 50 \u00b0C, any changes were seen as secondary crystallization and were less observable by optical microscopy.The melting and the crystallization temperatures of pure PE determined by optical microscopy are very close to the one observed on the DSC thermogram, except for TThe evolution of the PE spherulite radii and the spherulites growth rates as a function of the PE crystallization temperature are presented in The evolution of the PE spherulite growth rate as a function of the crystallization temperature is also presented in nM = 3600 g\u00b7mol\u22121 [Below 95 \u00b0C, spherulites are growing in a different plane from the one focused by the microscope and that growth cannot be recorded. The crystallization temperatures are very close to LDPE spherulitic crystallization observed between 117 \u00b0C  and 125  g\u00b7mol\u22121 . As the Two different morphologies of polyethylene crystalline structures are described in the literature: lamellas  or spherPE lamellas crystallizing at 2 \u00b5m/s were observed by Abo el Maaty . PE spheBoth PE and PEG homopolymers are semi-crystalline, and PE and PEG blocks are consequently able to crystallize in PE-b-PEG copolymers. PEG homopolymer presents a melting temperature lower than PE homopolymer and a very fine peak representative of a high crystalline organization. The aim of this part is to evidence the influence of each block on the crystallinity, the morphology and the growth rate of the crystalline structures of PE-b-PEG diblock copolymers.m max = 74 \u00b0C) to a lower melting temperature, indicating that the PE crystals are not as well organized as in the PE homopolymer. The DSC curves obtained for PE-b-PEG A show a distribution of less organized crystalline structures with different melting and crystallization temperatures compared to the DSC curves of the PE homopolymer, relevant to the influence of the PEG blocks on the PE blocks crystallization. Moreover, no single contribution at 52 \u00b0C or lower temperatures, characteristic of the PEG homopolymer melting, is observed in When the PE block is in the majority in the diblock, as is the case for PE-b-PEG A containing 77 wt. % PE, one very wide endothermic peak attributable to the PE crystalline phase melting is observed a. The meThe PE-b-PEG C copolymer containing a majority of PEG block in its composition was also analyzed by DSC in order to reveal the influence of the block ratio on the thermal properties and on the crystallinity of PE-b-PEG copolymers c. The DSm max for PEG is equal to 36 \u00b0C and Tm max for PE is equal to 65 \u00b0C.In order to more deeply understand the block ratio influence on PE-b-PEG copolymer crystallinity, a PE-b-PEG B (55 wt. % PEG) with an intermediate block composition was investigated by DSC and is presented in The same tendency is observed for the crystallization temperatures. The melting peak of the PEG blocks presents a melting enthalpy of 32 J/g corresponding to a crystallinity degree of 16% for the PEG blocks. The melting peak of the PE blocks presents a melting enthalpy of 77 J/g corresponding to a crystallinity degree of 27% for the PE blocks.m, Tc, cX) of each block are modified by the presence of the other block, the study of the crystalline morphologies can help us to understand the crystallinity behaviours of PE-b-PEG copolymers.An important effect of one block on the ability of the other block to crystallize is evidenced. When a block is not strongly preponderant in a PE-b-PEG copolymer, each block is able to crystallize. Since the crystalline characteristics  is low compared to the one observed by DSC (52 \u00b0C). As the PEG crystalline structures are less organized due to the PE block influence, they melt at lower temperatures and are thus more difficult to detect by optical microscopy.DSC curves of PE-b-PEG C c showed The morphology of PE-b-PEG A containing 77 wt. % PE was observed by optical microscopy under cross polarization light. In order to obtain information about the crystalline structures of a copolymer containing a high proportion of PE block, a heating and a cooling ramp have been applied successively to the copolymer while a camera records.During the cooling ramp, PE spherulite growth is observed by optical microscopy from 95 \u00b0C to 70 \u00b0C. Some pictures of PE spherulite growth during the crystallization ramp of PE-b-PEG A are presented in The PE block crystallization temperature window is narrow for PE-b-PEG A relative to PE-b-PEG C due to the melted PEG blocks hindering the crystallization of the PE blocks in PE-b-PEG C. The growth rate decreases as the crystallization temperature of the diblock decreases, as observed for PE-b-PEG C. No other crystallization event has been observed during the cooling of PE-b-PEG A. The PEG block crystallization was thus impossible to visualize by optical microscopy. As PEG blocks are in a reduced proportion (23 wt. % PEG), they either do not crystallize, or crystallize through the dense network developed by PE lamellas.The melting and the crystallization temperatures of each block in PE-b-PEG A have been determined by optical microscopy and compared to the temperatures obtained by DSC in c final is observed at 70 \u00b0C by microscopy, but the crystallization process should go on until 1 \u00b0C according to the DSC data. The same gap is observed for the initial melting temperature, as the secondary crystallization is not as visible as the primary one is by optical microscopy.TIn order to better understand the crystalline morphology of each block depending on the composition of the copolymer, PE-b-PEG B has been investigated by optical microscopy under polarized light. This copolymer has an intermediate composition relative to PE-b-PEG A and PE-b-PEG C. PE-b-PEG B presents a single melting peak and a single crystallization peak for each block.A heating and a cooling ramp have been applied successively to the copolymer, while a camera records these processes. During the cooling ramp, PE lamellas grow between 98 \u00b0C and 70 \u00b0C see . The PE PE lamellas reach an average length of 10 \u00b5m at their maximum in PE-b-PEG B, which is close to the average length of the PE lamellas in PE-b-PEG C containing a high proportion of PEG block. This means that over 50 wt. % of PEG, the PE blocks are not able to crystallize as spherulites, due to the PEG block hindrance. The growth rate of PE lamellas is very slow, lower than 0.02 \u00b5m/s compared to the growth rate of PE spherulites in PE-b-PEG A (0.04 \u00b5m/s) containing a high proportion of PE blocks. The mobility of PE lamellas is reduced due to the influence PEG blocks covalently linked to the PE blocks, and also due to the viscosity of the melted PEG blocks during the PE crystallization, revealing an important hindrance of the PEG blocks on the PE lamellas growth.The melting and the crystallization temperatures of each block in PE-b-PEG B have been determined by optical microscopy coupled to a hot-stage and compared to the temperatures obtained by DSC in nM = 2041 g\u00b7mol\u22121. The high crystallinity of PEG homopolymer was also observed for PEG with nM = 1000 g\u00b7mol\u22121 and nM = 2000 g\u00b7mol\u22121, presenting a crystallinity degree of 92.7% and 94% [nM= 20,000 g\u00b7mol\u22121 corresponding to a crystallinity degree of 99.2% [The effect of PE-b-PEG composition on the crystalline phase and the morphology of each block was observed by DSC and optical microscopy coupled to a hot-stage. The PEG homopolymer crystallinity was investigated and was compared with the PEG block crystallinity in PE-b-PEG diblock copolymer. The crystallinity degree of PEG homopolymer is equal to 95% for a molecular mass of  and 94% , respect and 94% , as descof 99.2% . These dnM = 1474 g\u00b7mol\u22121) is very crystalline, with a crystallinity degree equal to 82%, as described in the literature [In PE-b-PEG C containing 83 wt. % of PEG, the PEG block  increasing with the copolymer molecular weight. The PE homopolymer crystallinity was investigated and was also compared to the PE block crystallinity in the PE-b-PEG diblock copolymer, as presented in nM = 1630 g\u00b7mol\u22121. The crystallinity degree of PE with nM = 1700 g\u00b7mol\u22121 has been determined to be 68% in the literature [\u22121  of PEG, TThe PE crystallinity decreases when the PEG block ratio increases in the copolymers, and becomes negligible when the PE block ratio is equal to 17%, as in PE-b-PEG C. The PE block crystallinity decreases then as the content of PE decreases in copolymers, whereas the PE block length remains constant. A similar PE block length does not crystallize in the same way, as a function of the PEG block length. The longer the PEG block, the more numerous the PEG/PEG interactions , and the PE/PE interactions are consequently reduced, leading to a decrease of the PE blocks crystallinity. The strong hindrance of the PEG block length on the PE crystallization is then evidenced by these results.The previous explanations related to influence of PEG block length on PE crystallinity are also supported by the study of the morphology and the growth rate of the crystalline structures of each block, investigated by optical microscopy coupled to a hot-stage.The PEG block crystallization was not observable for PE-b-PEG with a PE block proportion higher than 20 wt. %, such as PE-b-PEG A and PE-b-PEG B. On the other hand, the PE block crystallization was observed by optical microscopy for all PE-b-PEG copolymers. For high PEG content in the copolymer, as was the case for PE-b-PEG C, PEG blocks crystallize freely in large spherulites of average diameter equal to 200 \u00b5m as observed for the PEG homopolymer. It is important to notice that the growth rate of these spherulites is more than four times slower in the diblock containing 17 wt. % of PE than in the PEG homopolymer. The PE block induces then a hindrance on the PEG crystallization due to the PE crystalline phase already solidified at the PEG crystallization temperatures. Moreover, the PE lamellas are smaller in PE-b-PEG C than the PE spherulites observed for PE-b-PEG A, and growth is also slower.When the PE ratio is majority in the copolymer, as is the case for PE-b-PEG A, PE blocks crystallize in very small spherulites of 16 \u00b5m radius, growing at 0.04 \u00b5m/s. It is important to notice that the growth rate of these spherulites is five times slower in the diblock containing 17 wt. % of PE than in the PE homopolymer. This rate is much slower than for the PE homopolymer due to the melted PEG sequences hindering the growth of PE crystalline structures. If the PEG ratio becomes more important, as is the case for PE-b-PEG B and PE-b-PEG C, PE is not even able to crystallize as spherulites anymore, but rather in lamellas.The strong competition between PE and PEG block organization greatly influences the crystallinity of PE-b-PEG copolymers, as shown with the study of a copolymer with an intermediate composition. Indeed, PE-b-PEG B contains 55 wt. % of PEG, and the molecular weights of each block are quite equivalent. DSC curves of this copolymer b showed Using DSC and optical microscopy coupled to a hot-stage, the bulk crystallinity of the PE-b-PEG diblock copolymers was investigated. According to the results presented in this paper, the effect of the blocks ratio and length was significant on the morphology and the growth rate of the crystalline structures. The crystallization of PE varies from spherulites to lamellas, depending on the proportion and the block length of PEG. When the ratio of one block is increased, the crystallization of that block is favored, while the crystallization of the other block is hindered, and could even be totally prevented.The optical microscopy under cross polarization and coupled to a hot-stage appeared to be an appropriate and complementary tool to the DSC in order to get information about thermal transitions. These results, relative to the bulk crystallinity of PE-b-PEG diblock copolymers, could be compared, in a further study, to the organization of the same copolymers adsorbed, as a thin layer, on a solid substrate."}
+{"text": "Glucose is the main fuel cell used for energy production via a series of enzymatic reactions in the presence of oxygen in a process known as aerobic respiration. The main steps in this process are glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS). Cancer cells rely mostly on glycolysis and less on OXPHOS for rapid production of energy and intermediate macromolecules that are required to sustain their increased proliferation rate. This metabolic reprogramming is considered one of the hallmarks of cancer and has been linked to tumor growth and progression, as well as to the development of therapy resistance. Cancer stem cells (CSCs) are a subset of tumor cells with self-renewal and differentiation capacities and have gained much attention due to their involvement in cancer initiation and resistance to conventional therapies. In contrast to the bulk of tumor cells, CSCs can switch between glycolysis and OXPHOS depending on stimuli from their microenvironment. This metabolic plasticity allows them to adapt and survive under various stressful conditions, maintaining, at the same time, their stemness properties, and, thus, contributing to the development of therapy resistance and tumor recurrence. Consequently, understanding the specific features of CSC metabolism is crucial for the successful elimination of these cells. In this review, we provide a concise description of the metabolic signatures of CSCs, emphasizing their metabolic plasticity and its involvement in drug resistance; we also draw attention to the potential of targeting CSC metabolism as a complementary therapeutic approach in cancer.Cancer stem cells (CSCs), a subpopulation of tumor cells with self-renewal capacity, have been associated with tumor initiation, progression, and therapy resistance. While the bulk of tumor cells mainly use glycolysis for energy production, CSCs have gained attention for their ability to switch between glycolysis and oxidative phosphorylation, depending on their energy needs and stimuli from their microenvironment. This metabolic plasticity is mediated by signaling pathways that are also implicated in the regulation of CSC properties, such as the Wnt/\u03b2-catenin, Notch, and Hippo networks. Two other stemness-associated processes, autophagy and hypoxia, seem to play a role in the metabolic switching of CSCs as well. Importantly, accumulating evidence has linked the metabolic plasticity of CSCs to their increased resistance to treatment. In this review, we summarize the metabolic signatures of CSCs and the pathways that regulate them; we especially highlight research data that demonstrate the metabolic adaptability of these cells and their role in stemness and therapy resistance. As the development of drug resistance is a major challenge for successful cancer treatment, the potential of specific elimination of CSCs through targeting their metabolism is of great interest and it is particularly examined.  Cancer is the second leading cause of death globally, accounting for an estimated 9.6 million deaths in 2018 according to the World Health Organization (WHO) . There aThe clonal evolution model maintains that all tumor cells are initially biologically equivalent. The accumulation of genetic and epigenetic alterations in some tumor cells may result in functionally and phenotypically distinct clones with different degrees of aggressiveness, invasiveness and/or therapy resistance . The secThe term \u201cmetabolism\u201d encompasses a large group of intracellular, complex chemical reactions that use nutrients for energy production and macromolecule synthesis and are indispensable for all cellular functions. Healthy and cancer cells share mostly common metabolic pathways ; howeverNotably, intra-tumoral heterogeneity is also manifested on the metabolic level, with subpopulations of tumor cells exhibiting distinct metabolic characteristics . ContradIn this review, we aim to provide a concise description of the distinctive metabolic traits of CSCs and the pathways that regulate them, as well as discuss how these are implicated in promoting stemness. We particularly highlight studies that demonstrate the metabolic plasticity of CSCs and its role in drug resistance, and we present research data that underline the promise of targeting CSC metabolism as a complementary therapeutic approach to alleviate the burden of cancer.Cells break down glucose to produce ATP for their energy needs through a series of chemical reactions that are collectively known as cellular respiration. The first step is glycolysis, and it takes place in the cytoplasm, where glucose is converted to pyruvate A. 2 and the release of three molecules of NADH, one FADH2, and one ATP (or GTP) (2 produced during the previous step are now utilized for electron transfer in a series of oxidation\u2013reduction reactions that ultimately lead to the generation of 36 ATPs/glucose molecules (The net energy production of glycolysis is two molecules of ATP and two of reduced nicotinamide adenine dinucleotide (NADH) per glucose molecule. In the presence of oxygen, the pyruvate is transferred to the mitochondria, where it is converted to acetyl-CoA, which, in turn, enters the tricarboxylic acid (TCA) cycle, a chain of chemical reactions that leads to its oxidation to CO(or GTP) B. The TColecules . Under holecules A. Cancer cells prefer to convert glucose to lactate, irrespectively of the presence of oxygen, a phenomenon first described by Warburg and known as aerobic glycolysis . NotablyWhereas the bulk of tumor cells mainly use aerobic glycolysis for glucose metabolism, CSCs can exhibit metabolic flexibility and switch between glycolysis and OXPHOS B, dependSeveral studies have shown that CSCs derived from various tumor types are even more glycolytic than non-CSCs, as they express higher levels of glycolysis-associated genes and lower levels of genes involved in OXPHOS ,23,24,25+ breast CSCs purified from MDA-MB-231 and MCF-7 mammospheres expressed higher levels of the glycolytic gene PDK1 and reduced levels of pyruvate dehydrogenase (PDH) compared to non-CSCs [PDK1 in MDA-MB-231 cells impaired the stemness properties of CSCs, leading to a decline in the ALDH+-subpopulation and reducing mammosphere forming efficiency (MFE) and downregulation in the expression of stemness genes [Indeed, ALDHnon-CSCs . Knock-dss genes . A diffess genes . Specifiss genes . These oss genes . The usess genes . + CSCs, isolated from the PLC/PRF/5 cell line, exhibited upregulation of the glycolytic genes GLUT1, HK2, PDK4, and PGM1 (phosphoglucomutase 1) and downregulation of the gluconeogenic genes G6Pase (glucose-6 phospatase) and PEPCK (phosphoenolpyruvate carboxykinase), leading to decreased cellular ATP levels compared to CD133\u2212 non-CSCs [+ CSCs, further supporting the idea that enhanced glycolysis plays a significant role in hepatic CSC maintenance [In hepatocellular carcinoma (HCC), the CD133non-CSCs . Glycolyntenance .ENO1 enhanced the glycolytic capacity, as well as the stemness properties, of the gastric CSCs. Glycolysis inhibition using 2-DG led to impairment of the self-renewal, migratory, and invasive capacities of these cells, further highlighting the link between stemness and glycolysis. Notably, high ENO1 expression was also associated with poor patient prognosis [Similar results were also obtained with cultures of PAMC-82 and SNU16 spheroids enriched in gastric CSCs, which showed high levels of the glycolytic enzyme enolase 1 (ENO1) . Overexprognosis .In accordance with the above observations, side-population cells with CS-like characteristics isolated from the non-small cell lung cancer (NSCLC) A549 cell line by flow cytometry also bore a hyperglycolytic profile, as indicated by the higher glucose uptake and lactate production, as well as by the higher expression of glycolytic enzymes (including PDK-1 and HK-1) compared to differentiated cancer cells . FurtherOverall, the above-described studies suggest that the high glycolytic activity in CSCs is interlinked with their stemness properties. Thus, targeting glycolysis could be a promising therapeutic approach to eliminate this aggressive cancer sub-population. An increased rate of glycolysis is not always the rule in CSCs, since a number of other studies have demonstrated a preference of these cells towards OXPHOS for energy production to sustain their survival ,31,32,33A prime example of CSCs that favor OXPHOS to meet their energy demands are the glioma stem cells (GSCs). GSCs isolated from neurospheres generated from the U87, GBM-146, and GBM-176 cell lines appeared less glycolytic than the differentiated glioma cells, as they consumed less glucose and produced more lactate, while they relied more on OXPHOS to yield higher ATP levels . + leukemic stem cells (LSCs) derived from patients with chronic myeloid leukemia, metabolic analysis revealed increased levels of OXPHOS compared to CD34\u2212 cells, while inhibition of this process resulted in their selective eradication in vitro [MYC, which in turn controls the transcription of the amino-acid transporter SLC1A5 that is also implicated in the regulation of glutaminolysis. Inhibition of any of the above proteins in LSCs led to reduced TCA cycle activity and inhibition of OXPHOS, establishing the STAT3-MYC-SLC1A5 axis as a regulator of energy metabolism in these cells. The authors also showed a potential therapeutic application of their data by using a small molecule STAT3 inhibitor, which led to the selective death of stem and progenitor cells isolated from AML patients, while sparing normal hematopoietic cells [Similarly, in CD34in vitro . In a diin vitro . STAT3 iic cells .+/CD44+ liver CSCs (LCSCs), derived from the HCCLM3 HCC cell line, and their differentiated counterparts, with the former exhibiting more robust levels of OXPHOS [PDHC, indicating the involvement of OXPHOS in the stemness potential of the cells. Inhibition of OXPHOS by the inhibitor of mitochondrial division Mdivi-1 led to downregulation of stemness genes and of CD133 and CD44, further supporting the role of this process in the maintenance of LCSCs [Metabolic heterogeneity has also been observed between CD133f OXPHOS . Indeed,f OXPHOS . This prof LCSCs . +/CD117+ CSCs, the overexpression of the OXPHOS genes PDHK1 and PDH and the higher mitochondrial activity suggested a preference for pyruvate fueling the TCA cycle and for OXPHOS over glycolysis [+/CD117+ CSCs without affecting the viability of CD44+/CD117\u2212 cells [Likewise, in patient-derived ovarian CD44ycolysis . OXPHOS 7\u2212 cells . KRAS, a well-known oncogene in pancreatic ductal adenocarcinoma (PDAC), in cancer maintenance [Even though the targeting of oncogene-driven signaling pathways represents a clinically validated therapeutic approach, a fraction of surviving cells leads to tumor relapse. Based on this observation, in an interesting study by Viale et al., the authors tried to illuminate the role of ntenance . They shntenance . Inhibitntenance . In small cell lung cancer, CSCs were isolated from the H446 cell line by sorting the urokinase-type plasminogen activator receptor (uPAR) positive cells, since this receptor is associated with CSC function . These cIn conclusion, even though CSCs lie in hypoxic niches, they may still prefer OXPHOS to glycolysis in some cases. This paradoxical phenomenon may be attributed to two reasons: a) the metabolic symbiosis of non-CSCs with CSCs could result in the use of the lactate produced via glycolysis by the former for the OXPHOS of the latter, leading to an impressively efficient way of fuel utilization; b) contrary to the bulk of tumor cells, CSCs are generally maintained in a quiescent state with low proliferative activity and, therefore, do not need glycolysis intermediates for macromolecule biosynthesis . As it was mentioned before, several studies have demonstrated that CSCs have the ability to alter their metabolic phenotype as a response to signals from the stromal niche or to external stressors, such as drug exposure .+ CSCs. Along the same lines, both spheres and CD133+ CSCs exhibited increased mitochondrial mass, but lower glucose uptake, lactate production, and ROS levels [MYC overexpression. MYC was downregulated in the drug-sensitive CSCs, and it was moderately expressed in the resistant PaCSCs, where it negatively regulated the peroxisome proliferator-activated receptor-gamma coactivator 1A (PGC1A), which is essential for mitochondrial metabolism [MYC expression could restore the resistance to metformin by enforcing PaCSC dependance towards OXPHOS through PCG1A upregulation. Thus, metabolic heterogeneity of PaCSCs should be considered for the design of efficient therapies against them [Metabolic heterogeneity seems to be a feature of pancreatic CSCs (PaCSCs) . In a veS levels . InteresS levels . HoweverS levels . Metformtabolism . Inhibitnst them .