{"text": "In premenopausal women with advanced breast cancer the luteinising hormone-releasing hormone agonist goserelin  will produce serum levels of oestradiol equivalent to those following surgical oophorectomy or the menopause. This paper reports our further experience of using this drug in 75 premenopausal patients with advanced breast cancer. In addition to response rates, duration of response is reported. An objective response was seen in 25 patients (33%), the median duration of which was in excess of 15 months. Seven patients (9%) showed a complete response to therapy; median duration greater than 37 months. There was no significant difference in time to disease progression  and probability of survival  between those patients assessed as having either static disease, or those showing a partial response at six months. Response to therapy correlates significantly with the oestrogen receptor status of the primary tumour . The modest side-effects, ease of administration and reversibility make this approach to therapy very attractive. This is to be remembered in that 53% of patients had disease progression whilst receiving goserelin. These patients thus avoided the unnecessary and irreversible morbidity associated with surgical oophorectomy. With the proven efficacy and minimal morbidity associated with goserelin we believe there is no current role for surgical oophorectomy in the management of premenopausal patients with advanced breast cancer."}
{"text": "Sixty patients with advanced breast cancer unresponsive to tamoxifen have been randomised to receive four course of mitozantrone, 14 mg m-2 (n = 30) intravenously every 3 weeks  or megesterol acetate, 160 mg bd (n = 30). One in three patients (11 from each group) had substantial disease control for a minimum period of 6 months i.e., lack of progression; seven patients (23%) showed objective response to mitozantrone compared to four (13%) receiving megesterol. Non-progressive disease occurred in all sites, including visceral metastases and receptor negative patients. There were no significant differences between treatment groups in the median time (5 months each) to disease progression response duration or survival  from commencing second-line therapy. Toxicity was considerably higher in the mitozantrone group. Second-line hormonal therapies can produce similar therapeutic results as those achieved from a short course of a 'short option' single agent cytotoxic in patients who were previously thought hormone insensitive. Provided that the patient does not have life threatening disease a trial of megesterol acetate is worth consideration in that it does not prejudice subsequent response to combination cytotoxic chemotherapy."}
{"text": "The mol­ecule is folded about the ethyl­ene bridge, adopting a staggered conformation such that the benzene ring of the tosyl group and the five-membered ring of the phthalimide moiety have a face-to-face orientation with a centroid-to-centroid separation of 3.7454 (12) Å. The crystal structure is stabilized by weak inter­molecular π–π inter­actions between symmetry-related five-membered rings of the phthalimide groups, with a centroid-to-centroid distance of 3.3867 (11) Å. The compound is used for the attachment of a suitable chelate functionality for radiolabeling purposes.In the title mol­ecule, C Å                           b = 12.5642 (12) Å                           c = 19.3194 (19) Å                           β = 107.121 (2)°V = 3173.8 (5) Å3                                                   Z = 8                           Kα radiationMo −1                        μ = 0.23 mmT = 90 (2) K                           0.40 × 0.35 × 0.30 mm                                 Bruker APEX CCD area-detector diffractometerSADABS; Brucker, 2007T                           min = 0.913, T                           max = 0.934Absorption correction: multi-scan (16184 measured reflections3865 independent reflectionsI > 2σ(I)3773 reflections with R                           int = 0.020                                                            R[F                           2 > 2σ(F                           2)] = 0.052                           wR(F                           2) = 0.122                           S = 1.25                           3865 reflections218 parametersH-atom parameters constrainedmax = 0.48 e Å−3                        Δρmin = −0.41 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008CrystalMaker (Palmer, 2006SHELXTL (Sheldrick, 2008Data collection: 10.1107/S1600536808037951/lh2734sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808037951/lh2734Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:"}
{"text": "Tuberculosis (TB) is the second leading cause of mortality associated with infectious diseases globally and the leading cause of death among people living with HIV. In 2010, there were an estimated nine million people with TB – a third of whom do not have access to TB services – and approximately 1.5 million deaths were attributed to this disease [This year, the STOP TB PARTNERSHIP is focusing on diagnosis, treatment and cure for all .Comparing the pathogenesis of TB with other infections, most infected people can remain asymptomatic for decades. The clinical definition of a latent infection is limited to a positive response to an injection of purified mycobacterial components. The spectrum of active disease is complex. Adult pulmonary TB is considered a public health problem, and extrapulmonary disease occurs in a significant proportion of co-infected patients. Also, paediatric manifestations are difficult to diagnosis. Moreover, many of the 3 million undiagnosed people live in the world's poorest and most vulnerable communities , 3.Treatment is complicated. The combination of drugs requires sequenced administration, unlike other antibacterial treatments that have to be administered for a minimum of six months. The complex interplays between the genetic strain of the bacteria, immune response of the host, and pathogenicity and acquisition of resistance increase the challenges posed by the global TB epidemic and require the development of appropriate approaches for the discovery of drugs and diagnostic techniques applicable in these settings. More research is needed in order to increase our understanding of, and evaluate, the natural history of TB in the context of co-infections, comorbidities and risk behaviours .New England Journal of Medicine in 2012 and based on findings from 4000 patients monitored by the Chinese Center for Disease Control and