+ CSCs) and exhibited increased OXPHOS; the second type was characterized by a quiescent, invasive mesenchymal-like state (M) that highly expressed CD44 (CD44+ CSCs) and was more glycolytic [In another interesting study, Luo et al. isolated two types of breast CSCs from the SUM149, HCC1806, MCF-7, and T47D cell lines that carried two distinct metabolic profiles . The firycolytic . A hypoxycolytic . Metabolic heterogeneity in CSCs has been an ongoing field of intense research, as the lack of a common pattern makes their metabolic characterization a challenging matter. The above results highlight the fact that elucidation of the metabolic signatures of all CSC subpopulations in a tumor is mandatory for their effective eradication; targeting both OXPHOS and glycolysis may constitute a better therapeutic strategy against them . Glycolysis is connected with the pentose phosphate pathway (PPP) that uses glycolysis intermediates for macromolecule biosynthesis to support cancer cell proliferation. The catalytic action of HK2 results in the phosphorylation of glucose with the product, glucose-6-P, entering the two phases of PPP in the cytosol. The first phase is oxidative and results in the conversion of glycose-6-P into ribulose-5-phosphate (Ru-5-P) and the production of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is essential for the maintenance of a redox balance under stress, as it is implicated in ROS elimination. Cancer cells use this as an antioxidant mechanism; in response to high ROS levels, they can enhance glycolysis and promote the PPP to generate more NADPH. The second phase of PPP is non-oxidative and results in the conversion of Ru-5-P either into ribose-5-phosphate, which is essential for nucleic acid synthesis, or into xylulose-5-phosphate that generates the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate, which are precursors of amino acid synthesis . KRAS mutant colorectal carcinomas in mice led to the enrichment of CUB-domain-containing protein 1 positive (CDCP1+) cells [+ CSCs showed increased oxidative PPP metabolite levels and de novo purine biosynthesis mediated by the inactivation of the glycolytic enzyme triosephosphate isomerase. The metabolic rerouting towards PPP protected CDCP1+ CSCs from the oxidative stress induced by chemotherapy, while targeting the oxidative phase of PPP resulted in increased chemosensitivity of the cells [CSCs exhibit increased glucose influx into the PPP, which serves to meet their high anabolic demands, to regulate oxidative stress and in the development of chemoresistance. Treatment with 5-fuorouracil (5-FU) or oxaliplatin of +) cells . CPCP1 i+) cells . The CDChe cells .+  and ER\u2212  cell lines [In an in vitro model of breast cancer, histone deacetylase (HDAC) inhibitors could reprogram non-breast CSCs into stem-like cells by promoting PPP metabolism . Specifill lines , suggestThe aforementioned studies draw attention to another metabolic route that may be enhanced in CSCs, shedding more light on the metabolic heterogeneity of these tumor cells. Glutamine (Gln) is a nonessential amino acid, as it is endogenously synthesized, and can either fuel the TCA cycle leading to other amino acid and glutathione (GSH) biosynthesis, or it can remain in the cytosol and be used for nucleotide synthesis and production of glutamate in the process . When th+ CSCs and tumorspheres also had increased levels of Gln; deprivation of the amino acid resulted in inhibition of sphere-forming properties, reduction in the ALDH+CSCs, diminished tumor-initiating capacity in vivo, and increased radiosensitization. In contrast, Gln depletion in the LNCaP cells did not have such severe effects. The metabolic reprogramming towards Gln has been associated with high MYC expression levels [A study conducted in prostate cancer cell lines revealed that Gln was significantly upregulated in radioresistant DU145, but not in radioresistant LNCaP cells, compared to the parental ones . Inhibitn levels , a findin levels . SOX-2 and ABCG2, both on the transcriptional and translational level. Gln replenishment reversed the effect of its deprivation in the stem-like SP population in the A549 cells, which recovered, while it also upregulated the expression of SOX-2 and ABCG2 [Another interesting study investigated the role of Gln metabolism on the stem-like side populations (SPs) of the A549 NSCLC and AsPC-1 pancreatic cancer cell lines . Gln depnd ABCG2 . Furthernd ABCG2 . The abond ABCG2 . +/CD24\u2212/low subpopulation compared to parental MCF-7 cells [+/CD24\u2212/low cells, while supplementation with Gln was accompanied by a significant increase in these cells [SLC1A5 supporting high Gln metabolism compared to parental MCF-7 cells, as well as the stem cell marker NANOG. Pharmacological inhibition of SLC1A5 reduced the CD44+/CD24\u2212/low subpopulation, indicating a Gln dependency for their survival, while it also promoted their escape from TIS [The association of Gln metabolism with the expression of stemness properties and the evasion of chemotherapy-induced senescence has also been examined in breast cancer . Specifi-7 cells . Gln depse cells . Furtherfrom TIS .+/CD44+ CSCs of certain cell lines (deemed metformin sensitive), but not in others (deemed metformin resistant). Further experiments showed that the metformin-induced AMPK (adenosine monophosphate-activated protein kinase)-dependent mTOR  pathway was involved in the regulation of the metformin-sensitive HT29 CSCs. The metformin-sensitive HT29 cells were also more dependent on OXPHOS than the metformin-resistant SW620 cells. The CSC-suppressing effect of metformin was induced in SW620 cells and enhanced in HT29 cells under Gln deprivation conditions, where tumorspheres from either cell line could not survive. The expression of the transporter ASCT2 was higher in the SW620 compared to HT29 cells, an observation that suggested a higher ability of the former to utilize Gln. Knock-down of ASCT2 in the SW620 cells significantly decreased the CD133+/CD44+ CSCs upon treatment with metformin, proposing that inhibition of the Gln pathway could be an effective complementary treatment to metformin to enhance its CSC-suppressing effect, especially in resistant cells [Finally, a new study used magnetic resonance imaging (MRI) to assess Gln uptake in mouse xenografts of HT29 colorectal cancer cells . Higher nt cells . Gln metabolism has emerged as an important metabolic pathway in the regulation of CSCs, suggesting that its inhibition or Gln deprivation could be an attractive therapeutic choice in cancer treatment. Lipids encompass a large heterogeneous family of organic compounds that are essential for a multitude of cellular functions, including energy production, membrane biosynthesis, and signal transduction. Lipid metabolism is dysregulated in cancer sustaining tumor growth, progression, and metastasis . Increas+CD24\u2212ESA+ breast CSCs isolated from the MCF10AT cell line and clinical specimens, and it could promote tumor progression [Indeed, the stearoyl-CoA desaturase 1 (SCD1) enzyme that regulates the conversion of saturated into monounsaturated fatty acids (MUFAs) was associated with stemness in ovarian, breast, and liver cancer, as its overexpression promoted CSC proliferation while preventing apoptosis . The stegression .Colon CSCs isolated from the HCT-166 cell line contained more unsaturated lipids and fatty acids than their non-CSC counterparts, and this lipid abundance was essential for maintaining their stemness properties . InhibitMoreover, MUFAs can affect CSC generation and their stemness properties . It has Similarly, the enzyme fatty acid synthase (FASN) that mediates fatty acid synthesis has been reported to be highly active in the GSC lines G144 and G179, as well as in tumorspheres generated from glioma tissue samples surgically resected from patients after their diagnosis . IncreasOverall, the alterations in the lipid metabolism of CSCs play an essential role in their survival and maintenance through the modulation of key signaling pathways, as it has been reviewed elsewhere . TargetiAutophagy is a highly conserved cellular process that involves the breakdown of intracellular components, including molecules and organelles, via lysosome-mediated degradation . The aut+ cells exhibited enhanced survival and reduced apoptosis compared to CD133\u2212 cells under glucose deprivation through the activation of autophagy-associated genes. Further investigation revealed that CD133 was more abundant in the cytoplasm in starvation conditions, whereas it was membrane-bound under normal glucose levels [\u2212 cells. The above study proposed that targeting CD133-signaling and autophagy in glioma could improve anti-cancer treatment. The expression of CD133, a well-known stem cell marker, was found to regulate autophagy in GSCs in a glucose-deprived environment . GSCs exe levels . This obAutophagy has also been linked to the metabolic mechanisms of the SCs in a study by Viale et al., in PDAC . TranscrMitophagy, the process by which aged and damaged mitochondria undergo autophagy, has been associated with CSC metabolic reprogramming and survival, especially under stressful conditions, such as hypoxia and chemotherapy . It is rISG15), an ubiquitination-like modifier, and the post-translational modification it regulates, known as ISGylation, were upregulated in PaCSCs [ISG15, stemness genes, and genes associated with mitochondrial processes, including OXPHOS [ISG15 in the PaCSCs using CRISPR led to accumulation of dysfunctional mitochondria, reduced OXPHOS and impaired glycolysis. Further experiments confirmed that loss of ISG15 led to an impairment of mitophagy and an increase in autophagosomes and autophagy flux, possibly as a compensatory mechanism [ISG15 loss were highly sensitive to the mitochondrial inhibitor in vitro and in vivo, indicating a diminished metabolic plasticity [An interesting study showed that interferon-stimulated gene 15 (n PaCSCs . RNA-seqg OXPHOS . Geneticechanism . The samechanism . Howeverasticity .Expression of the hepatitis B virus x protein (HBx) is a predisposing factor for HCC and promotes cancer stemness. In a recent study, the authors confirmed that expression of HBx induced a cancer stemness phenotype and promoted a metabolic shift towards glycolysis in HCC in vitro and in vivo . By inhiThe above studies support the idea that autophagy/mitophagy can promote CSC survival and stemness through metabolic reprogramming and suggest that blocking them may be a new therapeutic intervention against this highly tumorigenic population. As it was extensively reported in the sections above, the metabolic networks of CSCs are interlinked with their stemness properties. Several well known signaling pathways that support self-renewal and survival in CSCs, including Hippo, WNT/\u03b2-catenin, JAK/STAT, and Notch, seem to be also involved in the regulation of the metabolic reprogramming of these cells .Hippo signaling is an evolutionarily conserved pathway and a master regulator of cell proliferation and cell fate during organ development . The majGLUT3 and other glycolytic enzymes [GLUT3 overexpression in the HCT116 cells resulted in higher expression of stemness-related genes and increased tumorsphere formation, while GLUT3 silencing in the 116-LM cells suppressed their metastatic properties and reduced the expression of stemness-associated transcription factors [GLUT3-induced invasiveness and stemness properties were attributed to a YAP-depended mechanism, as silencing of YAP signaling suppressed these properties [It has also been demonstrated that YAP/TAZ activation regulates metabolism and metabolic reprogramming of CSCs . In the  enzymes . GLUT3 o factors . The GLUoperties . +CD24\u2212/low breast CSCs isolated from several cell lines had a high expression of a long non-coding RNA, lncROPM, which upregulated phospholipid metabolism and free fatty acid production, leading to activation of the Hippo pathway and maintenance of the stemness properties [2 (PLA2G16), leading to the production of free fatty acids and especially arachidonic acid. Knockdown of lncROPM in CSCs significantly decreased the expression of stemness-related genes and mammosphere size, while its overexpression in non-CSCs promoted the expression of such genes and increased the MFE. Lipidomic analysis of the lncROPM-knocked-down CSCs and the lncROPM-overexpressing non-CSCs revealed that arachidonic acid was the most significantly altered metabolite between the two groups. Arachidonic acid administration resulted in the expression of stemness-related genes in the knocked-down breast CSCs through the activation of both the Hippo/YAP and the Wnt/ \u03b2-catenin signaling [In breast cancer, YAP/TAZ activity has been linked to high-grade tumors and high CSC content, reflecting its correlation with aggressiveness . Bioinfooperties . More spignaling .The above described studies provide substantial evidence to support the regulation of CSC metabolism by the Hippo pathway, yet the underlying mechanisms are still unclear and need further investigation .The Wnt/\u03b2-catenin signaling cascade is also evolutionary conserved, as it is critical for numerous physiological processes including cell fate, proliferation, migration and polarity in development, and tissue homeostasis . DysreguPDK1 expression and by reducing the conversion of pyruvate into acetyl-CoA [The Wnt/\u03b2-catenin pathway is also involved in the reprogramming of cancer metabolism, where it directs cells into glycolysis and away from mitochondrial OXPHOS, through the regulation of etyl-CoA . Blockinetyl-CoA . Aberranetyl-CoA .Furthermore, the Wnt pathway seems to be a mediator of the effects of metabolic changes on CSC survival, as described in the following studies. MDA-MB-231 and MCF-7 mammospheres and PC3 and LNCaP prostate tumorspheres were grown in medium with no, low, or high glucose and were treated with the Wnt/\u03b2-catenin pathway inhibitor sFRP4 . The tumThe crosstalk of Wnt signaling with lipid metabolism was investigated in colon CSCs . PharmacThe Wnt/\u03b2-catenin pathway has also been linked with the stemness properties of CSCs through glutamine metabolism. Specifically, in the stem-like side populations isolated from the A529 NSCLC and the AsPC-1 pancreatic cancer cell lines, as well as in the GSC11 and GSC23 GSCs, glutamine activated the Wnt/\u03b2-catenin pathway through a ROS-mediated mechanism and upregulated the expression of stemness genes. Glutamine deprivation or inhibition of glutamine metabolism led to an increase in ROS levels and inactivation of \u03b2-catenin, decreasing stemness properties . The above studies suggest that the Wnt/\u03b2-catenin pathway can be modulated in CSCs by nutrient metabolism to affect the cells\u2019 viability and stemness. Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the phosphorylation of STAT proteins, leading to the translocation of STATs into the nucleus and the activation of gene expression. The JAK/STAT pathway is critical for a multitude of physiological processes involved in development and tissue homeostasis, such as hematopoiesis, stem cell maintenance, immunity, tissue repair, and inflammation . An incrSTAT3 knock-down reduced the expression of FAO genes, including CPT1B, in MDA-MB-468 tumorspheres and inhibited their self-renewal [CPT1B in breast carcinomas compared to healthy tissues, while it was also found elevated in recurrent tumors. High CPT1B levels also correlated with poor patient outcome and were negatively associated with therapeutic response [A recent study shed light onto the association between JAK/STAT3 signaling and breast CSC metabolism . Treatme-renewal . Interesresponse . Based oThe Notch signaling pathway is a highly conserved pathway that orchestrates cell fate decisions during development. It consists of the Notch cell-surface receptors that bind transmembrane ligands expressed on neighboring cells to mediate cell\u2013cell communication. Binding of the Notch ligands results in the proteolytic cleavage of the intracellular domain of the receptor, its translocation to the nucleus, binding to the CLS protein , and, finally, to the transcriptional activation of targeted genes . Notch shi GSCs were located in hypoxic niches and mainly relied on anaerobic glycolysis, while the Notchhi cells resided in perivascular niches using mostly OXPHOS for their energy needs [hi cells led to their metabolic reprogramming through suppression of anaerobic glycolysis, which rendered the cells vulnerable to hypoxia [In glioblastoma, metabolic adaptations that supported the survival and growth of GSCs in diverse niches were associated with heterogeneous activation of Notch signaling . The resgy needs . Ectopic hypoxia .A previously described study linking SCD1 inhibition with the suppression of the Wnt pathway showed that Notch signaling was affected, too . PharmacNotch signaling also regulates CSC survival in triple negative breast cancer (TNBC) through activation of mitochondrial metabolism . OncogenThe aforementioned studies reveal a new role for the Notch pathway in the regulation of CSCs through its intersection with CSC metabolism. Further work should elucidate the downstream effectors of Notch signaling on the metabolic network of these cells.Low oxygen level (hypoxia) is a major stressor to cells, which have developed adaptive mechanisms to manage it. Hypoxia-inducible factors (HIFs) are the key mediators of cellular adaptation to this condition and regulate the expression of genes involved in cell proliferation, apoptosis, metabolism, and invasion . HIF is HK2, PDK1, and ENO1). It also promoted stemness properties in these cells, which was manifested by an increase in tumorsphere formation and in drug resistance, as well as an enhanced migratory ability [The roles of ubiquitin-specific protease 22 (USP22) and HIF-1\u03b1 were investigated in promoting stemness and metabolic alterations in HCC in a recent study . Overexp ability . Knock-d ability . The aut ability . GLUT-1 and LDHA. Additionally, the glucose uptake and lactate production were decreased, while the endogenous cellular oxygen consumption was increased, confirming a switch towards OXPHOS in these cells [The association between HIF-1\u03b1, stemness, and metabolism was also studied in breast cancer . Knock-dse cells . The effse cells . The autse cells . high/CD24low subpopulation, and higher expression of pluripotency genes, including OCT4 and NANOG, in MDA-MB-231, MCF7, T47-D, and MDA-MB-468 breast cancer cells. It also enhanced the tumorigenic properties of the MDA-MB-231 cells in vivo. Knock-down of HIF-1\u03b1 significantly reduced the DKG-dependent induction of OCT4 and the CD44high/CD24low subpopulation [GLUT1 and PDK1), while the expression of genes associated with the mitochondrial electron transport chain was reduced [In a different study, the extended use of dimethyl-2-ketoglutarate (DKG), a cell membrane-permeable \u03b1-KG analogue that stabilizes HIF-1\u03b1, could reprogram breast cancer cells to acquire stem-like characteristics . Specifipulation . Further reduced . Thus, i reduced .These representative studies suggest that hypoxia and metabolic reprogramming can sustain CSCs and, thus, targeting the related factors  and/or pathways should be taken under consideration when devising therapeutic strategies against cancer.Increased drug resistance is a key feature of CSCs, and it has been attributed to several adaptive mechanisms that these cells have developed to survive the stress from drug exposure. These mechanisms include the upregulation of DNA repair mechanisms with overactivated cell-cycle checkpoints and overexpression of DNA damage repair proteins, as well as the increased expression of transmembrane drug-efflux pumps and the adoption of a quiescent state, where CSCs reversibly arrest in the G0 phase and exhibit basal metabolic activity . It has Since the metabolic rewiring of CSCs is closely interlinked with their enhanced drug resistance, deciphering their metabolic fingerprint to uncover potential targets has attracted much attention as a means for CSC elimination and coping with tumor recurrence. Interestingly, it has been reported by several groups that OXPHOS is the main regulator of drug resistance in CSCs from different types of cancer ,102,103.+ CSCs [The switch of CSC metabolism towards OXPHOS results in high levels of mitochondrial ROS and resistance to oxidative stress through anti-oxidant mechanisms . In an i+ CSCs .Similar results regarding the association of high mitochondrial mass with a stemness phenotype were obtained in breast cancer . High miMYC and the myeloid cell leukemia-1 protein (MCL1), an anti-apoptotic protein, were found to be overexpressed in drug-resistant TNBC patients after therapy, in paclitaxel-resistant MDA-MB-436 and SUM159PT cells and in CSC-enriched mammospheres generated from the parental cell lines . In a seSIRT1 was found to be overexpressed in the gefitinib (an EGFR-TKI) resistant PC9 and HCC827 lung cancer cells lines, which were also significantly enriched in CSCs compared to the parental cells [SIRT1 in the resistant cell lines reduced OXPHOS, the tumorsphere formation capacity, and the ALDH1+ CSC fraction and enhanced drug-sensitivity [SIRT1 promoted OXPHOS and subsequent CSC enrichment in TKI-resistant lung cancer. Clinical data from lung cancer patients indicated that higher expression of SIRT1 and OXPHOS-associated proteins correlated with tumor recurrence and poor survival, supporting that OXPHOS inhibitors could be a part of a combination therapy for better clinical outcome in these patients [Sirtuin 1 (SIRT1) is a NAD-dependent histone deacetylase that has a central role in regulation of gene expression, stemness maintenance, and metabolism . It is aal cells . The CSCsitivity . These rpatients .BCL-2 (B-cell lymphoma-2), a gene commonly found overexpressed in cancers [BCL-2 resulted in OXPHOS impairment and selective eradication of chemoresistant LCSs, without having any toxicity effect to normal cells from healthy donors [LSC-enriched populations, resistant to chemotherapy, were isolated from primary AML samples and were found to be metabolically quiescent with lower levels of ROS compared to non-SCs . Metabol cancers . Pharmacy donors .FoxM1 that controlled the ROS detoxification gene PRX3, as well as through an increased fatty acid oxidation-mediated NADPH regeneration. Both FoxM1 and the enhanced NADPH regeneration were shown to mediate drug resistance in CSCs [FoxM1 was associated with the prediction of poor survival in patients with different types of cancer. It was proposed that a mitochondrial ROS homeostasis-targeted approach in CSCs could constitute a therapeutic strategy against these therapy-resistant cells [Stem-like gastric cancer cells, generated through in vitro chronic metabolic stress, reprogrammed their metabolism towards increased OXPHOS compared to parental cells . Surpris in CSCs . Additiont cells .In pancreatic tumorspheres from PDAC cell lines  enriched in CSCs, the maintenance of a quiescent metabolic state with a reduced glycolytic activity was associated with increased chemoresistance . HoweverFinally, in chemoresistant HEP-G2 (rHEP-G2) cells with a CSC phenotype, metabolic reprogramming from glucose to glutamine dependency via mitochondria led to the adoption of a quiescent state from these cells . The useThe inability of common drugs to fully eradicate CSCs leads to tumor recurrence and poor patient survival, rendering efficient targeting of CSCs a matter of high importance. Inhibiting OXPHOS has gained a great interest as a means to overcome CSC drug resistance. A number of pharmacological agents that target OXPHOS are under investigation in ongoing clinical trials. Several FDA-approved agents, such as salinomycin, erythromycins, tetracyclines, and glycylcyclines, selectively eradicate CSCs through OXPHOS inhibition ,110. NotDrug resistance is still a major challenge for the treatment of cancer, and its association with cancer cells with stem-like properties is now well established. CSCs have developed a wide repertoire of mechanisms to evade chemotherapy; one of them is metabolic plasticity that allows them to switch between glycolysis and OXPHOS, depending on stimuli from their environment. Several signaling pathways, such as Hippo, Wnt/\u03b2-catenin, Notch, and JAK/STAT, are interlinked with the metabolic flexibility of CSCs, underscoring the complex regulation of this trait. However, more studies are needed for clarifying the role of metabolic plasticity of CSC in cancer progression, metastasis, chemoresistance, and tumor recurrence.Metabolic targeting of CSCs remains a challenging goal, as, in most cases, inhibition of one metabolic pathway leads the cells to enhance other metabolic processes for their survival. As CSCs cover their energy needs mainly through OXPHOS and glycolysis, simultaneous targeting of these two pathways could be an alternative and more effective therapeutic approach for their complete eradication."}
+{"text": "In the original version of the article , Xin LiuThe original article has been corrected."}
+{"text": "The original article has been corrected."}
+{"text": "The original version of this article unfortunately contained a mistake. The footnotes in Table The original article has been corrected."}
+{"text": "Patients with pituitary lesions experience decrements in quality of life (QoL) and treatment aims to arrest or improve QoL decline.To detect associations with QoL in trans-nasal endoscopic skull base surgery patients and train supervised learning classifiers to predict QoL improvement at 12 months.A supervised learning analysis of a prospective multi-institutional dataset (451 patients) was conducted. QoL was measured using the anterior skull base surgery questionnaire (ASBS). Factors associated with QoL at baseline and at 12-month follow-up were identified using multivariate logistic regression. Multiple supervised learning models were trained to predict postoperative QoL improvement with five-fold cross-validation.ASBS at 12-month follow-up was significantly higher  than preoperative ASBS . High preoperative scores were significantly associated with institution, diabetes and lesions at the planum sphenoidale / tuberculum sella site. Patients with diabetes were five times less likely to report high preoperative QoL. Low preoperative QoL was significantly associated with female gender, a vision-related presentation, diabetes, secreting adenoma and the cavernous sinus site. Top quartile change in postoperative QoL at 12-month follow-up was negatively associated with baseline hypercholesterolemia, acromegaly and intraoperative CSF leak. Positive associations were detected for lesions at the sphenoid sinus site and deficient preoperative endocrine function. AdaBoost, logistic regression and neural network classifiers yielded the strongest predictive performance.It was possible to predict postoperative positive change in QoL at 12-month follow-up using perioperative data. Further development and implementation of these models may facilitate improvements in informed consent, treatment decision-making and patient QoL. Pituitary adenomas are common and may be present in up to 10% of people with normal endocrine function . FunctioPatients with pituitary lesions experience worse QoL than the general population , 5, 6. TSupervised machine learning is a subdomain of artificial intelligence (AI) that involves the application of algorithms to identify complex patterns in large datasets enabling effective outcome prediction and classification \u201317. SupeThis study was conducted to detect clinical associations with QoL in trans-nasal endoscopic skull base surgery patients and train and test a collection of supervised learning classifiers to predict QoL improvement at 12 months. The study was guided by the following two research questions. (1) What clinical factors are associated with preoperative QoL in skull base surgery patients? (2) Can postoperative change in QoL be effectively predicted using supervised learning algorithms?This multi-institutional study involved an analysis of a prospectively collected dataset. Each patient was older than 18 years and underwent skull base neurosurgery to treat pituitary pathology. Patients were treated at three tertiary hospitals in Melbourne, Australia: St Vincent\u2019s Hospital, Monash Medical Centre, and The Royal Melbourne Hospital.The study was conducted under institutional review board (IRB) ethics approval . Patients provided their consent for the use of their data for quality improvement analysis. The IRB provided a waiver of consent for the use of the deidentified dataset for research.Covariates  included operating institution, gender, age, presentation history, co-morbidities, anatomic site of the lesion, histopathology, endocrine status, characteristics of the surgery, and intra- and postoperative complications. The primary outcome measure was QoL, as measured by the Anterior Skull Base Surgery Questionnaire (ASBS). Scores range from 35 to 175 and higher scores indicate a better state of QoL , 29. PreCovariates and outcomes were coded as binary variables. If datapoints were missing for >10% of cases for a given covariate, then it was excluded from the analysis. If <10% of cases for a binary covariate contained missing data, then it was assumed that the patient\u2019s clinical state with regard to that covariate was normal and the missing fields were filled with zeros. This was done to retain and include as many clinical covariates as possible in the analysis, maximising the use of the specialist dataset collected. In the postoperative machine learning dataset, this applied to five covariates: \u201cany postoperative complication\u201d (n missing = 15), \u201csecreting adenoma\u201d (n missing = 8), \u201cdevelopment of postoperative diabetes insipidus\u201d (n missing = 10), \u201cdevelopment of postoperative syndrome of inappropriate anti-diuretic hormone secretion (SIADH)\u201d (n missing = 15) and \u201creoperation\u201d (n missing = 4). Patients with missing outcome data were excluded from the analysis.The analysis involved two phases: (1) statistical analysis using multivariate logistic regression; and (2) training and testing supervised learning classifiers. The first phase involved applying multivariate logistic regression to detect significant associations between covariates and outcome variables. Covariates were included in multivariate models in a hypothesis-driven manner based on clinical relevance and the expertise of senior surgeons . CovariaNumerous supervised learning classifiers were trained and tested, including random forest (RF) , gradienA preoperative ASBS score was recorded for 451 patients. Mean patient age was 53.63 years (SD = 16.86). One hundred and ninety-nine patients had an ASBS score recorded at 12-month follow-up. Mean patient age was 52.85 years (SD = 17.31). There was no statistically significant demographic difference between the pre- and postoperative groups. Mean preoperative ASBS was 121.87 (SD = 25.72), while mean ASBS at 12 months was 132.19 (SD = 24.87), which was significantly higher . Mean change in ASBS score at 12-month follow-up was 7.5 points (SD = 25.79). Descriptive statistics are displayed in The two multivariate logistic regression models classifying the highest and lowest preoperative ASBS quartiles were both significant (LLR p<0.05). High preoperative ASBS scores were significantly associated with three covariates: institution, insulin dependent diabetes (negative association) and lesions at the planum sphenoidale / tuberculum sella anatomic site . PatientLow preoperative ASBS scores were significantly associated with five covariates. There were positive associations with female gender, a vision-related presentation history, insulin-dependent diabetes and secreting adenoma. A negative association was found for lesions at the cavernous sinus anatomic site . Women wThe multivariate logistic regression model predicting top quartile change in postoperative ASBS scores at 12 months was significant . Five coSupervised learning models were trained using two groups of covariates: (1) statistically selected covariates from the preceding multivariate logistic regression analysis, and (2) the top 27 most relevant covariates selected using RFE. Mean five-fold cross-validation performance metrics for each classifier are displayed in This multi-institutional study was designed to (1) determine associations between perioperative clinical factors and (a) preoperative QoL and (b) postoperative improvement in QoL in patients undergoing anterior endoscopic skull base surgery and (2) train supervised learning classifiers to predict postoperative change in QoL. Change in QoL 12 months after endoscopic skull base surgery in this sample was, on average, significant and positive. Mean change in ASBS score at 12-month follow up (7.5) was much higher than the established minimally important clinical difference (0.4) , suggestStatistical associations detected using multivariate logistic regression were well supported by existing literature. For example, lower cholesterol has previously been associated with higher QoL  and endoIt appears that QoL was influenced by lesion anatomic site. Patients with a lesion at the cavernous sinus site were less likely to report low preoperative QoL, while patients with a lesion at the planum sphenoidale / tuberculum sella site were much more likely to report high preoperative QoL than patients with lesions at other sites. Together, these results suggest that, overall, lesions at these sites tended to be associated with higher preoperative QoL. Patients with sphenoid sinus lesions were more likely to experience a positive change in postoperative QoL at 12 months. There may be numerous factors  associated with lesions at some sites that may influence preoperative QoL and make them more amenable to surgical treatment, resulting in more substantial QoL improvements. Lesions at the planum sphenoidale tend to be meningiomas, which are typically benign and usually cause visual disturbance rather than endocrine dysfunction . This maOverall, the highest performing machine learning classifiers yielded a moderate level of classification performance. These classifiers, nevertheless, demonstrated performance that was better than chance and as such may offer an additional useful input into the clinical decision-making process. We have, in this work, presented a benchmark for the field using some standard methods and a modest dataset. Interestingly, classifier performance appeared to be significantly affected by the dimensionality reduction and data augmentation methods applied. Dimensionality reduction using multivariate logistic regression appeared to yield superior classifier performance when compared with RFE. Furthermore, data augmentation applied as recommended  did not A salient issue in the field of clinically applied machine learning and machine ethics, which was intentionally addressed, is that of model interpretability or explainability . Neural Future research may consider the development of refined high-performance models that contain less covariates to facilitate more efficient system implementation, usability and generalisability. Analyses based on larger sample sizes from multiple institutions would facilitate a more detailed investigation of QoL associations at additional postoperative time points. Larger datasets would also allow for the use of one or more holdout datasets to more rigorously evaluate and validate classifier performance. The number of ASBS respondents at 12 months was lower than the number of preoperative respondents. Verification of results through replication and external validation is required as selection bias may have influenced results. Specialist clinical datasets are difficult and expensive to acquire and maximal use of data for beneficial research is an ethical issue. The research team is exploring the use of alternative classifier implementations that allow for missing data.Significant associations were detected between perioperative clinical factors, preoperative QoL scores and improvement in postoperative QoL scores at 12 months amongst patients undergoing anterior endoscopic skull base surgery. This study demonstrated that machine learning may be applied to predict changes in QoL at 12-month follow-up using perioperative data, facilitating optimisation of patient care and outcomes.S1 Checklist(PDF)Click here for additional data file."}
+{"text": "BackgroundReligious gatherings like the Hajj, an Islamic pilgrimage, attract millions of people to one place during the same time frame. Due to crowding, infectious diseases, specifically tuberculosis (TB), are very common during such events. This study investigates the knowledge, attitudes, and practices of the public in the western region of Saudi Arabia related to TB to better understand the situation.MethodologyAn observational, questionnaire-based, cross-sectional study was conducted over two months between January and March 2022. A survey of 29 questions was used to collect data from the general population. The study included any person who was a resident of Makkah. Individuals under 18 years of age and health workers were excluded. We used OpenEpi, version 3.0, for sample size calculation, which gave a result of 604 participants, and SPSS version 25.0  was used for data analysis.ResultsA total of 604 participants were included in this study; 64.7% of respondents showed poor overall knowledge, and 14.1% had good knowledge of TB. Concerning attitude, 89.9% of the respondents showed poor attitude, and only 2.3% had a good attitude. As for practice, 59.4% of respondents had poor knowledge of proper practices, and only 10.4% knew the right practices regarding TB. Upon further analysis of our results, women exhibited better knowledge of TB than men , 0.44-0.87). Participants over 50 years old had the lowest knowledge about TB compared with participants aged 18 to 28 years old . Non-Saudi residents had less knowledge compared with Saudi residents . Level of education also played a substantial role; university graduates had the most knowledge about TB compared with participants with below university or no formal education .ConclusionsParticipants with lower educational backgrounds were the most lacking in knowledge, attitude, and practice regarding TB. This lack of knowledge was more common among non-Saudi men over 50 years old. Information campaigns are needed to help reduce the prevalence of TB. Tuberculosis (TB) is a well-known contagious infection that spreads through the inhalation of droplets produced by the coughs or sneezes of an infected person. TB usually affects the lungs (pulmonary TB) but can also affect multiple other organs. In 2020, the number of people infected with TB was estimated to be 9.9 million worldwide, with a drop of 18%\u00a0from 2019 and an increase in mortality rate. Mortality reached almost 1.3 million deaths among human immunodeficiency virus (HIV)-negative patients from 1.2 million in 2019 . ConsequThe high incidence of TB cases coupled with the lack of knowledge globally and locally highlight the importance of population awareness, especially in regions such as Makkah, and bring forward the need for research to investigate the level of awareness among the general population. Locally, not enough studies have evaluated the knowledge of TB. In this paper, we provide an investigation of knowledge, attitudes, and practices of TB among the public in the western region of Saudi Arabia.This observational, cross-sectional study was conducted using a self-administered questionnaire. The study targeted the general population of the western region of Saudi Arabia and spanned two months between January and March 2022. We included all Makkah residents. Individuals younger than 18 years of age and health workers were excluded from the study. Participants were selected using a non-sampling convenience sampling technique. Based on a review of similar studies, the research team developed a 29-item questionnaire divided into four sections to accomplish the research objectives [Data analysisAfter data extraction and coding, SPSS Statistics version 25.0  was used for data analysis. Descriptive analysis was utilized and expressed as frequency and percentage for categorical variables. Participants\u2019 overall knowledge, attitude, and practice were categorized according to Bloom\u2019s cutoff point into good, moderate, and poor based on the total percentage score as follows: good for scores 80% and above, moderate for scores 60-79%, and poor for scores below 60% [Ethical considerationsThis study was approved by the Medical Research Ethics Committee at Umm Al-Qura University . Consent to participate was taken from participants electronically.The survey included 604 participants. The sociodemographic characteristics are presented in Table Results regarding participants\u2019 knowledge showed that only 65.6%\u00a0had heard about TB, with the majority relying on family and friends and media as their primary source of information . We found that 64.7%\u00a0of the participants had overall poor knowledge, 21.2%\u00a0had moderate knowledge, and 14.1%\u00a0had good knowledge of TB. Additional data on participant knowledge are presented in Table Upon investigating the attitude and stigma related to TB, most respondents considered TB a somewhat serious disease 43.2%). In relation to their specific area, the majority thought TB to be a somewhat serious problem (34.4%). When asked how they would react if they were to be diagnosed with TB, fear and surprise were the most frequent choices , and hopelessness was the least (7.1%). The overall attitude was poor in 89.9%\u00a0of the participants. For more data on attitude, refer to Table .2%. In rRegarding participant practices pertaining to TB, most respondents said they would seek professional help by visiting a healthcare facility if they experienced symptoms of TB 61.4%), while others said they would go to pharmacies, traditional healers, or pursue self-treatment options . Most of those who chose options other than healthcare facilities explained that their behavior was due to not knowing where to go, not wanting to find something wrong, and their inability to leave work . Therefore, 59.4%\u00a0of the respondents had poor knowledge of the proper practice to follow, 30.1%\u00a0had moderate knowledge, and only 10.4%\u00a0had good knowledge of the right practices for TB. More data on practice are provided in Table %, while Participants\u2019 gender played a major role in the results, as women were less likely to have\u00a0poor knowledge in comparison to men . Participants over 50 years old had substantially less knowledge about TB compared with participants 18-28 years old . In addition, non-Saudi residents had less knowledge compared with Saudi residents . Level of education also played a substantial role; university graduates had the most knowledge about TB compared with participants with no formal education and other education levels . For more data on variables predicting poor TB knowledge, see Table This study demonstrates that most participants (64.7%) had poor knowledge of TB, and only 21.2%\u00a0had a moderate level of knowledge. In comparison, a cross-sectional study done in the eastern and western regions of Saudi Arabia reported poor knowledge of TB among 81.9% of participants. Another study in the country had a similar result at 74.9%\u00a0,11. ReseOn overall knowledge of practice, 59.4%\u00a0of the participants showed poor results, while only 30.1% had moderate knowledge of good practice. Results showed that 20.9%\u00a0were unsure where to go if they had TB symptoms, and another 20%\u00a0feared knowing that they had the disease. Similar reasons were mentioned in southwest Ethiopia and Vietnam ,25. HoweStudy limitationsThis study has a few limitations. First, recall bias may have influenced results.\u00a0Second, the study included only one region in Saudi Arabia and, hence, cannot be generalized to the whole country.Poor knowledge appears to be more prevalent among men than women and individuals over 50 years old. Moreover, more non-Saudi residents have poor knowledge, attitudes, and practices related to TB. More campaigns, whether in real life or through online platforms in association with the Ministry of Health, should be considered to raise awareness regarding TB."}
+{"text": "Tuberculosis (TB) is a high-burden respiratory infectious disease. There was a sharp decline in the number of confirmed TB cases during the pandemic; this is likely to be influenced by the COVID-19 pandemic response, with under-reporting due to resource diversion. There are typically 13,000 tuberculosis-associated deaths in Afghanistan annually, with significant problems posed by drug-resistant TB.A cross-sectional descriptive study was conducted in Afghanistan on Kabul residents who visited the adult outpatient departments of public hospitals for any health-related reason from 1st January to 20th March 2022. The study scored their knowledge, attitude, and practices (KAP) toward tuberculosis. The sample size was calculated using Epi-Info, and the minimum sample size was 385. The sampling method is chosen the non-probability convenient sampling for data gathering. Data were analyzed using SPSS version 28, and we used the Mann-Whitney test, Chi-square or fisher extract test, spearman correlations, and binary logistic regression model.P-value = 0.009, 0.000, 0.003, and 0.009), respectively. A positive attitude was expected to have good knowledge 6.035 times more than a negative attitude (95% CI: 1.572\u201323.167).Of 829 participants, 450 (54.3%) were males and 379 (45.7) females. The median age was 28 years, and 63.3% were married. Most participants were unemployed (75.5%), but 54% had a monthly income >3,000 Afghanis, indicating the reliance on family. By TB knowledge score, 727 (87.7%) participants had good knowledge, and 800 (96.5%) participants had a positive attitude toward treatment and control. Only 2 participants reported poor practices regarding prevention. Regarding the binary logistic regression, young age, being a male, belonging to the \u201c1,000\u20133,000\u201d Afghani monthly income category, and having a positive attitude were significant predictors of good TB knowledge (The study findings highlighted that outpatients in Kabul had good knowledge, attitude, and practice toward TB. More studies are needed to highlight KAP in different Afghan populations, including in other parts of the country. Data collection took place from 1st January to 20th March 2022 from public health facilities such as .For this study, inclusion and exclusion criteria were all patients 18 years of age or above and were Outpatient departments referral. Exclusion criteria were patients under 18 and were not Outpatient departments referral.The survey questionnaire was adapted from a similar study conducted in Ethiopia . The queThe study asked basic sociodemographic questions such as age, gender, marital status, educational status, occupation, and average monthly income (afghani), which were categorized into three classes, namely, <1,000 (afghani), 1,000\u20133,000 (afghani), and >3,000 (afghani). The median monthly income in Kabul is 5,000 Afghani, equating to ~57.14 US dollars.We ask about TB mode of transmission, TB preventability, and TB symptoms. The knowledge grade was calculated by giving a (correct answer a score of 1 and a wrong answer a score of 0). We had nine points, so the maximum score was nine, and the minimum score was zero (0-9). We categorized the knowledge as follows\u2014Poor knowledge (0\u20135) and Good knowledge (6\u20139). This scoring system was based on a similar study done in Ethiopia. The area under the receiver operator characteristic (ROC) curve (AUC) results of the knowledge were considered good and equalled 0.86.We ask whether participants considered TB a dangerous disease for the community, whether TB transmission from human to human and the presence of stigma for TB patients. The attitude grade was calculated according to (true answer = one and wrong answer = 0). We had three points, so the maximum score was three, and the minimum score was zero (0\u20133). We categorized the attitude as follows [Positive attitude (2\u20133) and Negative attitude (0\u20131)]. The area under the ROC curve (AUC) attitude results were considered good and equalled 0.801.We ask about participant knowledge around good practices for controlling TB, including opening house windows regularly, opening car window's during traveling, TB screening, having TB health education, having TB, and action after having TB. The practice grade was calculated according to (true answer = one and wrong answer = 0). We had eight points, so the maximum score was eight, and the minimum score was zero (0\u20138). We categorized the practice as follows [Poor practice (0\u20134) and Good practice (5\u20138)]. The area under the practice's ROC curve (AUC) results were considered excellent and equalled 0.998.All participants fulfilling the inclusion criteria were invited to participate. The sample size was calculated using Epi-Info. Kabul's population has an estimated 4 million 400 thousand with a 95% confidence level, 50% expected frequency, 1.0 effected size, and 5% margin of error. The measured required minimum sample size was 385 participants. The sampling method is chosen the non-probability convenient sampling for data gathering.The questionnaire was pre-tested on a small sample of participants (70 participants) to confirm the practicability and clarity of questions. Expert researchers reviewed the questionnaire for content validity and considered their comments. For reliability, Cronbach's alpha was 0.753 for every questionnaire section, indicating an acceptable level of consistency. Observations of the pilot study were not included in the final analysis.Ethical approval was obtained from the Microbiology Department of Kabul University of Medical Sciences Research Committee (KUMS-MD-2241). Informed consent was taken from the participants before they participated in the study.n) and percentages (%) for categorical data with comparisons made using the Chi-square or Fisher extract test. A value of p < 0.05 was considered statistically significant. The association between knowledge, attitude, and practices toward tuberculosis was tested using spearman correlations. The binary logistic regression model was interpreted using odds ratio (OR) and 95% confidence interval (CI).The knowledge scoring was based on the three knowledge questions. The extracted data was presented in an excel sheet, and data were analyzed using SPSS version 28. The optimal cutoff point was evaluated using the ROC curve. The area under the ROC curve (AUC) results were considered excellent for AUC values between 0.9 and 1, good for AUC values between 0.8 and 0.9, fair for AUC values between 0.7 and 0.8, poor for AUC values between 0.6 and 0.7 and failed for AUC values between 0.5 and 0.6 . NormaliOf the 829 participants, 450 (54.3%) were males, 379 (45.7) were females, and the median age was 28 years (IQR = 20) and ranged from 9 to 80. The majority of our sample was unemployed participants (75.5%), but 54% of the participants had a monthly income >3,000 Afghanis (34.29$) Overall, 727 (87.7%) participants had good TB knowledge, and 800 (96.5%) participants had a positive attitude toward TB treatment and control. Moreover, only 2 participants reported poor practices regarding TB prevention. The baseline characters are shown in n = 369 and 358, respectively) . Belonging to the \u201c>3,000\u201d Afghani monthly income category significantly had better knowledge more than other categories   . A positive attitude was significantly associated with good knowledge .Males significantly had better knowledge more than females , respectively . Being employed was predicted to have good knowledge 1.177 times more than being unemployed (95% CI: 0.660\u20132.100). Belonging to the \u201c <1,000\u201d Afghani monthly income category expected to have good knowledge 2.171 times more than other categories  (95% CI: 1.306\u20133.608). A positive attitude was expected to have good knowledge 6.035 times more than a negative attitude (95% CI: 1.572\u201323.167) .n = 427 and 373, respectively) . Good knowledge was significantly associated with a positive attitude   .P-value = 0.018, 0.027, and 0.024), respectively . Overall, very good practices against the prevention of TB were noted throughout (All single participants showed good practices against TB 279 (100%). Males had non-significant good practice more than females , Young participants were expected to have a good practice 1.013 times more than old participants (95% CI: 0.911\u20131.128). A male was predicted to have a good practice 1.188 times more than a female (95% CI: 0.074\u201319.055). Being employed was predicted to have a good practice 3.094 times more than being unemployed (95% CI: 0.193\u201349.691) .P-value = 0.001]. A low positive correlation was noted between practice grading and practice score . A strong negative correlation was noted between attitude grading and attitude score  (A strong positive correlation was noted between knowledge grading and knowledge score [Rho (R)-value = 0.696 and = 0.001) .In this study, most participants had good knowledge (87.7%) and attitude (96.5%) toward TB. Similarly, participants ranked highly overall for TB practice. Higher monthly income and being female were observed predictors for higher scores.However, this may not genuinely reflect the extent of TB KAP among the participants, as the country is going through unique circumstances. The Taliban regime has come into power. The dominance was followed by sanctions from different countries and international organizations, which resulted in funding suspension. Since then, the already-fragile healthcare system has not been functioning properly. The country's largest healthcare services provider \u201cSehatmandi\u201d program, has lost its full functionality . The proThe finding of this study regarding knowledge about TB is reported higher than in a study done in the Mecha district of Ethiopia , where 54% of participants had good knowledge about TB . HoweverRegarding the attitude, 96.5% of the participants had a good attitude toward TB. Females had a better attitude than their male counterparts; this is in line with a study conducted in Shingle town, eastern Ethiopia, where half of the participants considered TB a very serious disease . In anotIn this Afghanistan study, those participants with a better attitude had good practice toward TB. Only a few participants reported poor practice regarding TB. Furthermore, male participants had better practice, but it was not statistically significant. Observed differences may reflect social dynamics, environment, and perceived access to healthcare facilities. These patients were interviewed in the health settings of Kabul, the capital of Afghanistan, where access to TB medication is available. However, this does not apply to all country contexts, especially rural ones.In this study, 98% of the participants stated that TB is preventable in our study. This high number could be attributable to Kabul's awareness program and widely available treatment centers. The finding is similar to what is reported in Shinali town, where 79.3% of the participants responded that transmission of TB would be preventable . HoweverMost participants (96.6%) stated cough as the main symptom, followed by fever and loss of appetite. Similar symptoms were reported by other participants in other studies , 20, 23.Gender and level of education were associated with better knowledge of TB; this is in line with a study conducted in Khyber Pakhtunkhwa, Pakistan, where females and education level was correlated with better knowledge of TB .Our study has several limitations. The study was conducted only in the capital, Kabul. The findings cannot be generalized to the whole country. Further studies are needed to provide a more in-depth assessment of TB in the country. Moreover, the study was done in hospital settings. Hence, there could be differences among participants in the community. Thus, another study could provide greater insight into this.The study concluded that participants had good knowledge, attitude, and practice toward TB. A recommendation for policymakers is that TB knowledge is high in this study population, which can be used as a stepping-stone for future health promotion activities. Future awareness-raising campaigns will need to be tailored to the needs and socio-economic circumstances of the target communities. This study can contribute to the evidence base that informs those campaigns. Further research is warranted to provide additional data from other Afghanistan populations to allow comparisons with the findings from this study; this should include vulnerable individuals who find it difficult to access healthcare, residents in rural areas where healthcare is especially limited, and internally displaced or refugee Afghan populations. These are all groups at higher risk of both tuberculosis and drug-resistant tuberculosis.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Microbiology Department of Kabul University of Medical Sciences Research Committee (KUMS-MD-2241). The patients/participants provided their written informed consent to participate in this study.ME and AN conceived the concept of the paper. ME, SA, and MK wrote the first draft and performed the analysis. KR, RN, and TD contributed data collection. AN and MH edited the second draft. All authors read and approved the final draft.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "RI) is as follows: Hg > Cd > As > Pb > Cu > Ni > Cr > Zn, with noncarcinogenic chronic risks of Cr > As > Cd > Pb > Ni > Cu > Hg > Zn; furthermore, dermal contact is the main pathway of exposure causing health risks. The total carcinogenic risks caused by heavy metals were as follows: Cr > Cd > As > Pb; and the risks posed by Cr, Cd, and As were higher than the threshold value (1.0 \u00d7 10\u22124); people face a higher threat to heavy metals in soils in Zhenxiong, Ludian, Huize, Weixin, and Zhaoyang. The evaluation result of the EPA PMF model shows that the soil heavy metals are mainly composed of five sources, of which basalt, Permian, and Triassic carbonate rock parent material constitute the natural background source, while the mining activities of lead\u2013zinc mines and the emissions of coal burning by residents constitute the anthropogenic source. The contribution was ranked in order of lead\u2013zinc mining (26.7%) > Triassic carbonate (23.7%) > basalt (20.9%) > coal burning and automobile emissions (16.1%) > Permian carbonate (12.6%).A total of 28,095 surface soil samples were collected in areas with high natural background levels; the potential ecological risk is generally low, and the high-risk area is small and mainly affected by lead\u2013zinc mines. The contribution to the potential ecological risk factor ( Ecological risk assessment is an important part of environmental risk assessment, and it is the process of assessing the possibility of negative ecological effects occurring because of exposure to single or multiple pollution factors . Health Northeast Yunnan is a typical area with high geochemical levels of heavy metals in southwestern China. Compared with the background value of soil (layer A) in China ,15, ther2, accounting for 5.2% of the survey area, and the second-class quality spans 86.5% of the survey area, which shows that the heavy metals in the soil in the area pose a high potential risk. It is of great significance to carry out surveys of heavy metals in soil and ecological health risk assessments in northeast Yunnan.Basalt and carbonate rocks are widely distributed in the area with high geochemical background levels in northeastern Yunnan, and those rocks have become the main soil-forming parent materials. The special geographic location and topography determine the diversity of land-use types in the study area. Therefore, these factors cannot be ignored when conducting soil ecological geochemical assessments in the study area; specifically, it is believed that the heavy metal sources of soil are caused by different land-use types and different parent materials. The results of the comprehensive classification of soil environmental quality show that the first-class environmental quality of soil in northeast Yunnan spans an area of 1456 kmThis article refers to a relatively mature risk assessment model in the \u201cEcological Risk Assessment Guide\u201d from the US Environmental Protection Agency (USEPA), combined with the land-use types of the study area. The research focuses on eight heavy metals, including As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn, and the risk of chronic intake and carcinogenicity of soil in the high geochemical background area of heavy metals of northeast Yunnan were evaluated under three exposure methods: dietary intake, unintentional inhalation, and dermal contact. At the same time, the positive matrix factorization model (PMF) proposed by the USEPA  was used2. The administrative division is under the jurisdiction of Zhaotong and Qujing  was used to test the Cd, Cu, Ni, Pb, and Zn, and full-spectrum direct reading inductively coupled plasma-emission spectroscopy (ICP-OES) was used to complete the Cr test. Samples were decomposed with aqua regia, potassium permanganate solution was added for oxidation treatment, and the solution was diluted with oxalic acid solution. Then, potassium borohydride was used as a reducing agent to be pre-reduced by thiourea-ascorbic acid. Atomic fluorescence spectrometry (AFS) was used to measure the As with HG-AFS. Finally, the remaining solution was directly diluted to measure the Hg with CV-AFS using SnClQuality assurance and control measures were used throughout the testing process, and the national first-level standard substances  were used as the standard reference materials to monitor the testing process. During the analysis, four pieces of password samples were inserted for each batch (approximately 50 samples) for external and internal quality controls. Two blank tests were carried out in an analysis batch to control the blank changes. The results showed that the overall pass rate of the first-level standard substance samples for monitoring accuracy was 100%, the overall pass rate for the precision monitoring samples was 100%, the total element reporting rate was 99.97%, and the overall pass rate of randomly selected 5% repeatability test samples was 99.5%. The analysis results met the quality requirements of the technical specification for multipurpose regional geochemical survey DZ/T0258-2014 .PERI) was used to evaluate the potential ecological risk of heavy metals in soil. The PERI of a single heavy metal (RI) were calculated by Equations (1)\u2013(3) [i; i in the soil sample; i; In this study, the potential ecological risk index ( (1)\u2013(3) :(1)Cfi=CPERIs of eight heavy metals, including As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn, in soil were calculated. Hakanson [RI) into four risk levels: extremely strong risk (600 \u2264 RI), strong ecological risk (300 \u2264 RI < 600), moderate ecological risk (150 \u2264 RI < 300), and low ecological risk (RI < 150), which helps researchers analyze the risk more intuitively.The Hakanson  divided \u22121; \u22121; EF) selects different EFs according to different land-use types in the study area: the farmland and residential land EF is 350 day\u00b7a\u22121; the industrial and mining land EF is 250 day\u00b7a\u22121; the forestland, grassland and other land-use type EF values are equal to 40 day\u00b7a\u22121 [ED) is divided into noncarcinogenic chronic risk and carcinogenic risk. The exposure period of noncarcinogenic chronic risk is 24 years [The health risk assessment models provided by the USEPA were used in this study to evaluate noncarcinogenic risk ,35,36. T day\u00b7a\u22121 . Exposur24 years .HQ) and characterized by the ratio of the amount of heavy metals ingested by the human body to the specified reference dose (RfD) of the pollutant under different exposure pathways. The hazard index (HI) is the sum of chronic risks. In this study, the HI represents the health risks of chronic diseases in humans caused by the three exposure pathways of the eight heavy metals, including As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn. Chronic diseases may occur when the HI is greater than 1, and the risk of diseases increases with an increasing HI [HQ and HI of heavy metals can be expressed with Equations (7) and (8):ith heavy metal through the jth exposure pathway, mg\u00b7(kg\u00b7d)\u22121; ith heavy metal under the jth exposure pathway, mg\u00b7(kg\u00b7d)\u22121; and the reference dose corresponding to a single heavy metal used in this study is listed in Heavy metals in the soil can be ingested by the human body through multiple exposure pathways, causing the risk of chronic diseases. It is defined as the hazard quotient (asing HI . The HQ CSF) through the individual exposure to soil carcinogens by various exposure pathways [jth exposure pathway, jth exposure pathway, mg\u00b7(kg\u00b7day)\u22121; and jth exposure pathway. The carcinogenic slope factors used in this study are shown in \u22126, which is equivalent to the probability of cancer occurring in one out of 1,000,000 people, the risk can be considered to be negligible, and when it is higher than 1.0 \u00d7 10\u22124, it is recognized that the carcinogenic risk is unacceptable [\u22126 has been recognized as the threshold for determining carcinogenic risk [Soil carcinogenic risk is characterized by the probability that an individual will experience cancer through lifelong exposure to carcinogens in the environment, and this value is obtained by establishing a cancer slope factor . The concentration of each heavy metal in this study is greater than the MDL; therefore, the uncertainty can be expressed as follows:C is the concentration of element j, mg\u00b7kg\u22121; and MDL is the method detection limit.The PMF is a reliable and effective pollution source identification method proposed by the USEPA . It is urally 5% ,29,56,57Statistical analysis of characteristic parameters was performed by SPSS (version 19.0)  software. The spatial distributions of heavy metal concentrations adopted the power exponential weighting method using the software GeoIPAS (version 3.2) , and the ecological health risks were determined using the software ArcGIS (version 10.2) . The identification of heavy metal sources was performed using PMF (version 5.0) .\u22121 and 0.56 mg\u00b7kg\u22121, respectively, and their enrichment coefficients are 3.2 and 5.8, respectively, which are values indicative to the typical soil heavy metal enrichment area in southwest China. The coefficients of variation of Cu, Hg, Cd, and As in the area are 72%, 53%, 50%, and 48%, respectively, with a wide variation range, which shows that the sources of these heavy metals are complex. Based on the ratio of geochemical background value to reference value (background/referenceK) in the study area, it is clear that As, Cd, Hg, and Pb are strongly enriched in the surface soil relative to the deep soil, and more than 80% of the surface soil in the study area displays enriched Cd; compared to the deep soil, the surface soil enrichment degree is 3.5 times greater. These characteristics indicate that most of the heavy metals in the surface soil are affected by natural or human activities during the process of soil formation. As a result, the distribution pattern of heavy metals in the surface soil changes, especially the biophilic elements and environmental elements, which are the most significantly affected. These elements show the characteristics of regional enrichment, while others show local enrichment or depletion affected by fractional factors ; moderate ecological risk areas (150 \u2264 RI < 300) account for 20.7% of the study area and are mainly distributed in Huize, Ludian, Yongshan, Yiliang, and Zhenxiong; high ecological risk areas (300 \u2264 RI < 600) account for 2.72% of the study area and are interspersed with moderate-risk areas; extremely high ecological risk areas (600 \u2264 RI) account for less than 1% of the study area and can be found in Huize, Ludian, and Yongshan. It is worth noting that the distribution of extremely high ecological risk areas is consistent with lead\u2013zinc mining in the study area (The udy area a. It is PERI (RI) is Hg (RI) in Ludian, Yongshan, and Zhenxiong is higher than 150, which shows a high potential ecological risk of heavy metals in these three counties. The RI values of the other counties are all greater than 100 except for that in Suijiang and Shuifu (The distribution of the single indicator s (ErAs) a. Hg, Cds (ErAs) a. Ludiand Shuifu a, indicaThe potential ecological risk of heavy metals in the soil of the study area has obvious differences between various land-use types . Figure  in soil , and it CDI), mg\u00b7(kg\u00b7day)\u22121. The results of several studies on the accumulation of heavy metals in soils under different land uses show that the diversity of land-use patterns directly affects the exposure frequency and CDI of adults exposed to the soil environment, which leads to unreasonable evaluation results of human health risks under different exposure methods [EF is 350 day\u00b7a\u22121; industrial and mining land EF is 250 day\u00b7a\u22121; and forestland, grassland, and other land-use type EF values are equal to 40 day\u00b7a\u22121 [CDIs of eight heavy metals were calculated through dietary intake, inadvertent inhalation, and dermal contact, and the calculation results of the hazard quotient (ijHQ) and the hazard index (HI) of 28,095 samples were based on the known reference dose (RfD), mg\u00b7(kg\u00b7day)\u22121 [The exposure of heavy metals in soil can be characterized by the chronic daily intake \u22121 ,46,47.HI < 1.0), accounting for 74% of the study area; moderate-risk areas (1.0 \u2264 HI < 1.5) are distributed in Huize, Ludian, and Yiliang, accounting for 0.28% of the study area. Huize and Ludian are the main areas with moderate risk, which may be related to the widespread existence of Cd-rich strata causing the accumulation of the heavy metal Cd in the soil. High-risk areas (1.5 \u2264 HI) are scattered in Huize, Ludian, and Daguan counties, of which the Ludian\u2013Lehong and Zhehai areas are more concentrated , and the remaining counties had values between 0.1 and 0.2  and Q (true) values obtained from the PMF model were the smallest and most stable when the number of running factors was 5, and the correlation coefficient between the observed and predicted concentration value (r2) of each heavy metal was the largest, which indicated that these five factors could comprehensive and reasonably reflect the information in the original data [The distribution of potential ecological risks and human health risks shows that the soil heavy metals have multiple sources. To further determine the source of heavy metals and the contribution of each heavy metal to the pollution source in the surface soil of the study area, this research used the PMF  to identnal data . Figure Factor 1 has higher factor contributions of Cu, Zn, and Ni . Over 20% of the study area has exposed basalt, and the distribution range of Cu and Ni anomalies in the soil is consistent with the widely exposed basalt in the area . ExistinFactor 2 is mainly related to Hg, As, and Pb . Studies have shown that over 40% of Hg emissions in China are from anthropogenic sources caused by coal combustion , and atmFactor 3 has the strongest correlation with Cd (the contribution rate is 85%), and the remaining heavy metals contribute less than 10% to this factor. Compared with the background value of soil (layer A) in China ,15, Cd i4 tons, and Zhaotong and Qujing are the third and fourth largest cities, respectively, of lead and zinc resource reserves in Yunnan Province [Factor 4 is dominated by Pb, As, Zn, and Hg . Commonly, soil from lead-zinc mining areas is enriched with Pb, Zn, and As from the mining of deposits ,71,72,73Cr, Ni, and Zn are associated with Factor 5 . Some researchers believe that the amounts of Cr, Ni, and Zn in the soil are controlled by the diagenetic parent rock and are related to the diagenetic composition ,68. The In summary, the sources of heavy metals in the study area are mainly reflected by the above five factors, of which Factor 1, Factor 3, and Factor 5 are all natural sources, indicating information on the basalt and carbonate rocks in the study area; in contrast, Factor 2 and Factor 4 reflect anthropogenic sources, which indicate information on mining activities and atmospheric precipitation caused by coal burning and traffic emissions. The total contribution of the five factors followed a descending order: Factor 4 > Factor 5 > Factor 1 > Factor 2 > Factor 3 . Lead\u2013ziHeavy metals show different levels of potential ecological risks in different land-use types, and the source of heavy metals in the area is complex and more or less restricted by types of land use. More importantly, the impact of industrial and mining land and farmland on ecological risk assessment is significant and it cannot be ignored.HQ followed a descending order: As > Cd > Pb > Ni > Cu > Hg > Zn > Cr, and the three exposure pathways showed the following: dermalHQ > ingestHQ > inhaleHQ. Cd and As are the main contributors to chronic health and carcinogenic risk in the study area; the heavy metals in the soil that cause these risks have multisource characteristics.Mining activities were one of the important factors affecting health risks posed by the soil. The contribution of the eight heavy metals to the The soil heavy metals in the study area mainly comes from five sources, of which parent material such as basalt, Permian, and Triassic carbonate rock constitutes the natural geological background source, the mining activities of lead\u2013zinc mines and the emissions of coal burning and automobile exhaust from residents constitute an anthropogenic source. The accumulation of heavy metals by mining activities contributed the most to the soil. The total contribution of the five sources followed a descending order: lead\u2013zinc mining (26.7%) > Triassic carbonate (23.7%) > basalt (20.9) > coal burning and automobile emissions (16.1%) > Permian carbonate (12.6%).The ecological risk assessment of areas with high geochemical background should focus on the analysis and understanding of the geological background, combined with the comprehensive judgment of the results of pollutant source analysis. Based on the different exposure pathways, the impact of human activities on health risk assessment is crucial. These results provide basic information on the study of heavy metals and guidance on the selection of natural source treatment options in the high geochemical background area of southwest China."}
+{"text": "EF) showed that As, Co, Cu, and Pb were not contaminated in these soils, while Cd, Cr, Hg, Ni, and Zn were lightly contaminated. The index of geoaccumulation (geoI) for the soda soils indicated that Co and Pb were uncontaminated, and Cr, Cd, Ni, Zn, Hg, Cu, and As were lightly contaminated. The potential ecological risk index (RI) indicated there were no or low risks for As, Co, Cr, Cu, Ni, Pb, and Zn. Although the concentrations of Cd and Hg in the soil were low, the two heavy metals exhibited moderate\u2013high ecological risk because they have high biological toxicity. Cd in the soils from Hetao Plain in Bayannur is mainly exchangeable and reducible fractions. The other heavy metals in these soda soils are mainly in residue fraction, implying that their mobility is low and not easily absorbed and used by plants. Heavy metal fractions, principal component analysis (PCA), and correlation analysis showed that As, Co, Cr, Cu, and Pb were mainly from natural sources, while Ni, Cd, and Zn were mainly from anthropogenic discharge-related irrigation, fertilizers, and pesticide application, and Hg was mainly from winter snowfall in the study area. Naturally sourced metal elements have obvious sediment properties, and their adsorption by clay minerals and coupling with organic matter along with sediment transport sorting. The salinity and pH of soda soils in the study area have a highly positive correlation, hence the influence of factors on the concentrations of soil heavy metals are consistent. For anthropogenically imported heavy metals, increasing salinity and pH promote the precipitation of metallic elements in water. Cd is present as an exchangeable and reducible fraction, while Ni and Zn are mainly sequestered by organic matter and clay minerals.Soil is an important natural resource in the agricultural areas of northwest China. The heavy metal concentration and ecological risk assessments are crucial for food safety and human health. This work collected 35 surface soil samples and focused on a typical soda soil quality of the Hetao Plain in Bayannur, which is an important grain production base in northern China. The concentration and composition of heavy metal (arsenic (As), cobalt (Co), copper (Cu), lead (Pb), cadmium (Cd), chromium (Cr), mercury (Hg), nickel (Ni), zinc (Zn)), soluble salts, total organic carbon (TOC), and minerals of the surface soils were analyzed to assess the biotoxicity, ecological risk, sources, and influencing factors of heavy metals in these soda soil from this region. The enrichment factors ( Soil is not only a natural resource for human survival but one of the most important components of the ecosystem. However, the heavy metal pollution that accompanies economic development and the high-intensity agricultural activities of modern society has become a serious problem worldwide ,2. AlthoEF), Index of Geo-Accumulation (geoI), Potential Ecological Risk Index (RI), and Risk Assessment Code. Heavy metals, minerals, and ions were also investigated by principal component analysis and correlation analysis for heavy metal sources and influencing factors. The article aims to provide a scientific basis for the prevention and control of potential pollution of arable soils in the study area.At present, many agricultural lands in China are threatened by various kinds of pollution, and the area of arable land with moderate or heavy pollution is about 3.33 million hectares, and the production and ecological risks caused by soil pollution in many areas are already very serious. The exceedance rate of inorganic pollutants accounted for 82.8% of the total number of sites, while the exceedance rate of cadmium sites was 7% ,18,19. LThe study area is the southern Hetao Plain in Bayannur City, Inner Mongolia Autonomous Region, China, under a typical mid-temperate continental monsoon climate, with an average annual temperature from 3.7 \u00b0C to 7.6 \u00b0C and rainfall of 130\u2013285 mm ,22. The A total of 35 surface soil (0\u201320 cm) samples were collected from farmland in the study area . The sam2 content was measured to calculate the TOC [TOC was determined by an elemental analyzer  for the organic carbon content of the samples. The samples were ground, passed through a 100 mesh sieve, and the organic carbon in the soil samples was cauterized by burning at a high temperature of 1100\u00b0 to release carbon dioxide, and then the CO the TOC . For min\u2212, +, Na+, Ca2+, and Mg2+ were determined by ion chromatography , and The soil samples were air-dried, ground finely, and sieved through an 18-mesh sieve. A 2.5:1 water to soil ratio leachate was prepared, and the pH of the soil was determined using a Sartorius standard pH meter PB-10 . A 5:1 witration .3-HF-HClO4, and after complete digestion, the digested material was transferred to a clean volumetric flask and diluted to 1000 times the weight of the samples using ultrapure water, and the determination of the major elements was performed using an inductively coupled isotope emission spectrometer  for determination of major elements and inductively coupled plasma mass spectrometer  for determination of trace elements [The samples were weighed 20~30 mg (accurate to 0.01 mg) through a 200-mesh sieve, and the samples were digested in a PTFE digestion tank using HNO3COOH was used for the exchangeable fraction (F1), 30 mL of 0.1 mol/L NH2OH-HCl was used for the reducible fraction (F2); the oxidizable fraction (F3) was extracted with 5 mL of 8.8 mol/L H2O2 followed by 25 mL of 1 mol/L CHCOONH4, residual fraction (F4) was the extraction with HNO3-HF-HClO4 [The 12 soil samples were selected and weighed 1.00 g in 100 mL polypropylene centrifuge tubes, and the soil heavy metal forms were analyzed by a modified BCR procedure. Then, 30 mL of 0.11 mol/L CHHF-HClO4 . The Cd,Total organic carbon analyzer can detect TOC concentration detection error of \u00b11%. The detection limit and detection error of ion chromatography detection of anion and cation are 1 mg/kg and \u00b15%, respectively. During the test procedures for heavy metals, standard reference samples (GSS-18), blank samples, parallel samples, and study samples were analyzed similarly to control the quality of the entire analytical procedure and ensure comparable detection results. The repeatability of each element of the sample was greater than 95%, and the recovery of the standard sample ranged from 90% to 105% .EF) is an essential indicator for evaluating the level of contribution of anthropogenic and natural sources to the elemental content of particulate matter and quantitatively evaluating the degree and source of pollution [iC is the concentration of soil heavy metal element i and refC is the reference element content. Al was chosen as the reference element in this study as human activities have a small impact on the content of Al. The background element is the alkaline soil elements in the background value from Chinese soil elements [EF was classified into three levels, EF < 1.5 as no contamination, 1.5 \u2264 EF < 3 as weak contamination, and EF \u2265 3 as moderate contamination.The enrichment factor , commonly referred to as the Muller index [iC is the measured content of heavy metal i; k is the correction index (k = 1.5); iB is the soil background value of heavy metal i. According to the ground accumulation index geoI, the pollution level is divided into 7 levels [geoI < 0, no pollution; 0 \u2264 geoI < 1, no pollution to moderate pollution; 1 \u2264 geoI < 2, moderate pollution; 2 \u2264 geoI < 3, moderate pollution to strong pollution; 3 \u2264 geoI < 4, strong pollution; 4 \u2264 geoI < 5, strong pollution to very strong pollution; geoI \u2265 5, very strong pollution.The geological accumulation index  was proposed by Hakanson [RI evaluated by the grading method with a comparable equivalence property index. Its calculation Formula (3) is as follows:fiC is the contamination factor of heavy metal i; iC is the measured concentration of heavy metal i; and niC is the geochemical background value of heavy metal i. riE and riT are the ecological risk index and the toxicity coefficient of heavy metal i, respectively. The toxicity coefficient of heavy metal are the values of Zn = 1, Cr = 2, Co = Ni = Cu = Pb = 5, Hg = 40, and Cd = 30. Since the heavy metal species in this study were different from Hakanson [RI limits corresponding to each ecological risk level were adjusted according to the heavy metal species and their toxicity coefficients, and the adjusted evaluation criteria were: RI < 150 for minor potential ecological risk; 150 \u2264 RI < 300 for medium potential ecological risk; 300 \u2264 RI < 600 for strong potential ecological risk; where RI < 150 is a slight potential ecological risk; 150 \u2264 RI < 300 is a medium potential ecological risk; 300 \u2264 RI < 600 is a strong potential ecological risk; RI \u2265 600 is a very strong potential ecological risk [The potential ecological risk index  method was proposed by Perin et al.  based onThe RAC indices can be grouped into the following five categories: RAC < 1%, no risk; 1% < RAC < 10%, low risk; 11% < RAC < 30%, considerable risk; 31% < RAC < 50%, high risk; RAC > 50%, very high risk.Statistical analyses were performed using the SPSS 19.0 software . Correlation analysis and correlation analysis heat map by origin 2020 . The sampling site map  was draw+, Na+, Ca2+, Mg2+, Cl\u2212, + and Ca2+, while the anions were Cl\u2212, +, Na+, Ca2+, and Mg2+ ranged from 2.41 to 105.14, 12.32 to 285.01, 15.76 to 167.16, and 7.56 to 120.23 mg/kg, respectively; the Cl\u2212, The EC, pH, KThe TOC and minerals in the soda soil samples from the study area are shown in The statistics of heavy metals in the soils of the study area and other agricultural areas in China are shown in The BCR extraction method classifies soil heavy metals into four chemical forms: acid extractable; reducible (Fe-Mn oxide bound); oxidizable (organic bound); and residue. The acid extractable form covers the exchangeable and carbonate-bound forms, which are highly mobile and can be directly absorbed and used by organisms. Heavy metals in the residue form exist mainly in the crystalline matrix of primary and secondary minerals and silicates, which are the most stable and cannot be easily released under normal natural conditions. The composition of heavy metals in soils is closely related to the migration, transformation, cycling processes, and environmental impacts of heavy metals in the surface soil. Therefore, the composition of heavy metals is more likely to accurately assess the environmental impact of soil heavy metals as opposed to the assessment of heavy metal concentrations in soil.The results of the heavy metal composition of these soils showed that the residual fraction (F1) of As, Co, Cr, Hg, Ni, and Zn was higher than 85% of the heavy metal of the soil, indicating that these heavy metals are mainly embedded in the mineral lattice and cannot be easily migrated for plant uptake. The content of the oxidizable fraction (F2) of soil heavy metals is low. The reducible fraction (F2) of Cd, Cu, and Pb in the soil is relatively high, accounting for about 20%, followed by As, Co, Ni, and Zn, accounting for about 10%, and Cr and Hg are low, accounting for less than 5%. The weakly acid-extracted fraction (F1) of Cd in soils is higher than all other heavy metals, accounting for more than 50% . Since tEFs of the nine heavy metals in the soils were in the order of Cr (2.87) > Cd (2.34) > Ni (2.27) > Hg (1.97) > Zn (1.68) > Cu (1.45) > As (1.33) > Co (1.13) > Pb (0.87). The EFs of As, Co, Cu, and Pb were less than 1.5, indicating no contamination, and the EFs of Cd, Cr, Hg, Ni, and Zn had EF values between 1.5 and 3, which are moderately contaminated. According to this standard As, Co, Pb is no pollution; Cd, Ni, Hg, Zn, Cu is no pollution\u2013minor pollution; Cr is minor pollution\u2013moderate pollution  > Cd (0.27) > Ni (0.23) > Zn (0.14) > Hg (0.11) > Cu (0.08) > As (0.03) > Co (\u22120.03) > Pb (\u22120.16). The geoI values of Co and Pb are negative, indicating no contamination. The range of geoI values of Cr, Cd, Ni, Zn, Hg, Cu, and As are between 0 and 1, indicating that the study area is low contamination.The ollution a. FigureIn this work, the summation of potential ecological risk coefficients of heavy metal contaminants for 36 soil samples showed that most of the samples were of medium ecological risk. Only one sample showed very high ecological risk, two samples showed high ecological risk, and two samples showed low ecological risk . The val Pb > Zn a. Cd andEF and geoI focus on the enrichment and accumulation of heavy metals relative to background soil, while RI assesses ecological risk based on the biotoxicity of different heavy metals, and RAC emphasizes the mobility of heavy metals, i.e., the components that can be taken up by plants. Therefore, it could be concluded that most of the soils in the study area have low levels of heavy metals and are non-contaminated or mildly contaminated. However, as the most biotoxic Hg and Cd, the soil in the study area has a high ecological risk. Owing to the contribution of these two heavy metals, the RI of the sampling sites in the study area exhibits a medium risk\u2013high risk. It is also notable that low levels of Cd are highly biotoxic and mobile, which should be passivated or adsorbed in various conditions at the pH < 7.The risk assessment code (RAC) of the 12 selected heavy metals in the study area are shown in p < 0.001), indicating that the application of PCA was appropriate. The first principal component (PC1) and the second principal component (PC2) of the soil samples that explained the variance accounted for 45% and 16% of the total variance, respectively. PC1 was significantly positively loaded with Al (0.74), Fe (0.96), Ca (0.66), Mn (0.96), As (0.88), Co (0.85), Cr (0.41), Cu (0.84), Pb (0.95), clay (0.88), and TOC (0.88) and negatively loaded with Quartz (\u22120.76) and Plagioclase (\u22120.58). PC2 was significantly positively loaded with Cd (0.83), Ni (0.93), and Zn (0.86).For the purpose of identifying the sources of heavy metals and their influencing factors, principal component analysis (PCA) and correlation analysis were used for As, Cd, Co, Cr, Cu, Hg, Ni, Pb, Zn, pH, EC, Quartz, Plagioclase, and clay . The PCACombined with the composition of heavy metal in the soda soils, it is concluded that there are three sources of heavy metals as follows: (1) As, Co, Cr, Cu, and Pb have a highly positive correlation with clay minerals , Fe, andThe bioavailability, toxicity, and mobility of heavy metals were affected by soil or water pH, soluble salt composition, organic matter, and clay minerals. A fine-grained fraction of minerals plays a significant role in the quality of water and sediment, especially in the adsorption of metals , and eve\u2212 to form exchangeable and reduced state components, while the higher content of Ni and Zn was mostly chelated by organic matter and clay minerals, except for a small amount combined with \u2212.In the process of transporting and sorting the clastic particles from the mountain front to the downstream, the fine-grained sediments are gradually enriched in the downstream area, and these metal elements and organic matter are adsorbed with the clay mineral surface and interlayer, forming a complex of organic matter, metal elements, and clay minerals. The salinity of soda soils in the study area has a good positive correlation with pH, so the influence of these two factors on the content of soil heavy metals is consistent. For heavy metals of anthropogenic origin, the increase in salinity and pH favored the precipitation of metal elements in the water column, and the lower content of Cd combined with +, K+, and Ca2+, while the anions were Cl\u2212 and EF and geoI indicated that these heavy elements are non-contaminating or of low contamination levels, with a relatively low degree of anthropogenic contamination. RI and the Risk Assessment Code indicated Cd and Hg responded to moderate ecological risk, while As, Ni, Cu, Cr, Co, Pb, and Zn all belonged to a slight ecological risk. In particular, since Cd is a mainly exchangeable and reducible fraction in the study area, appropriate measures should be taken for passivation or adsorption under an alkaline environment.In this paper, the pH, salinity, and mineral composition of the agricultural soil in Hetao Plain of Bayannur were first analyzed. As a typical sodic soil, it has a pH greater than 8, and the cations in the soluble salt of the surface soil are Na\u2212 combine to form the weak acid exchangeable fraction or reduced fraction, while Ni and Zn are adsorbed by clay minerals to form the residue. Hg originated mainly from a source of atmospheric fallout, and probably from the recorded winter snowfall in Hetao Plain of Bayannur. The Hg levels in these soda soils do not appear to be related to clay minerals, pH, or salinity.The compositions of heavy metals, principal component analysis, and correlation analysis showed that these heavy metals come from three sources. As, Co, Cr, Cu, and Pb mainly came from the natural accumulation of sediments from the alluvial plains. The contents of these metal elements in soil are controlled by particle size-sorting effect and usually exist as residues combined with clay minerals and organic matter. Ni, Cd, and Zn are mainly derived from anthropogenic discharge-related irrigation, fertilizers, and pesticide application. Due to the synergistic influence of pH and salinity, these metal elements precipitate rapidly after entering water; Cd and"}
+{"text": "The Pb content of 50% of the wheat grain samples exceeds the lead limit in the standard. The evaluation results of the single factor pollution index and geoaccumulation index show that the pollution degree of heavy metals in the soil is Cd\u2009>\u2009Pb\u2009>\u2009Cu\u2009>\u2009Zn\u2009>\u2009Ni. The potential ecological risk index in the study area is 288.83, and the soil heavy metal pollution is at a moderate-considerable ecological risk level. The average value of Cd's single-factor environmental risk index is 233.51, which belongs to the high environmental risk and is the main influencing factor. Cd and Pb in soil are significantly disturbed by the production activities of heavy metal processing enterprises around the farmland. It is speculated that there are two primary sources of soil heavy metal pollution in the study area. Cd, Pb, Zn, and Cu are mainly industrial and mobile sources, and Ni is primarily agricultural and natural sources.Studying the pollution status, spatial distribution characteristics, and sources of heavy metals in farmland soil in Anxin County will provide a method basis for the next step of soil remediation. This study investigates the contents of Zn, Cu, Pb, Cd, and Ni in wheat grains and soil samples. Moreover, different methods are used to evaluate soil heavy metal pollution. The results show that the soil in the study area is weakly alkaline. Cu, Zn, and Ni contents in the ground are lower than the risk screening values for soil contamination of agricultural land. In comparison, Cd and Pb contents are higher than the screening value of soil pollution risk of agricultural land, and the proportion of points lower than the control value of soil pollution risk of agricultural land are 64.58% and 16.67%, respectively. The farmland with high Cd and Pb content is mainly distributed near roads and factories and concentrated primarily on 0-20\u00a0cm topsoil. The Cd content in wheat grains meets the standard, but 4.17% of the samples are close to 0.1\u00a0mg kg Heavy metal pollution in the soil can lead to the destruction of the balance of the ecosystem. Some crops cannot grow and develop normally, resulting in reduced agricultural output and a decline in quality. At the same time, it enters the human body through the food chain and other channels and accumulates in the human body, endangering human health4.Soil is a necessary guarantee for the survival and development of human society. The soil environmental quality of cultivated land directly affects food security and the quality of agricultural products and is closely related to people's health and social development. With the continuous development of the economy and society, human activities have led to increasing pollution of heavy metals in the soil5, sewage irrigation7, agricultural production8, mining9. Farmland around heavy metal enterprises is threatened by heavy metal pollution10. A nationwide survey in China revealed that little pollution was found in typical farmland, far from noticeable anthropogenic emissions, but Cd and Hg in mining-smelting and industrial areas significantly accumulated in soil11.With the development of industry, more and more pollutants enter farmland soil through various ways, such as atmospheric settlement12. The effects of heavy metals in soil on plants, soil microorganisms, and soil animals have been well documented13. The excessive accumulation of heavy metals in soils has led to phytotoxic metabolism and inevitably poses risks to human health via the food chain16. Long-term exposure to heavy metals contributed to mental and behavioral disorders and increased the risk of cancer17. Therefore, it is necessary to conduct soil heavy metal pollution assessment.There is a consensus that heavy metals have great harm to the ecological environment18. Geoaccumulation index method combines the changes of background value caused by natural diagenesis in the evaluation of soil heavy metal pollution19, the potential ecological risk index method comprehensively considers the toxic effects of heavy metals20, these two methods have been widely used in the evaluation of soil pollution. Many research scholars have also studied the bioavailability of heavy metals21. The existing soil pollution evaluation methods are limited to whether the content exceeds the standard, and there are few studies on the analysis of the background value of soil elements and the source analysis of heavy metals. There is no comprehensive evaluation of soil heavy metal pollution.The single factor index method and Nemerow pollution index method often are used to assess soil heavy metals pollution22. Extensive development will significantly impact the soil environment25. So far, few articles have conducted comprehensive research on farmland soil ecological environment in Anxin County, and the risk of heavy metal pollution is unknown. On December 25, 2018, the State Council of China issued and implemented the Reply of the State Council on the General Planning of the Hebei Xiong\u2019an New District (2018\u20132035), requiring effective control of rural non-point source pollution. Studying the pollution situation, spatial distribution characteristics, and sources of heavy metals in farmland soil in Anxin County will provide a method basis for the next step of soil remediation.On April 1, 2017, China's State Council decided to establish Xiong\u2019an New Area in Hebei Province of North China. The planning scope of Xiong\u2019an New Area covers Xiong County, Rongcheng County, Anxin County, and some surrounding areas in Hebei Province of China. It is located in the hinterland of Beijing, Tianjin, and Baoding City. It is 105\u00a0km away from Beijing. Anxin County is the most extensive footwear production base, waste metal distribution center, and distribution center in North China. Metal smelting enterprises mainly focus on lead-containing non-ferrous metal smelting. Due to the lack of unified planning and management, they are mainly small enterprises, and the distribution is relatively scattered. Some enterprises still have high energy consumption, backward production technology, and low comprehensive utilization rateAnxin County is located in the central part of Hebei Province Fig.\u00a0, with 7326. The county's arable land area is 480,000 mu, the soil with higher terrain develops into cinnamon soil, and the soil with lower terrain develops into flavor-aquic soil27. The main crops are wheat and corn, which are ripened twice a year. Anxin County is the most extensive footwear production base, waste metal distribution center, and distribution center in North China. Overall, industrial development is lower, and the ecological environment is better in the region. The main survey area of this study is the farmland near the heavy metal enterprise in Anxin County, with a total survey area of about 200 mu.According to the seventh census data, as of 0:00 on November 1, 2020, the permanent population of Anxin County was 453,723Technical rules for monitoring of environmental quality of farmland soil (NY/T 395-2012)28, the study area was divided into cells of 50\u00a0m\u2009\u00d7\u200950\u00a0m, and soil samples in the plow layer (0\u201320\u00a0cm) were collected in the cells. 48 soil samples were collected by the plum-shaped distribution method , D1 , D2 , D3 . D0 was no heavy metal-contaminated farmland soil in the previous survey, and D1, D2, and D3 were heavy metal-contaminated farmland soil in the study area. Dig a soil profile with a length\u2009\u00d7\u2009width\u2009\u00d7\u2009depth of 2.0\u00a0m\u2009\u00d7\u20091.0\u00a0m\u2009\u00d7\u20091.5\u00a0m in the soil layers of 0\u201320\u00a0cm, 20\u201340\u00a0cm, 40\u201360\u00a0cm, 60\u201380\u00a0cm, and 80\u2013100\u00a0cm sampling. Sample preparation was as described above.The wheat sampling points corresponded to the soil sampling points and were collected by the plum-shaped distribution method one week before the wheat harvest. A total of 48 wheat samples were collected. After the collected wheat ears were placed on the balcony to dry naturally, the outer skin was manually rubbed to obtain the grain part, which was pulverized with a pulverizer, passed through a 0.15\u00a0mm nylon sieve, packaged in a self-sealing bag, and placed in a desiccator for storage. In this study, 48 soil and 48 wheat grain samples were collected in the study area on July 10, 2019  and available heavy metals (by DTPA [pH\u2009=\u20097.3] extraction)29. The air-dried soil samples sieved through 0.15\u00a0mm were tested for total Cu, Zn, Pb, Cd, and Ni (HCl-HNO3-HClO4-HF digestion). Put 0.1\u00a0g of air-dried soil samples sieved with 0.15\u00a0mm into a digestion tube, and add a few drops of water to moisten. Then add 3\u00a0mL of HCl and 1\u00a0mL of HNO3, cover with a small funnel, and place it in a fume hood to soak overnight. The next day, it was put into the SH220F graphite digestion apparatus, and the temperature was gradually raised to 130 \u00b0C for digestion for 2\u00a0h. When the remaining 2\u20133\u00a0mL was evaporated, it was removed and placed in a ventilated place to cool. Add 1\u00a0mL of HClO4 and 2\u00a0mL of HF, cover with a small funnel, heat up to 160\u00a0\u00b0C in steps, continue to digest for 2\u00a0h, then open the lid and heat to remove silicon. When heated until white smoke of perchloric acid appears, cover it to make the black organic substances on the inner wall of the digestion tube disappear, open the cover, and steam until the content is viscous but not flowing, then remove and cool. After cooling completely, rinse the inner wall and lid with deionized water, transfer the total amount and make up the volume to a 50\u00a0mL volumetric flask. The heavy metal concentrations on the filter were analyzed using the HK-8100 inductively coupled plasma emission spectrum and TAS-990AFG atomic absorption spectrophotometer.The air-dried soil samples sieved through 0.84\u00a0mm were tested for pH , and kept overnight (\u223c18\u00a0h). Afterward, the beaker was placed on a hot plate and heated at 100 \u00b0C for 0.5\u00a0h. After cooling down, the sample was extracted with perchloric acid (5\u00a0mL); the heating process continued until the solution was colorless, transparent, and evaporated to a final volume of 2\u00a0mL. Finally, two drops of concentrated nitric acid were added to the solutions. The heavy metal content in the samples was determined using the HK-8100 inductively coupled plasma emission spectrum and TAS-990AFG atomic absorption spectrophotometer.Soil quality-Analysis of soil heavy metals-atomic absorption spectrometry with aqua regia digestion (NY/T 1613\u20132008)30, and available Pb and Cd by Soil quality-Analysis of available lead and cadmium contents in soils-Atomic absorption spectrometry (GB/T 23769-2009)31 for determination, the detection limit is Cu 2\u00a0mg kg\u22121, Zn 0.4\u00a0mg kg\u22121, Pb 5\u00a0mg kg\u22121, Cd 0.01\u00a0mg kg\u22121, Ni 2\u00a0mg kg\u22121. Cd in wheat samples is determined according to the National Food Safety Standard-Determination of Cadmium in Food (GB 5009.15\u20132014)32, the detection limit is 0.001\u00a0mg kg\u22121, and the quantification limit is 0.003\u00a0mg kg\u22121. Pb in wheat samples is determined according to the National Food Safety Standard-Determination of Lead in Food (GB 5009.12-2017)33 graphite furnace atomic absorption spectrometry, the detection limit is 0.02\u00a0mg kg\u22121, and the quantification limit is 0.04\u00a0mg kg\u22121.Heavy metals in soil samples are determined by 34. The analytical data's reporting rate, accuracy, and precision reached 100%. The analytical data is reliable.For elemental analysis, three replicate samples, and national first-class reference materials (soil standard sample GBW08303 and shrub dead leaf sample GBW07603) were used for quality monitoring. The quality of sample analysis and test and the detection limit of each element index meet the technical specification requirements for sample analysis of ecological geochemical evaluation35. The damage degree of heavy metal pollutants in soil was determined by the single factor pollution index method. Soil environmental quality-Risk control standard for soil contamination of agricultural land  (GB15618-2018)36 issued by the Ministry of Ecological and Environment of China was as an assessment standard and shown in Table A) means that the pollutant content in the soil of agricultural land is equal to or lower than this value, and the risk to the quality and safety of agricultural products, the growth of crops or the soil ecological environment is low and can be ignored in general. If the value exceeds this, there may be risks to the quality and safety of agricultural products, crop growth, or soil ecological environment, and soil environmental monitoring and coordinated monitoring of agricultural products should be strengthened. In principle, safe use measures should be taken. Risk intervention values for soil contamination of agricultural land (B) means that if the content of pollutants in the agricultural land exceeds this value, and the edible agricultural products do not meet the quality and safety standards, the risk of agricultural land soil pollution is high. In principle, strict control measures should be taken. The pollution index of heavy metals was calculated with Eq.\u00a0. Si is the standard assessment value of heavy metal i (mg kg\u22121), the value is risk screening values for soil contamination of agricultural land (A) of Soil environmental quality\u2014Risk control standard for soil contamination of agricultural land  (GB15618-2018). Pi is the pollution index of heavy metal i in soil. According to the Pi value, when Pi is less than or equal to 1, the soil does not exceed the national standard. 1\u2009<\u2009Pi\u2009\u2264\u20092 indicates light pollution, 2\u2009<\u2009Pi\u2009\u2264\u20093 indicates moderate pollution, Pi\u2009>\u20093 indicates heavy pollution37.In this study, the single pollution index method was used to evaluate the contamination of heavy metals in soil. The single pollution index method is one of the most widely used methods to determine the degree of pollution at home and abroad, and it can reflect the pollution degree of different pollutants to the environmentTrial) GB5618-201838. RI was calculated with Eq.\u00a0, Cni is the corresponding background value of heavy metal i in soil (mg kg\u22121) of Hebei Province39. Tri is the toxicity coefficient of heavy metal i in soil, Eir is the single factor ecological risk index of heavy metal i in soil, and RI is the comprehensive ecological risk index of heavy metal i in soil. Tri of Cu, Zn, Pb, Cd, and Ni is 5, 1, 5, 30, and 5, respectively40. The Eir and RI values are classified with a system described by Hakanson et al., and the classification is shown in Table The potential ecological risk index (RI) was used to evaluate the ecological risk of heavy metals in soilwith Eq.\u00a0:2\\docume41. It was calculated with Eq.\u00a0. Bi is to evaluate the geochemical background values of heavy metal i. Igeo is the geoaccumulation index of heavy metal i in soil. In this study, Bi is selected from the average value of the Chinese soil element background. Table 42.The geoaccumulation index method was proposed by Muller in 1969, which was mainly used to evaluate the pollution degree of heavy metals in sedimentswith Eq.\u00a0:3\\documeCni and Bi selected the average value of every single element in layer A soil of Hebei Province as the element background value39, and the element background values of Cu, Zn, Pb, Cd, and Ni are 21.8\u00a0mg kg\u22121, 78.4\u00a0mg kg\u22121, 21.5\u00a0mg kg\u22121, 0.094\u00a0mg kg\u22121, and 30.8\u00a0mg kg\u22121, respectively.In formula  and 3),,3), Cni 43. Transfer coefficient was calculated with Eq.\u00a0, while Cs is the content of heavy metal i in soil (mg kg\u22121).The element transfer coefficient can reflect the plant's ability to accumulate heavy metals and is the ratio of the content of an element in the plant sample to the content of the corresponding element in the soil. It can, to some extent, explain the enrichment of heavy metals in the plant bodyThe experimental data analysis was carried out with Microsoft Office Excel 2010 software, ArcGIS Desktop 10.2 was used for Kriging analysis of spatial points, Origin 2017 software was used for mapping, and Adobe Photoshop CC 2018 was used for image merging.Regulation for the collection of genetic resources (HJ 628\u20132011), The Technical Specification for soil Environmental monitoring (HJ/T 166\u20132004), the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. We have permission to collect wheat seeds. We guarantee that all samples are used for scientific research only.All plant samples of this study complied with the Specification of land quality geochemical assessment (DZ/T 0295-2016)44 for the definition of soil acidity, the Kriging interpolation analysis of soil acidity is conducted by using ArcGIS, and the results are shown in Fig.\u00a045. When plants absorb heavy metals, they will cause a decline in crop yield and quality and endanger human health through the food chain.According to Fig.\u00a0\u22121, respectively. The average values of Cu, Zn, Pb, Cd, and Ni are 2.92 times, 2.19 times, 6.36 times, 7.78 times, and 1.34 times of the background values of soil elements in Hebei, respectively. The contents of Cu, Zn, and Ni in the soil are all less than the risk screening value, and the risk to the quality and safety of agricultural products, crop growth, or soil ecological environment is low and can be ignored in general. The soil Pb and Cd content percentages between the risk screening value and risk intervention value are 16.67% and 64.58%, respectively. The problem of exceeding the standard of Pb and Cd is more serious, and there may be risks to the quality and safety of agricultural products, crop growth, or soil ecological environment, and safe utilization measures should be taken.Table 46. By analyzing the characteristics of heavy metal content in the study area, it can be seen that the distribution of the five elements in the local soil is moderately variable, and the CV of Pb and Cd is large. It shows that the distribution of Pb and Cd is uneven, and it is affected to some extent by human activities.The coefficient of variation (CV) is the ratio of standard deviation to the average value, which can characterize the degree of dispersion of data. The CV is classified into a weak variation (CV\u2009<\u20090.1), moderate variation (0.1\u2009<\u2009CV\u2009<\u20090.9), or high variation (CV\u2009>\u20090.9)It can be seen from Table 47.Figure\u00a048 but also closely related to human activities49. The same element may have multiple distribution types in different sections of soil profiles, but the probability of a specific type is relatively high. In the four soil profiles of D0, D1, D2, and D3 studied in this work, the vertical distribution characteristics of heavy metals Cd and Pb on the profiles have both commonalities and differences. According to Figs.\u00a050, including perennial agricultural farming, road transportation, and the production activities of the surrounding heavy metal processing industry. These factors have significant effects on the vertical migration and enrichment of heavy metals in the soil. The vertical distribution trends of heavy metal Cd and Pb elements in the four soil profiles are the same, but the contents in the 0\u201320\u00a0cm layer are different. The main reason is that the four soil profiles are located in different blocks. D1, D2, and D3 are heavy metal-contaminated farmland soils in the study area.In the vertical section of the soil, the distribution of elements is not only restricted by its geochemical properties, soil-forming parent material, and soil-forming processes under natural conditions\u22121 and 10.00 to 75.10\u00a0mg kg\u22121, respectively, and the average effective content of Cd and Pb is 0.47\u2009\u00b1\u20090.18\u00a0mg kg\u22121 and 28.69\u2009\u00b1\u200913.75\u00a0mg kg\u22121, respectively. By analyzing the dispersion degree of the available content of Cd and Pb in the study area, it can be seen that the distribution of Cd and Pb in the local soil is moderately variable. At the same time, the coefficient of variation of Pb is more significant, indicating that Pb is affected by human activities to a certain extent. The effect is more vital than Cd.Table 51. The study area is mainly dominated by Cd and Pb pollution, so the practical content of Cd and Pb at 48 sites in the study area is analyzed 53 stipulates that the limit values of Cd and Pb in wheat grains are 0.1\u00a0mg kg\u22121 and 0.2\u00a0mg kg\u22121, respectively. Among the 48 wheat samples investigated, the Cd content meets the standard, but 4.17% of the samples are close to 0.1\u00a0mg kg\u22121 (more than 0.09\u00a0mg kg\u22121). The content of Pb in 50% of the samples exceeds the standard, and the Pb in the wheat grains exceed the standard seriously.Table 54.The evaluation of five heavy metal elements by the single pollution index method is shown in Fig.\u00a0Figures\u00a055.The potential ecological risk index method Fig.\u00a0 shows thAccording to Fig.\u00a0The proportion of soil sample pollution classification shows no Ni pollution in the study area, and the pollution rates of the four elements of Cu, Zn, Pb, and Cd all reach 100%. Among them, for Pb, 52.08% of the soil is at a middling pollution level, 43.75% of the soil is at a moderate pollution level, and 4.17% of the soil is at a biased pollution level; For Cd, 29.17% of the soil is at a middling pollution level, 66.67% of the soil is at a moderate pollution level, and 4.17% of the soil is at a biased pollution level; For Cu, 58.33% of the soil is at a light pollution level, and 41.67% of the soil is at a middling pollution level; For Zn, 97.92% of the soil is at a light pollution level, and only 2.08% of the soil is at a middling pollution level. Cu and Zn contribute less to soil pollution, while Pb and Cd contribute the most. The significant standard deviation of Pb and Cd indicates that the Pb and Cd have a high degree of dispersion in the geoaccumulation index and a high variation coefficient. The two elements in the soil are significantly interfered with by the production activities of heavy metal processing enterprises around the farmland, which leads to severe pollution of Pb and Cd in the local soil. According to the evaluation results of the geoaccumulation index Fig.\u00a0, it can 58. Using SPSS software to perform KMO test on the data, the obtained statistic value is 0.742, and the accompanying probability of the Bartlett sphericity test is 0.000. Therefore, principal component analysis and discriminant classification techniques can be used to study pollution types of farmland around the heavy metals industry in Anxin County, Hebei Province of China, and the analysis results are shown in Table The principal component analysis is a multivariate statistical analysis method that converts multiple indicators into a few unrelated comprehensive indicators and classifies the comprehensive indicators according to specific rules. It is often used to identify the source of elements in the medium and obtain the contribution rate of different sources to the same element59, and the accumulation of industrial waste is also an essential source of Cd pollution61. Most cars use leaded gasoline as fuel, and long-term use has caused the accumulation of soil Pb63. Because of its high corrosion resistance and high thermal conductivity, Cu is an essential component of vehicle braking systems and automotive radiators64. The wear of automobile parts will cause copper to enter the surrounding environment, so It is also often used as the identification element of the source of traffic66. Maximilian et al.67 found that Zn can also be a marker element for mobile sources. Cu, Pb, and Zn will enter the surrounding environment as the car parts wear out during the driving process69. PC1 is closely related to Cd, Zn, Cu, and Pb and has the characteristics of a traffic source and an industrial source. Therefore, it is speculated that PC1 may be a traffic source and an industrial source, mainly affected by vehicle traffic, exhaust emissions, and industrial production.Cd, Pb, Zn, and Cu show higher loads on the first principal component, respectively 0.859, 0.845, 0.840, and 0.738. The four elements are significant within the 95% confidence interval, indicating that the four elements have the same pollution source. The spatial distribution of the four heavy metal elements is relatively similar. The areas with higher content are concentrated in the southeast of the study area, the distribution area is close to the road, and there is a heavy metal processing plant area around. The average value of the four heavy metal elements is higher than the soil background value, indicating that it is significantly affected by human activities. Related research shows that metal smelting and other production activities will increase the content of Cd in the atmosphere, Cd sedimentation causes pollution to the soil70. The evaluation result of the soil accumulation index of Ni is pollution-free, and the contribution of human activities to Ni is relatively low, mainly natural sources71. The content is mainly affected by the soil-forming parent material. Therefore, it is speculated that the pollution sources represented by PC2 are natural sources and agricultural sources.Ni exhibits a higher load on the second principal component, which is 0.990. The Ni in the soil mainly comes from rock weathering, atmospheric dust reduction, irrigation water (including nickel-containing wastewater), farmland fertilization, and the decay of plant and animal residues78. Compared with the study by Zhou et al.73 in Hebei Province, the average content of Cd and Pb is slightly lower, but the difference is not significant because the selected study area is different, and there are differences in human interference. Due to the different soil properties in different plots, the results obtained are also different, but overall, there is serious Cd and Pb pollution. The results of this study are consistent with those of previous studies.In order to better analyze the pollution characteristics of heavy metals in different types of farmland soils, the results of this study were compared with the average values of heavy metal contents obtained by other studies. The comparison results are shown in Table In summary, this paper reviews the heavy metal pollution of farmland soil in Anxin County, Hebei, China. The main points obtained through the analysis are as follows:National Food Safety Standard\u2014Limits of Contaminants in Foods (GB2762-2017), the Cd content in wheat grains all meets the standard, and the Pb content in 50% of the wheat grain samples exceeds the standard lead limit. There is a risk of Cd and Pb contamination in the study area.The soil in the study area is weakly alkaline, and the contents of Cu, Zn, and Ni in the soil are all lower than the risk screening values for soil contamination of agricultural land. In comparison, the contents of Cd and Pb are higher than the risk screening values for soil contamination of agricultural land and lower than the risk intervention values for soil contamination of agricultural land. The proportions of the values points are 64.58% and 16.67%, respectively, and the pollution is mainly concentrated in the topsoil of 0\u201320\u00a0cm. According to the The evaluation results of the single pollution index, potential ecological risk index, and geoaccumulation index show that the order of soil heavy metal pollution risk in the study area is Cd\u2009>\u2009Pb\u2009>\u2009Cu\u2009>\u2009Zn\u2009>\u2009Ni. The potential ecological risk index in the study area is 288.83, and the soil heavy metal pollution is at a moderate-considerable ecological risk level. The average value of Cd's single-factor environmental risk index is 233.51, which belongs to the high environmental risk and is the main influencing factor. Pb and Cd pollution is more severe than the other elements, and the heavily polluted areas are mainly distributed in the southeast of the study area. Cd and Pb in soil are significantly disturbed by the production activities of heavy metal processing enterprises around the farmland.Principal component analysis/absolute principal component score (PCA/APCS) receptor model analysis results show two primary soil heavy metal pollution sources in the study area. It is speculated that source 1 may be industrial and mobile sources, and source 2 may be agricultural and natural sources. Cd, Pb, Zn, and Cu are mainly industrial and mobile sources, and the sources of Ni are mainly agricultural and natural sources.While building Anxin County, environmental management and restoration must be done well. Focus on strengthening the attention to Pb and Cd in the soil of the Anxin County while carrying out soil remediation work and doing an excellent job of prevention and control, urging heavy metal processing enterprises to improve production processes, shutting down some heavily polluting enterprises to avoid developing in a more serious direction. In the future construction process, soil restoration is still an arduous task."}
+{"text": "Scientific Reports 10.1038/s41598-022-26813-8, published online 04 January 2023Correction to: The original version of this Article contained an error in the Funding section,\u201cThis work was supported by Basic Science Research Program through the Chungbuk Techno-Park funded by Chungcheongbuk-do, Korea. The funders had no role in the study design, analysis, decision to publish, or preparation of the manuscript.\u201dnow reads:\u201cThis research was supported by \u2018Regional Innovation Strategy (RIS)\u2019 through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021RIS-001).\u201dThe original Article has been corrected."}
+{"text": "Neurosarcoidosis is an autoimmune disorder of unknown etiology. We report a case of a 27-year-old African American male presenting with fever, vomiting, and seizure. Initially, bacterial meningitis was suspected, and empiric antibiotics with dexamethasone were started. Workup revealed negative cultures, leptomeningeal enhancement, and cavitary lung nodules with hilar lymphadenopathy on imaging\u00a0and elevated angiotensin-converting enzyme levels on cerebrospinal fluid (CSF) analysis. Neurosarcoidosis was then suspected, and a lung biopsy was performed. The results were inconclusive, but the patient\u2019s condition improved. He was discharged on prednisone. Our case demonstrates the diagnostic difficulty of neurosarcoidosis while displaying the importance of early initiation of glucocorticoids in the acute inpatient setting. Sarcoidosis is an immune-mediated disorder of unknown etiology, characterized by granulomatous inflammation of multiple organ systems -7. PathoPatient informationA 27-year-old African American male presented to the emergency department with nausea, vomiting, and a severe headache for 12 hours. His headache began insidiously the night prior, but due to worsening pain, he drove himself to the emergency department. During triage, the patient experienced expressive aphasia and would only respond with head nodding. Shortly after this, the patient was witnessed having a tonic-clonic seizure. On further investigation, the patient\u2019s girlfriend provided a history that included chills, night sweats, and unintended weight loss for six months leading up to the patient\u2019s current presentation.This patient had a history of legal incarceration occurring two years ago and a past medical history of childhood asthma. The patient reported that he frequently smoked marijuana but denied the use of other drugs, tobacco, or alcohol. He was not familiar with tuberculosis and thus has no knowledge of exposures. Further history from his family revealed a family history of thyroid disease in his maternal grandmother but denied any autoimmune conditions, including sarcoidosis, systemic lupus erythematosus, or rheumatoid arthritis.The patient had previously been to the emergency department three times in the four months leading up to his current presentation. Each encounter was secondary to complaints of nausea and vomiting, which were resolved with fluids and metoclopramide.Clinical findingsDirectly after the patient\u2019s witnessed seizure, abortive therapy was attempted with a 2 mg intravenous (IV) push of lorazepam without success. A second attempt with a 2 mg IV push of midazolam was successful, and the patient was intubated for airway protection with concern for status epilepticus. Initial vitals signs showed a temperature of 100.6\u00b0F, heart rate of 88 BPM, blood pressure of 125/72 mmHg, respiration rate of 23, and SpO2 of 100% on room air.\u00a0On physical examination, the patient was intubated and sedated to a Richmond Agitation Sedation Scale (RASS) of -5. Cranial nerve examination revealed midline gaze, 2 mm pupils, and reactive to light. Lower extremities had positive clonus bilaterally, along with spontaneous contractions in bilateral upper extremities. No rashes or skin changes were noted. Physical examination was limited due to sedation but did not reveal any other abnormalities of the cardiovascular, pulmonary, gastrointestinal, or vascular systems.Diagnostic assessmentInitial laboratory findings included a complete blood count with differential, comprehensive metabolic panel, and thyroid function tests that were unremarkable. Electroencephalogram (EEG) was performed for 15 hours continuously while the patient was sedated and revealed severe nonspecific diffuse or multifocal cerebral dysfunction with no epileptic activity. Following this, a computed tomography angiography (CTA) of the head and neck with Omnipaque 350 mg/mL contrast revealed severe/critical narrowing of the right distal M1 through proximal M2 branches as well as an incidentally noted 1.5-cm thick-walled cavitary lesion in the right lung apex and additional scattered nodules. Due to the patient\u2019s history and presentation, a lumbar puncture was performed on hospital day 2 and revealed low glucose, elevated protein, elevated neutrophil count, and increased opening pressure\u00a0 on hospital day 3. By hospital day 8, acid-fast smears, CSF gram stains, and PCR were confirmed negative, and all antibiotic therapy was ceased. Based on the increasing suspicion of sarcoidosis or another autoimmune cause, the patient underwent endobronchial ultrasound with biopsy (EBUS) for a sarcoidosis workup. The patient was transitioned to oral prednisone 50 mg two times a day as well as levetiracetam 500 mg daily. He continued to have good clinical improvement at this time with no further seizures. The patient was ambulating unassisted with no complaints of headache or dizziness.Follow-up and outcomesFollowing the patient\u2019s consistently negative bacterial cultures and continued clinical improvement, he was deemed safe and stable for discharge from the hospital on day 9. Following discharge, he followed up with rheumatology and neurology for this presumed diagnosis of neurosarcoidosis despite the inconclusive biopsy. The patient\u2019s steroid dose was then decreased from 50 mg twice daily to 40 mg once daily, and he was maintained on levetiracetam 500 mg daily.\u00a0He continued to experience intermittent headaches, nausea, and tremors. The patient was then scheduled to obtain a positron emission tomography (PET) scan and follow up for outpatient visits in 4-6 months. However, the patient was brought to the emergency department a month and a half following the initial admission for unwitnessed seizure activity, despite reported adherence to his medical regimen. Upon this second seizure and admission, the patient\u2019s steroid dose was increased to 60 mg daily pending further imaging with PET and CT scans.Sarcoidosis most commonly affects the lung, skin, eyes, liver, and lymph nodes -3,5-7. TNeurosarcoidosis, while rare, was found to be the first manifestation of disease in 52% of patients diagnosed with sarcoidosis , which iDue to the extensive workup, it was not until hospital day 7 that neurosarcoidosis became top of our differential. CSF ACE levels have low sensitivity and specificity for neurosarcoidosis . Our patGlucocorticoids are first-line therapy -3,5,7. EOver 80% of patients with neurosarcoidosis relapse, and patients with encephalic involvement are at increased risk [Prompt workup and the start of therapy are important to avoid potentially permanent neurological deficits. Fortunately for our patient, glucocorticoids were started early, and further neurological deficit was avoided. His initial condition improved without the need for second- and third-line therapies. It is difficult to say that neurosarcoidosis should be high on any physician\u2019s differential, but when the possibility is identified, initiating glucocorticoid therapy may provide some benefit in the acute inpatient setting, as demonstrated by our case."}
+{"text": "Reproducibility is a cornerstone of scientific progress. In epigenome- and transcriptome-wide association studies (E/TWAS) failure to reproduce may be the result of false discoveries. Whereas multiple methods exist to control false discoveries due to sampling error, minimizing false discoveries due to outliers and other data artefacts remains challenging. We propose a robust E/TWAS approach that outperforms alternative methods to improve reproducibility such as split-half replication. Furthermore, robust E/TWAS results in only a minor loss of power if there are no outliers and can in the presence of outliers, likely a more realistic scenario, even be more powerful than regular E/TWAS. Reproducibility is a cornerstone of scientific progress. In the k equal and non-overlapping folds, (ii) perform separate association studies in each fold, (iii) perform a signed meta-analysis across all k folds using Stouffer\u2019s Z-score method[In this article, we propose a method for eliminating false discoveries due to outliers and other data artefacts that we call robust E/TWAS. This method involves (i) partitioning the sample in re method after tr\u03b1=0.05, meaning that the null hypothesis should be rejected in 5% of the simulations if false discoveries are controlled properly. For the sake of comparison, we also study the impact of these outliers on regular association testing that analyzes the entire sample at once, and \u201csplit-half replication\u201d where the sample is randomly split in a discovery and replication part. For split-half replication, in simulations where the P value is smaller than 0.05 in the discovery sample we perform a \u201creplication\u201d using a one-sided test in the replication sample assuming the same direction of effect as in the discovery sample.We performed simulations to evaluate the robust E/TWAS method. We studied either univariate or bivariate outliers. Univariate outliers affect both the outcome or the biological marker  but not both variables in the same individual. Bivariate outliers involve outlying values for both the outcome and the biological marker in the same individual. We perform 10,000 simulations for a marker with a sample size of 250 and assuming that the outcome and marker are not associated. We either assume five univariate outliers or one bivariate outlier (bivariate outliers will be rarer than univariate outliers). Significance tests are performed allowing for a Type I error of k\u226510 that accurately controls the Type I errors at the 0.05 level. The bivariate outlier appears to have a much bigger impact. The split-half replication method is most sensitive to this outlier. Thus, the bivariate outlier will be present in either the discovery sample or the replication sample. If present in the discovery sample, it will have an increased probability (> 0.05) of being tested in the replication sample where it is expected to \u201creplicate\u201d in 5% of the simulations (\u03b1=0.05). If not present in the discovery sample, it will be tested in 5% of the replication samples (\u03b1=0.05) where it will have an increased probability (> 0.05) to \u201creplicate\u201d due to the outlier being in the replication sample. \u03b1=0.001 as the threshold for declaring significance. We only studied power in the absence of outliers and in the presence of univariate outliers as these are the common scenarios. Having a bivariate outlier for a marker with a true effect will be rare where, depending on whether the outlier is in line with the association trend, the outlier can either make it more or less likely that the association will be detected. When there are no outliers, robust E/TWAS results in only a slight loss of power compared to analyzing all data at once , which is determined by the statistical power. To study power, we repeated the simulations but now assuming a correlation of 0.3 between the outcome and biological marker and use  at once . In addik=5 folds. The lambda of 1.083 (\u22125). Two of the three sites identified as driven by outliers in the regular MWAS were no longer among these 24 robust MWAS, suggesting it successfully eliminated findings potentially caused by outliers. Two of the four sites that were not flagged as outlier-driven in the regular MWAS were still among the top 24 robust MWAS findings, suggesting it retained part of the sites that were not caused by outliers. The fact that not all four findings that were retained in the regular MWAS were among the top 24 robust MWAS findings may mean that (i) the MAD outlier detection algorithm is not perfect or (ii) because of lower power in the robust MWAS.To illustrate the method with real data we reanalyzed HumanMethylation450 array data for 691 individuals . After performing quality control as described elsewhere[of 1.083  was compIn this real data example, a variety of covariates were regressed out. Part of the reason for reduced power in the robust MWAS power may therefore be the loss of the degrees of freedom caused by regressing out covariates in each fold separately. We therefore tested a variant of the robust method that first regressed out effects of covariates in the entire sample, and then performed association testing in each fold separately with residualized outcome scores while charging the loss of degrees of freedom equally across the folds. However, this approximation resulted in deflated P values. We also tried a log data transformation to handle outliers as that will also avoid a loss of degrees of freedom. However, consistent with previous observations that results for transformed data are often not relevant for the original non-transformed data, we no lIn summary, robust E/TWAS provides an efficient method to improve the reproducibility of findings from high dimensional biological association studies by eliminating false discoveries due to outliers or other data artefacts. It results in only a minor loss of power if there are no outliers but can in the presence of outliers, likely a more realistic scenario, even be more powerful than regular E/TWAS. As it turns a single sample into multiple subsamples, the negative effect of outliers on false discoveries and power is essentially diluted when the association evidence from the affected subsamples is combined with the evidence from the subsamples that are not affected by the outliers. Robust E/TWAS clearly outperformed split-half replication in terms of false discoveries and power. It also provides a more systematic and statistically motivated method for handling outliers compared to traditional methods for outlier detection that would need to be performed post-hoc on a per site basis and involve multiple arbitrary choices. Finally, robust E/TWAS is easy to implement and can be used with any type of association test.Supplement 1"}
+{"text": "Isopropyl alcohol molecules, as a biomarker for anti-virus diagnosis, play a significant role in the area of environmental safety and healthcare relating volatile organic compounds. However, conventional gas molecule detection exhibits dramatic drawbacks, like the strict working conditions of ion mobility methodology and weak light-matter interaction of mid-infrared spectroscopy, yielding limited response of targeted molecules. We propose a synergistic methodology of artificial intelligence-enhanced ion mobility and mid-infrared spectroscopy, leveraging the complementary features from the sensing signal in different dimensions to reach superior accuracy for isopropyl alcohol identification. We pull in \u201ccold\u201d plasma discharge from triboelectric generator which improves the mid-infrared spectroscopic response of isopropyl alcohol with good regression prediction. Moreover, this synergistic methodology achieves ~99.08% accuracy for a precise gas concentration prediction, even with interferences of different carbon-based gases. The synergistic methodology of artificial intelligence-enhanced system creates mechanism of accurate gas sensing for mixture and regression prediction in healthcare. Isopropyl alcohol can play a significant role in anti-virus diagnosis but generally yields limited response in conventional detection. Here, authors propose a methodology of AI-enhanced ion mobility and mid-infrared spectroscopy for accurate gas identification despite presence different carbon-based gases. Many virus-perishing methods are investigated for combatting pandemic or cold diseases, such as managing indoor air, increasing gas ventilation, optimizing humidity levels, maximizing air filtration efficiency, and the widespread use of IPA. IPA is a flammable and colorless liquid with a fruity odor and a slightly bitter taste. It is used to clean electronic components and to remove the thermal paste from integrated circuit chip packages. Due to its extensive use, various healthcare problems may occur along with the exposure or inhalation of the IPA molecules, e.g., skin irritation, nervous system illness, and respiratory damage. As a result, rapid and accurate detection of IPA molecules becomes critical. The methods for IPA sensing include chemiresistive, gas absorption-based, two-dimensional(2-D) material-based, ion mobility, electrochemical, and other sensors16. However, such IPA sensors can only measure the total gas concentration among various volatile organic compounds(VOCs), and they have limitations such as slow response, poor selectivity, and insufficient accuracy. Thus, a sensing system with fast response, good selectivity, anti-interference, and high sensitivity for IPA identification is critical.The need for health and safety has been increasing the demand for isopropyl alcohol (IPA) as a vital element of anti-virus hand sanitizers23. Mid-infrared spectroscopy is a non-destructive method applied to identify the vibrational modes of gas molecules, and it has widespread applications in environmental monitoring, pharmacy, chemical analysis, bio-sensing, and the pharmaceutical industry. Regarding the sensitivity enhancement in mid-infrared spectroscopy, it is found that the instantaneous strong electrostatic field could be further tailored to the molecular vibration along with the optics refection response, thereby enhancing the absorption effect of infrared spectroscopy. In short, conventional ion mobility sensing has the constraint of the requirement of a strict measurement environment, whereas conventional mid-infrared spectroscopy is limited by poor response during low-concentration identification. Overall, both methods have drawbacks in IPA sensing at this point.Ion mobility and mid-infrared spectroscopy are two well-known analytical techniques for identifying of gas-phase compounds. Scientists focus on ion mobility and investigated many approaches to increase sensing ability, e.g., producing ions by \u03b2-radiation, ultraviolet (UV) irradiation, and others. However, this approach requires a high-voltage source and a strict operating environment , yielding expensive and bulky equipment33 was discovered in 2012, allowing for an additional high-voltage output from mechanical vibrations. With the aid of triboelectric, Wang et al. reported respiration health based on AI-assisted diagnosis by multi-self-calibrated parameters from TENG with accuracy >95.21%33. However, anti-virus VOCs diagnosis from hybrid mechanism (mid-infrared spectroscopy) and data-enhanced algorithms still need to be investigated. For example, the ion power source from the triboelectric nanogenerator could be greatly optimized and provide ion mobility in cold environments and ambient air pressure. Thirdly, artificial intelligence (AI) is a hot research topic dramatically promoted with the fifth-generation cellular network technology (5\u2009G). A cloud server would easily handle plenty of data based on deep learning algorithms in a short time39. Providing feasible big data processing using machine learning and deep learning techniques (t-distributed stochastic neighbor embedding (t-SNE), linear discriminant analysis (LDA), principal component analysis (PCA), synthetic minority oversampling technique (SMOTE), and deep neural networks (DNN) regression), which may reinforce the characteristic IPA data-sets from ion mobility and mid-infrared. Thus, the AI-enhanced methodology would be an ideal solution for IPA detection in factories or personal healthcare, where identifying IPA molecules requires rapid response, high accuracy, good selectivity, anti-interference, and high sensitivity.Herein, three critical issues need to be addressed in order to improve the response of IPA molecules using existing ion mobility or mid-infrared spectroscopy. Firstly, focusing on the interaction of molecules with infrared irradiation, high-voltage plasma induces a strong electromagnetic field, yielding a coupling effect, which amplifies the mid-infrared absorption response. Therefore, plasma-enhanced infrared absorption spectroscopy enables the ultrasensitive detection of molecules with the aid of an existing high-voltage power source (ion mobility). Secondly, regarding ion mobility, a triboelectric nanogeneratorR2 score by DNN regression, which can be improved to ~0.98 R2 score using SMOTE before DNN regression. What\u2019s more, it is also demonstrated that the data preprocessing method could remove the background calibration with high accuracy in feature classification. In the perspective of sensing fusion between IMMS, the regression performance in concentration prediction can be enhanced to a ~0.99 R2 score. Moreover, even in the case of different gas interferences, this synergistic methodology of IMMS reaches ~99.08% accuracy in our study using the AI-enhanced method and high-voltage triboelectric cold ion mobility.To realize the IPA detection with good selectivity, fast response, and high sensitivity, we propose an artificial intelligence (AI)-enhanced chemical detection based on IMMS assisted by a multi-switched triboelectric nanogenerator. It provides an additional high-voltage power source for ion mobility and plasma enhancement for mid-infrared sensing. The plasma-enhanced mid-infrared response in IPA sensing exhibits a good concentration prediction with ~0.87 Voc) and the accumulative open-circuit charges (Qoc) are presented in Figs. SAs shown in Fig.\u00a037. Thus, the AI-enhanced approach can process multi-modal data from the collected data, and it can be further improved with the synergistic methodology to realize the fast response, wide detection range, and accurate detection of a gas mixture.The IPA response mechanism is from the characterizations in absorption and reflection by mid-IR. The external-provided strong electric field dramatically improves the sensitivity of the IPA detection due to the high-voltage fluctuation from the plasma discharge onto the vibrational mode of the molecule structure (coupling effect). Thus, the synergistic mechanism of ion-mobility sensing and enhanced-mid-IR reflection enables chemical sensing to achieve fast response times and accurate detection (two kinds of data in computing). Furthermore, the data processing with an AI-enhanced approach would combine the advantage of both ion-mobility sensing and enhanced-mid-IR response resulting in extra high accuracy and wide-range detection. The detailed experiment photos of the AI-enhanced IMMS can be found in Fig. SThe ion mobility system is constructed in a tip-plate electrode configuration, high-voltage component, and current collector, as shown in Fig.\u00a0R2 score is as follows:In order to enhance the accuracy of IPA detection, we adopted raw data analysis both with and without SMOTE\u2009+\u2009DNN regression for an in-depth study of the dark current pattern, as illustrated in Fig.\u00a0R2 score for the test dataset was 0.96, which represents a good regression performance.Here the \u22121, characteristic for CO2, a ~1\u20133 times higher response is observed than without plasma . The AI-enhanced methodology for mid-infrared spectroscopy is shown in Fig.\u00a0\u22121~3500\u2009cm\u22121. The relationship between different IPA concentrations with almost a linear numerical relationship is shown in Fig. S42. To accurately evaluate the noise in real measurement, the 0 ppm IPA molecules are chosen to extract the signal fluctuations of these spectra. By plotting the total noise and the output signal with SMOTE\u2009+\u2009DNN regression together, it can analyze the LOD of the Ai-enhanced IMMS. The SMOTE\u2009+\u2009DNN regression of the signal above the noise at 0 ppm indicates that the LOD of our IPA could reach a prediction of ~40 ppm based on the estimated mean error using an improved IMMS technique . However, the manual calibration before the real measurement is inconvenient and inefficient. To avoid this manual process (labor-consuming), we adopt an artificial intelligent method to provide the right concentration measurement. The raw data without data preprocessing is shown in Fig.\u00a038 and a decision tree. As shown in Fig.\u00a0Figure 45. To further demonstrate the IPA detection in mixture gases, as shown in Fig.\u00a0\u22121, clearly implying that the peak response of different gas species can be well-identified. The gas species are determined by the response of molecules with their mode at a certain wavenumber, whereas their concentrations are indicated by the response value. IPA mixture gas detection based on the plasma-enhanced mid-infrared spectrum is represented in Fig. SR2 score, which is improved compared with a single measurement as illustrated in Fig.\u00a0IPA as one of the important elements in VOCs family can affect human health and be used for the assessment of various diseases. For example, IPA has been widely used as antiviral hand sanitizer. Also, many studies have indicated that IPA levels can reflect the severity of lung cancer, serving as a specific biomarker for early diagnosis, which deserves attention and further researchIn conclusion, we propose a synergistic IMMS mechanism for AI-enhanced chemical sensing to achieve rapid response and accurate detection of a gas mixture. A triboelectric nanogenerator for high-voltage plasma is proposed to overcome the environmental pressure constraints to ion mobility and the weak response and reflection detection of gas molecules, limiting the use of mid-infrared spectroscopy. The reported self-powered ion mobility reaches up to almost two times higher accuracy with the aid of AI-augmentation by automatic extracting of the specific features than the conventional method. It is also shown that cold plasma from the triboelectric generator enhances the mid-infrared response for IPA sensing with a good linear prediction by deep learning. Moreover, the data processing could remove the background calibration based on our observation with a labor-consuming effect. Due to the challenges in IPA detection, the AI-enhanced approach is successfully demonstrated by extracting the features from the IMMS data with ~99.08% accuracy. This method can process multi-modal data from collected data, and can be combined with existing methodology to realize the synergistic mechanism.2. For power output, a positive triboelectric polymethyl methacrylate (PMMA) dielectric is connected with negative fluorinated ethylene propylene (FEP). The laser cutting machine is used to cut the acrylic plate for component assembly. The conductive nickel textile is used to connect and separate with/from an electrode via the action of \u201con\u201d or \u201coff\u201d.The multi-switched triboelectric nanogenerator contains the bottom pair of plates and a top movable pair of plates with a contact area of 49 cmVoc, and Qoc parameters. The infrared spectra are recorded using an FTIR spectrometer (Agilent Cary 610 Series), which operates at wavelengths from 4 to 8 \u03bcm. The measurement environment is at room temperature, exhibiting the tolerance of the test environment. The calibration sensor of the IPA is a commercial device.A programmable electrometer (Keithley model 6514) is adopted for The platform is made of the multi-switched triboelectric nanogenerator, the pre-mixture IPA chamber, and the FTIR equipment. The pre-mixed chamber is constructed to mix different gases. The calibration sensor (SKY2000-VOC) is used to identify the specific concentration of IPA. An oscilloscope is used to monitor the dark current from the collection electrode to record the dark current response along with the gases. A computer is used to record FTIR data for further machine learning. The gas chamber is sealed during the operation to improve the accuracy of the IPA concentration measurement. The oscilloscope is connected to the plate electrode collector to record the dark current response during the sliding of the multi-switched triboelectric nanogenerator.Synthetic minority oversampling technique is a synthetic data algorithm with comprehensive sampling, which can be used to solve the problem of unbalanced data distribution. Owing to the limitation of data collection, it is difficult to ensure that the information of sampled IPA data in different concentrations is distributed evenly, yielding worse performance in the predictive model. Thus, SMOTE which realizes in MATLAB R2022a is adopted for each concentration of IPA data. The data of each concentration should be equal in amount so that it can be used in the classification or regression model. The deep neural network is developed for the gas feature regression and IPA concentration estimation. The proposed DNN is developed on python 3.8 using the Keras and sci-kit-learn packages. Our DNN model is specifically constructed by multiple connected layers: dense  \u2192 dropout (0.2) \u2192 dense  \u2192 dense . The batch size is 20. The epoch number is 300. The loss function between the predict value and the true value is a mean-squared error. The optimizer is Adam. The ratio settings for train/test split are 70:30, 75:25, and 80:20. The result with different ratio settings are shown in Figs. SThe t-SNE is used in exploratory data analysis and for nonlinear dimensionality reduction. t-SNE reserves the local characteristics by transforming the data distance relationship into a probability distribution. When implementing t-SNE, the same amount of low-dimensional data is randomly generated, and then the loss function measures the difference between two probability distributions. The gradient descent method is used to update this batch of data, and the low-dimensional data satisfying the requirements is obtained. The t-SNE is performed in MATLAB R2022a to intuitively visualize the feature space in two and three dimensions. The support vector machines by the \u201clibsvm\u201d package in MATLAB 2022a are implemented to compare with LDA.Further information on research design is available in the\u00a0Supplementary InformationReporting Summary"}
+{"text": "Cellulose nanofibrils (CNFs) are multiscale hydrophilicbiocompatiblepolysaccharide materials derived from wood and plants. TEMPO-mediatedoxidation of CNFs (TO-CNF) turns some of the primary hydroxyl groupsto carboxylate and aldehyde groups. Unlike carboxylic functional groups,there is little or no information about the biological role of thealdehyde groups on the surface of wood-based CNFs. In this work, wereplaced the aldehyde groups in the TO-CNF samples with carboxyl groupsby another oxidation treatment (TO-O-CNF) or with primary alcoholswith terminal hydroxyl groups by a reduction reaction (TO-R-CNF).Rat mesenchymal stem/stromal cells (MSCs) derived from bone marrowwere seeded on polystyrene tissue culture plates (TCP) coated withCNFs with and without aldehyde groups. TCP and TCP coated with bacterialnanocellulose (BNC) were used as control groups. Protein adsorptionmeasurements demonstrated that more proteins were adsorbed from cellculture media on all CNF surfaces compared to BNC. Live/dead and lactatedehydrogenase assays confirmed that all nanocellulose biomaterialssupported excellent cell viability. Interestingly, TO-R-CNF samples,which have no aldehyde groups, showed better cell spreading than BNCand comparable results to TCP. Unlike TO-O-CNF surfaces, which haveno aldehyde groups either, TO-R-CNF stimulated cells, in osteogenicmedium, to have higher alkaline phosphatase activity and to form morebiomineralization than TCP and TO-CNF groups. These findings indicatethat the presence of aldehyde groups (280 \u00b1 14 \u03bcmol/g)on the surface of TEMPO-oxidized CNFs might have little or no effecton attachment, proliferation, and osteogenic differentiation of MSCs. BNC has attractive physicochemical properties  analogous to those ofcollagenous fibers in bone tissues.5 Morphologically,BNC fibers are comparable to collagenous nanofibers of native tissues,and therefore, BNC has shown great potential in tissue engineeringapplications.7 Like collagen scaffolds, BNC wasreported to effectively promote adhesion, proliferation, and osteogenicdifferentiation of osteoblast-like cells.8 Still, biomaterials without microbial or animal-derived componentsare preferred immunologically. Considering this, great interest inplant-based CNFs has grown recently. Because of their large aspectratio and flexibility, these nanofibrils can easily form hydrogelseven at low concentrations.9 Lately, CNF hydrogelswere used as tissue engineering scaffolds to support proliferationand differentiation of embryonic stem cells, liver cells, and mesenchymalstem/stromal cells (MSCs).12 CNFs, even at low concentrations,have favorable shear thinning properties because of their high aspectratio, and therefore, they are usually added to bioinks used in bioprintingof stem/stromal cells to improve their rheological properties.15Over the last decade, cellulose nanomaterialshave emerged as promisingbiomaterials in the field of tissue engineering and regenerative medicine.l-oxyl) radical-mediated oxidation or carboxymethylation.16 These chemical pretreatment methods often change the surface propertiesof the extracted fibrils. Surface properties of biomaterials are knownto regulate protein adsorption and subsequently cell responses.17 Surface functional groups, charges, wettability,topography, and stiffness are interconnected factors that can dictatecell adhesion, proliferation, and differentiation.21 In particular, functional groupshave been found to be an important cue for osteogenic differentiationof stem cells.22 It was reported that surfaceswith hydroxyl (OH) and amine (NH2) groups can upregulateosteoblast-specific gene expression, alkaline phosphatase (ALP) activity,and matrix mineralization compared with surfaces with carboxyl (COOH)and methyl (CH3) groups.23 Freeneutral surfaces with CH3 and OH support less protein adsorption,cell spreading, and adhesion but greater chondrogenic differentiationof stem cells than charged surfaces with COOH and NH2.18 Moreover, human umbilical vein endothelial cells(HUVECs) were shown to adhere well to surfaces with COOH but not tosurfaces with CH3. Arima and Iwata demonstrated that adhesionof HUVECs was improved by a CH3/OH surface (contact angle\u03b8 = 40\u00b0), whereas HeLa cells adhered best on CH3/OH and CH3/COOH surfaces (\u03b8 = 50\u00b0).20Wood-based CNFs are usually produced by mechanical treatmentcoupledwith some pretreatment strategies such as TEMPO  CNF-coated surfaces with and withoutaldehyde groups were first cultured with different protein solutionsto determine the total protein adsorption. Second, rat bone marrow-derivedstem/stromal cells (BMSCs) were cultured on the CNF surfaces, andtheir viability, morphology, proliferation, and osteogenic differentiationwere evaluated. Overall, this study links the role of mixed functionalsurface groups  of wood-based nanocelluloseto protein adsorption and subsequently to their biological performancesfor the use in bone regeneration applications.Previously, we investigated the effectof two chemical pretreatmentsof CNFs, TEMPO-mediated oxidation (TO-CNF) and carboxymethylation(CM-CNF), on the adhesion of mouse fibroblasts. TO-CNF had aldehydegroups (211 \u00b1 60 \u03bcmol/g) and carboxyl groups (764 \u00b160 \u03bcmol/g). CM-CNF had carboxymethyl groups (346 \u00b1 26 \u03bcmol/g)and less carboxyl groups (58 \u00b1 1 \u03bcmol/g).24 Briefly, wood pulp (110 g) wassuspended in water (8.25 L) containing 1.37 g of 2,2,6,6-tetramethylpiperidinyl-1-oxyl and 13.75 g of sodium bromide (NaBr). The pHwas adjusted to 10.5 by adding 0.5 M NaOH. After that, 2.5 mmol NaClO/gdry cellulose was added slowly to the slurry, and pH was kept constantat 10.5. After 50 min, the pH was adjusted to 7 and methanol was addedto remove TEMPO left in the slurry. Finally, the cellulose was washedthoroughly until the conductance of the filtrate was below 5 \u03bcS/cm.To prepare samples without aldehyde groups, selective oxidation andreduction of aldehyde groups were carried out. The TO-CNF was oxidizedagain (TO-O-CNF samples) with sodium chlorite (NaClO2)in water at pH 4\u20135 for 48 h at room temperature. TEMPO-oxidizedcellulose  was suspended in water (5 L) and mixed withNaClO2 (90.5 g) and 5 M acetic acid (1 L). The selectivereduction of the TEMPO-oxidized CNFs (TO-R-CNF samples) was performedby adding 5 g of sodium borohydride (NaBH4) to the slurryat pH 8 for 48 h at room temperature. After washing, all nanocellulosesamples were homogenized using a Rannie 15 type 12.56X homogenizer. The bacterial nanocellulose (BNC) waspurchased from JeNaCell (Germany) and used without any chemical modifications.TEMPO-mediated oxidized cellulose nanofibril gel-like samples (TO-CNF)were produced by chemical and mechanical treatments of fully bleached,never-dried softwood kraft pulp that was kindly donated by S\u00f6draCell , according to a previously describedmethod.25 Furthermore, to confirm the absence of aldehyde groups in TO-O-CNF,TO-R-CNF, and BNC samples, 2,3,5-triphenyltetrazolium chloride (TTC)was used. Nanocellulose suspensions were mixed with 0.3 M KOH and0.01 M TTC in water bath at 80 \u00b0C for 10 min. Oxidation of aldehydesleads to the reduction of TTC to the red compound 1,3,5-triphenyltetrazoliumformazan (TTF) under alkaline conditions.Total carboxylatecontent in all CNF samples was determined by the electric conductivitytitration method. Briefly, 0.3 g of dry CNFs was added to water (450mL) and 0.1 M NaCl (5 mL), and the pH was adjusted to 2.5\u20133.0using 0.1 M HCl. The solution was stirred for 30 min before 0.05 MNaOH was added at a rate of 0.1 mL/min up to pH 11. During the titration,the conductivity of the solution was measured using the 856 ConductivityModule (Metrohm). The carboxylate content of cellulose was determinedfrom the conductivity and pH curves. To determine the aldehyde contentof the TO-CNF, samples were further oxidized with sodium chlorite.The carboxylate groups formed by this second oxidation were formedfrom aldehyde groups originally present in the TEMPO-oxidized CNFs.Ra) was obtained from images (10 \u03bcm\u00d7 10 \u03bcm) using NanoScope Analysis software (version 1.9).For the viscositymeasurements, a DV2TLV viscometer (Brookfield) was used along withRheocalc software. Each CNF sample (0.4% CNF suspension) was evaluatedin three parallels, and the viscosity of each parallel was measuredfrom 0.1 to 100 rpm. For the nanostructures, 0.01% nanocellulose suspensionsof each sample (25 \u03bcL) was added to clean the mica surface,left to dry in air, and then imaged with a Dimension ICON atomic forcemicroscope (AFM) using NanoScope 9.4 Software (Bruker). Surface roughness was used. CNF suspensions werepumped through a flow cell where the fibers were photographed witha resolution of 4 \u03bcm. The images were further used to analyzethe mean length and width of the fibers. To evaluate the microstructureof all cellulosic materials, tissue culture plates  were covered with cellulosicsuspensions and stored at 37 \u00b0C for 24 h to dry. Films were analyzedusing an optical microscope  afterstaining with crystal violet.2 humidifiedatmosphere. For single protein solutions, 5% bovine serum albumin in phosphate-buffered saline (PBS) and for complexprotein solution, 100% fetal bovine serum  solutions were used. To mimic cell culture conditions, 10% FBSin \u03b1-MEM was used. The wells were then washed with PBS (LifeTechnologies) to remove weakly adsorbed proteins and incubated with2% sodium dodecyl sulfate  for 24 h to dissolvethe adsorbed proteins. The total amount of proteins was measured bya commercial protein assay kit  according to manufacturer\u2019s instructions.The zeta (\u03b6) potential of the nanocellulose samples (0.08 g/L)was measured using a Zetasizer NANO ZSP  apparatus with aZetasizer software (version 7.11) cell culture medium  containing 10% FBS (pH 7.3).To study the effect of surface modifications on the wettability ofthe materials, the contact angle was measured with a DAT 1100 dynamicabsorption tester (FIBRO system ab) at 23 \u00b0C and 50% relativehumidity. Nanocellulose suspension (0.26%) was added to Petri disheswith a diameter of 5.6 cm and air-dried to make films for the contactangle measurement. A droplet of water (4 \u03bcL) was deposited onthe specimen surface. A series of images were captured and analyzedby DAT3 software. The dynamic wetting (contact angle) was measuredas a function of time between 0 and 175 s. To evaluate the effectof surface chemistry on protein adsorption, 500 \u03bcL of nanocellulosesuspension (0.26%) of each sample was added to a 24-well plate andthen incubated at 37 \u00b0C for 24 h. Uncoated TCP wells were usedas controls. After that, nanocellulose-coated tissue culture plateswere incubated with 500 \u03bcL of either single or complex bovineprotein solutions for 4 h at 37 \u00b0C in a 5% CO26 Briefly, themetaphyseal ends of the femur were excised, and the marrow cavitywas flushed with complete \u03b1MEM. The cells were resuspended infresh \u03b1MEM medium containing 1% PS and 10% FBS and plated incell culture flasks. After flow cytometry characterization, cellswere negative for cluster of differentiation (CD) 34 and CD45 andpositive for CD73 and CD90. Cells from passage 4 were used in thestudy. For cell seeding, wells of 24-well plates were covered with500 \u03bcL of 0.26% nanocellulose suspension before drying overnight at 37 \u00b0C to evaporate the water. The plates were sterilizedunder ultraviolet (UV) light for 1 h. Uncoated tissue culture plate(TCP) wells were used as controls. The cell seeding density was 5000cell/cm2. In all experiments, fresh medium was suppliedtwice a week.The isolation of rat cells was approvedby the Norwegian Animal Research Authority . Bone marrow-derived mesenchymal stromal/stem cells wereharvested from the femur of Lewis rats and characterized as describedin a previous publication.To evaluatethat the chemical pretreatments are cellularly safe and that the washingof the nanocellulose materials was sufficient to remove any harmfulchemicals, an indirect cytotoxicity assay was conducted. Nanocellulosehydrogels were first sterilized using an autoclave and then incubatedin \u03b1MEM (1 g/5 mL) at 37 \u00b0C with constant shaking (60 rpm)for 24 h, and then, the extracts were filtered (0.2 \u03bcm). Ascontrol samples, \u03b1MEM without hydrogels was kept at the sameextraction conditions. Cells were plated and incubated in completemedium for 24 h to attach, and then, the media were replaced withextracts supplemented with 10% FBS. After 24 h, cell viability andmitochondrial activity were assessed utilizing live/dead and AlamarBlue assays . For live/dead stain,cells were incubated in a working solution containing ethidium homodimer-1(stains dead cells red) and calcein AM (stains living cells green)for 30 min and then imaged with a fluorescence microscope . For Alamar Blue, 50 \u03bcL of the reagent wasadded to each well and incubated for 4 h. The fluorescence was thenmeasured utilizing a Varioskan LUX microplate reader (Thermo FisherScientific).For direct cytotoxicity assessment, cells werecultured directly on nanocellulose-coated surfaces and TCP controlsand then analyzed by live/dead staining after 24 h. To evaluate thetoxicity of CNFs, lactate dehydrogenase (LDH) release was measuredusing a colorimetric kit . The enzymatic assaywas conducted in accordance with the manufacturer\u2019s instructionsfrom the medium corresponding to the live/dead assay. For cell morphology,the cells cultured on nanocellulose-coated TCP were fixed with 4%paraformaldehyde after 4 and 24 h before being incubated in a solutionof phalloidin-Atto488/PBS  for 45 minin the dark at room temperature. At last, 4\u2032,6-diamidino-2-phenylindole in PBS (1:2000) was added for 5 min to labelthe nuclei. The cell geometry was visualized with a fluorescence microscope,and the cell surface area and maximum cell length were analyzed utilizingImageJ software (1.46r).p-nitrophenyl phosphate(Sigma-Aldrich) for 15 min at room temperature. The absorbance wasmeasured at 405 nm using a microplate reader, and the values werenormalized to the DNA amount determined by the proliferation test.To study the late stages of osteogenic differentiation, Alizarin redS staining was performed after 21 days of culture in osteogenic medium.Samples were fixed in 4% PFA and then incubated with Alizarin redS (Sigma-Aldrich) solution for 15 min at room temperature. After washingand air-drying, images were taken with an optical microscope . For quantification, the dye was extracted with 100mM cetylpyridinium chloride (Sigma-Aldrich) at room temperature, andthe absorbance was measured at 540 nm.To evaluatecell proliferation, double-stranded DNA (dsDNA) was quantified usinga Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) in accordance withthe manufacturer\u2019s protocol. Briefly, after 1, 7, and 14 daysof culture in growth or osteogenic medium , cells were lysed with 0.1% Triton-X100 buffer before freezing (\u221280 \u00b0C). After two freeze\u2013thawcycles and sonication for 60 s, samples of each lysate (20 \u03bcL)were mixed with 180 \u03bcL of working solution in 96-well plates,and the fluorescence at 480/520 nm was measured with a microplatereader. To evaluate the early osteogenic differentiation of the MSCs,the release of alkaline phosphatase was measured from the same celllysate after incubation with n \u2265 3) areexpressed as the mean \u00b1 standard deviation (SD). Differenceswere considered statistically significant at p \u22640.05.Statistical comparisons were conductedby one-way ANOVA with a Tukey\u2019s post hoc multiple comparisonusing SPSS software (IBM). Data (2 oxidation or NaBH4 reduction treatmentswas used to produce different viscous suspensions of CNFs at a lowsolid content (1%) as present in In thepresent study, CNF hydrogels with and without aldehyde surface groupswere produced by various chemical pretreatments prior to mechanicalfibrillation of wood pulps. TEMPO-mediated oxidation, followed byeither NaClO27 As a result, negative chargesare introduced to the surface of the fibers and facilitate their electrostaticrepulsion.16 Therefore, this enables thedisruption of the fibers into nano- and microfibrils.28 Our results confirmed the introduction of significant amountsof carboxyl (804 \u00b1 3 \u03bcmol/g) and aldehyde (280 \u00b1 14\u03bcmol/g) groups by the TEMPO-mediated oxidation . Consequently, TO-O-CNFhad the highest carboxyl content (992 \u00b1 24 \u03bcmol/g), whilethe TO-R-CNF had the least carboxyl content (675 \u00b1 14 \u03bcmol/g).The detection of aldehyde groups in the different nanocellulose sampleswere investigated by color change after reacting with TTC. Oxidationof aldehydes leads to the reduction of TTC to the red compound ofTTF.27 The amount of aldehyde groups wascalculated from a calibration curve prepared with glucose solutionsof different concentrations. Except for TO-CNF, all nanocellulosesamples demonstrated absorbance values lower than the linear rangeof the standard curve. However, it is clear from the color that thealdehyde contents of TO-O-CNF, TO-R-CNF, and BNC are lower than thatof TO-CNF and close to zero as shown in It has been reported that the C6 primary hydroxylgroup of cellulosecan be oxidized to aldehyde and carboxyl groups using TEMPO-mediatedoxidation.xidation 1. The se28 The flow behavior of the prepared CNFs is presentedin 29 As shear increases, these entanglements are broken, and as a result,the fibers are separated and aligned with the flow, which explainsthe decrease in the viscosity. Moreover, Besbes et al.56 reported that the increase of carboxylic groupsdecreases the viscosity of CNF hydrogels, which is in line with ourresults. TO-O-CNF had the highest carboxyl content (992 \u00b1 24\u03bcmol/g) and the lowest viscosity value. The presence of suchhigh levels of carboxyl groups on the surface of TO-O-CNF samplesis likely to increase the electrostatic repulsion and reduce the extentof the interactions among CNFs, resulting in a lower viscosity ofthe suspension. Importantly, these results confirm not only the abilityof the prepared CNF hydrogels to flow easily under high shear stressconditions but also the ability to tailor their flow behavior by chemicalpretreatments. This is of great significance for the development ofinjectable hydrogels for drug delivery and for the development ofbioinks for stem cell bioprinting in tissue engineering.13Regarding the viscosity, CNFs form viscous hydrogelsdue to theirhygroscopic nature, high aspect ratio, and high specific surface area,resulting in their strong interactions at low concentrations.30 It appears thatall CNF samples showed fibers of a few hundred micrometers in length,irrespective of the chemical pretreatment. In between the relativelylarge fibers, there was a dense ultrathin network observed by thecrystal violet staining was extracted from AFM measurements. BNC demonstrated the smallest Ra (31 nm), while the TO-O-CNF group had thelargest Ra (180 nm). These structuraland morphological results are in line with the data obtained fromthe viscosity test. As the degree of fibrillation increases duringthe extraction process, the size of the fibrils generally decreases,and therefore, fibril\u2013fibril interactions increase, resultingin a greater suspension viscosity.31 Thiscan explain the low viscosity of TO-O-CNF due to the large size ofthese fibers and decreased fibril\u2013fibril interactions.The number of the microscale fibers are presentedin 32 Wu et al. reported that thewettability of CNF-based films correlated to the roughness values;the contact angle increased with the increase of surface roughness.32 In line with this, the contact angle of BNCin the current study was significantly smaller than those of all CNFgroups due to their lower Ra (31 nm).Another possible explanation of the hydrophilicity of BNC is the presenceof the high density of hydroxy polar groups on the surface.33 This can be explained by the presence of FBSproteins in the cell culture medium, which mask the surface of thenanofibers and generate a new interface.To investigate the charge properties of the nanocellulosematerials,\u03b6-potentials were measured in cell growth culture medium (pH= 7.4), and the respective data are included in 34 Adsorption of BSA, whichis a negatively charged protein, was varied as a function of the strengthof the electrostatic repulsion.35 SinceCNFs have more negative charges, their surfaces adsorbed less BSAthan BNC. Also, it has been reported that the protein adsorption onfibrous polymeric materials increases as the fiber diameter decreases.36 So, it is likely that BNC and TO-CNF, whichhave smaller diameters as confirmed by the AFM, adsorbed more amountsof BSA than TO-O-CNF and TO-R-CNF. Moreover, the aldehyde groups onTO-CNF can also explain the high BSA adsorption on TO-CNF as theycan react with the amines of the adsorbed proteins via a Schiff baselinkage. Yet, protein adsorption is a competitive phenomenon becauseany physiological environment involves multiprotein systems.37 Adsorption of a single protein on a biomaterialsurface is affected by the presence of other proteins in the solution.This can explain the change in the adsorption behavior of nanocellulosesurfaces in the case of FBS. Albumin, which is a small protein witha very high concentration in serum, tends to adsorb first, but itis partially replaced by larger proteins such as fibronectin and fibrinogen.38 Hasan et al. reported a linear increase in adsorbedFBS proteins with increase in surface hydrophobicity.19 This might explain why CNF groups (more hydrophobic surfaces)adsorbed more 10% FBS proteins than BNC samples. Generally, proteinadsorption is a complex phenomenon controlled by biomaterial surfaceproperties  and proteinsolution properties .39Adsorption of proteins on any biomaterialsurface is the first event that occurs in biological systems. Evaluatingthe adsorption of nonspecific proteins onto nanocellulose surfacesis important for their tissue engineering applications. Protein adsorptionwas greatly influenced by protein solution (type and concentration).When nanocellulose samples were soaked in 5% BSA solution, a significantlymore protein amount was adsorbed on the surfaces of TO-CNF and BNCthan that on the TO-O-CNF and TO-R-CNF 5A. In thThe potential releaseof any toxic products from the CNF materials after the chemical pretreatmentswas investigated by extract cytotoxicity test. The cellular responseto the extracts of different nanocellulose materials was examinedusing the live/dead stain after 24 h 6. All na9 Similarly, Hua et al.reported that the cell viability of wood-based nanocellulose extractswas comparable to cell viability in regular culture medium after 24h.40 Similarly, other reports demonstratedthat the extracts of wood-based nanocellulose prepared by differentchemical pretreatments had no negative effects on mitochondrial activityof different cells.42In our previous study, we demonstrated that, independentlyof thechemical treatments, CNF hydrogels showed no toxic effects againstmouse fibroblasts.44In the case of direct cytotoxicityassessment, cell viability wasassessed utilizing live/dead stain and the LDH assay 7. RegardThe first events of cell\u2013materialinteractions when cells are in direct contact with biomaterials areprotein adsorption and cell adhesion. The cell morphology was visualizedafter fluorescence staining of F-actin in green and nuclei in blueafter 4 h (data not shown) and 24 h 8.43After 4 h, cells on all surfaces showed round morphology.After24 h, cells on TCP and TO-CNF  surfacesdemonstrated the largest surface areas, while cells on TO-O-CNF (withOH and COOH groups) and BNC (with the OH group) had the smallest surfaceareas. Replacement of aldehyde groups with hydroxyl groups in TO-R-CNFstimulated cells to spread with the surface area comparable to TCPand TO-CNF. These findings suggest that not only the type of the functionalgroups but also their amount is critical for directing cell response.Hua et al. demonstrated that human fibroblasts and osteoblast-likecells presented poor cell adhesion on nanocellulose without carboxylicsurface groups. Specifically, they reported that carboxyl groups \u2265260\u03bcmol/g improved cell adhesion and morphology.18 The ability of carboxyl-terminatedCNFs to facilitate cell adhesion is due to their affinity to adsorbcell adhesion proteins such as fibronectin and vitronectin.45 In contrast, hydroxyl-terminated surfaces, suchas BNC, exhibit poor protein adsorption due to their neutral chargeand hydrophilic character.18 Our proteinadsorption results demonstrated that the physicochemical propertiesof the BNC surface, including small Ra (31 nm) and small contact angle (27\u00b0), adsorbed more of thenon-cell-adhesive BSA, which may explain the smaller cell surfacearea on the BNC surface.34 Moreover, CNFmaterials have a fibrous hierarchical structure of nano to microscale,which was reported to improve stem cell adhesion and osteogenesis.46 The absence of microfibers in BNC samples canbe another possible explanation of the decreases in the cell areaand cell length of the long axis.47 Thiscould be attributed to the size of cell focal adhesions that are upto a few micrometers in size.48 Therefore,it is suggested that microscale topographical features may providea stronger influence on focal adhesion formation than nanoscale features.47In general, cells cultured on all CNF surfaces showedsimilar orslightly more elongated cells than cells cultured on TCP controls.Nevertheless, the maximum length of cells cultured on TO-CNF and TO-R-CNFsurfaces was significantly higher than the length of cells on BNC.Regarding the surface chemistry, Cao et al. reported that mesenchymalstem cells exhibited more spreading morphology and larger surfaceareas on surfaces with carboxyl groups when compared to surfaces withhydroxyl groups.19 This speculation agreed well with other reportssuggesting that cell adhesion gets optimum on surfaces with contactangles of 50\u201380\u00b0.45 However, the TO-O-CNFgroup has a similar contact angle (63.0 \u00b1 6.6), but the cellscultured on its surface had significantly less surface areas thanTO-CNF. Even though the physicochemical features of biomaterials arepotent regulators of cell functions and can dictate their performance,it appears that the biological responses cannot be attributed to individualsurface parameter alone.Interestingly, TO-R-CNF, which has noaldehyde groups, showed comparableresults to TCP and TO-CNF, indicating that the improved cell adhesionon TO-CNF samples is likely not related to the presence aldehyde groups.Compared to TO-CNF, TO-R-CNF has a larger contact angle (63.8 \u00b17.5), which can explain the improved cell adhesion. Surfaces witha contact angle around 60\u00b0 were reported to enhance cell adhesion.46Generally, allnanocellulose surfaces supported cell proliferation in growth medium9A,C. At 46 Kumar and colleagues showedthat, in the absence of osteogenic supplements, polymeric nanofiberswere able to drive the cells down the osteogenic lineage.49 In the case of the osteogenic medium, ALP productionsignificantly increased from day 1 to 14 in all groups. The amountof ALP was always significantly higher in BNC and TO-R-CNF groupscompared to that in TCP at any time point. Among the CNF groups, cellscultured on TO-O-CNF produced significantly less ALP than cells onthe TO-CNF surface at day 7 and TO-R-CNF at day 14.ALP, an early marker of osteogenic differentiation,was analyzedto assess the osteogenic potential of all nanocellulose surfaces 9B,D. Aft50 Integrins bind to their ligand as a heterodimer of two \u03b1 and\u03b2 subunits. There are several \u03b1 (\u03b1) and \u03b2(\u03b2) subunits indicating that, through their different possiblecombinations, several cellular signaling pathways can be activated.50Moreover,the ability of BNC and TO-R-CNF to significantly supportosteogenic differentiation of the cells, when cultured in osteogenicmedium, was confirmed by Alizarin red S staining, as shown in 51 Incontrast, other reports suggested that OH-terminated surfaces supportedsignificantly more osteoblastic gene expression, ALP activity, andmatrix mineralization than COOH-terminated surfaces.23 This can explain the effect of BNC, which has more OH-terminatedsurfaces. Interestingly, TO-R-CNF has both hydroxyl and carboxylicgroups but showed more biomineralization than other groups. This findingindicates that the osteogenic differentiation might be related notonly to surface chemistry. Both BNC and TO-R-CNF demonstrated thesmallest Ra values ,which may explain their promoted mineralization results. In agreementwith this speculation, Khang et al. suggested that the nanoroughnessis influential in promoting osteoblast differentiation through integrinactivation and expression of cyclins.52 Faia-Torres et al. confirmed that changes in polymer roughness (Ra) may affect the commitment and degree of MSCosteogenic differentiation.53 Moreover,surface chemistry, wettability, and surface charges were reportedto affect cell adhesion and differentiation not only by the adsorptionof adhesive proteins  but alsoby changing their conformations.54 For example, it was shown that the biomineralizationwas promoted on negatively charged substrates with carboxyl groupswhen the integrin \u03b23 subunit was blocked.55 While it is hypothesized that the binding of \u03b1v\u03b21integrin promotes osteogenic differentiation, \u03b1v\u03b23 integrin,on the other hand, suppresses bone mineralization.54 The negative charges from COOH may cause a conformationalchange in the adsorbed adhesive proteins, which promotes the bindingof both \u03b15\u03b21 and \u03b15\u03b23 integrins. Such conformationalchanges may alter the integrin binding affinity and subsequently activatedifferent signaling pathways that leads to different stem cell differentiationresults.54 However, most of the data publishedto define the relationship between surface chemistry and MSC differentiationis based on single monolayer or hybrid systems with one or two functionalgroups involved.51 Indeed, the multiscale fibrillar nature of the cellulosic materialswith their mixed functional groups and different wettability and chargesmakes it difficult to identify a direct correlation between theirchemistry and cell responses. Importantly, the interaction betweencells with a biomaterial surface is a complex bidirectional process,and further investigations should be considered.At the level of surface chemistry, biomaterialswith carboxylicand hydroxyl groups have been shown to promote and maintain chondrogenesisbut did not support osteogenesis.In this study, the role of aldehyde functionalgroups of TEMPO-mediatedoxidized CNFs was investigated in vitro with rat bone marrow mesenchymalstem/stromal cells, in terms of morphology, proliferation, and osteogenicdifferentiation. Removal of aldehyde groups from TEMPO-oxidized CNFsdid not negatively affect cell responses. This indicates that theintroduction of aldehyde groups (280 \u00b1 14 \u03bcmol/g) throughTEMPO-mediated oxidation to the surface of CNFs might have littleor no effect on attachment, proliferation, and osteogenic differentiationof rat MSCs. Compared to TEMPO-oxidized and postoxidized groups, replacementof aldehyde groups with alcohols via reduction treatment decreasedcarboxylic groups and increased hydroxyl groups onto the surface ofTO-R-CNF samples. This was beneficial not only for cell adhesion andspreading but also for the osteogenic differentiation of the cells.It is concluded that TO-R-CNF coating can have a promising potentialto improve the biological performance of scaffolds for bone tissueengineering."
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