Prevention showed that in newly diagnosed TB cases, 34.2% had some form of drug resistance, 5.7% had MDR TB and 0.5% had XDR TB. Among those previously treated, 54.5% had some form of drug resistance, 25.6% had MDR TB and 2.1% had XDR TB [Microbial resistance has existed since the dawn of the antibiotic era. Multidrug-resistant (MDR) TB requires treatment for up to two years, with success rates of about 65–75%. Extensively drug-resistant (XDR) TB involves additional resistance to two second-line drugs; it is more difficult to treat and may be incurable. One study published in the The sputum smear misses diagnosis in a half of those infected and provides no information about drug resistance. In low-income and middle-income countries, where the global burden is heaviest, sputum, solid culture and chest radiography are scarcely available for diagnosis. However, these tests do not have sufficient sensitivity and specificity, and they are usually not used or available at the periphery of the health system.The ideal test for TB is a true point-of-care test that enables accurate diagnosis and the detection of drug resistance within the time of clinic consultation.The biggest challenge today is to progress in the development of new diagnostic assays.Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance, was endorsed by the World Health Organization (WHO) in December 2010. This assay was specifically recommended for use as the initial diagnostic test for suspected drug-resistant or HIV-associated pulmonary TB. Although the development of the Xpert MTB/RIF assay is undoubtedly a landmark, the clinical and programmatic effects and cost-effectiveness remain to be defined [Rapid progress has been made in the development of new diagnostic assays for TB in recent years. New technologies have been developed. The Xpert MTB/RIF assay, which enables simultaneous detection of  defined .Future advances in molecular diagnosis should build on this success. Despite recent developments, progress in nucleic acid amplification is still weak and should be strengthened.A rapid, accurate point-of-care diagnostic test that is affordable and can be readily implemented is urgently needed. Investment in the TB diagnostics pipeline should remain a major priority for funders and researchers.Another objective of the World TB Day is treatment and cure for all. New treatments and regimens for patients with drug-resistant as well as drug-sensitive TB are needed. The long duration of treatment and poor tolerability represent important challenges.As stated, the incidence of MDR in certain regions, such as eastern Europe, southern Africa and central Asia, is a current problem, as is retreatment as compared with new cases. Shorter regimens are essential. With new drugs for TB, multidrug regimens will require considerations of interactions, pharmacokinetics, toxicity, drug metabolism and the thresholds for the development of resistance. The recognition that patients with TB frequently present with co-infections and comorbidities, such as HIV or other viral infections , must be assessed at the earliest possible stage in clinical development .There are new drugs in a pipeline that is more robust than ever. Advancing the planning and coordinated assessment of treatment combinations is a high priority. Trials of a novel nitro-dihydro-imidazooxazole (delamanid), together with a background regimen of second-line drugs for MDR TB, resulted in culture conversion at eight weeks, increasing from 30% in placebo versus 45% in the delamanid group. A study of another novel agent, bedaquiline, showed similar results to the delamanid study, although the placebo group showed substantially lower results (9% efficacy). This may be due to the inclusion of more potent fluoroquinolones in the delamanid study [The REMoxTBtrial13 replaced either isoniazid or ethambutol with the fluoroquinolone moxifloxacin (M) in two experimental, four-month regimens (2HRZM/2HRM and 2MRZE/2MR); results are expected shortly. If the findings from the REMoxTB trial are positive, regulatory approval will be sought in 2014 and a national launch could start as early as 2015 . MoxifloReduce sexual transmission of HIV.Halve the rate of HIV infection among persons who inject drugs.Eliminate new HIV infections among children.Increase the number of persons on life-saving treatment to 15 million.Reduce by half the number of TB-related deaths in persons living with HIV.Finally, eliminating TB and TB/HIV in the 21st century is a priority for various high-level partners. This has been delineated by UNAIDS, world leaders and the United Nations General Assembly, who are committed, by 2015, to [In summary, World TB Day on 24 March 2014 brought with it the vision and possibility of achieving “a TB test, treatment and cure for all.”"}
{"text": "Although brief alcohol intervention (BI) is widely studied, studies from psychiatric outpatient settings are rare. The aim of this study was to investigate the effects of two variants of BI in psychiatric outpatients. By using clinical psychiatric staff to perform the interventions, we sought to collect information of the usefulness of BI in the clinical setting.Psychiatric outpatients with Alcohol Use Disorders Identification Test (AUDIT) scores indicating hazardous or harmful drinking were invited to participate in the study. The outpatients were randomized to minimal  or BI . Measurements were performed at baseline and at six and 12 months after the intervention. The primary outcome was change in AUDIT score at the 12-month follow-up.In all, 150 patients were enrolled and received either a minimal intervention (n = 68) or BI (n = 82). At 12 months, there was a small reduction in AUDIT score in both groups, with no significant differences in outcome between groups. At 12-month follow-up, 21% of participants had improved from a hazardous AUDIT score level to a nonhazardous level, and 8% had improved from a harmful level to a hazardous level (8%).Brief alcohol interventions may result in a reduction of AUDIT score to a small extent in psychiatric patients with hazardous or harmful alcohol use. Results suggest that BI may be of some value in the psychiatric outpatient setting. Still, more profound forms of alcohol interventions with risky-drinking psychiatric patients need elaboration. Over the past two decades, numerous investigations of the effectiveness of brief intervention (BI) for hazardous or harmful drinking have been performed,2. BriefBrief intervention is a method of addressing alcohol problems in an early stage. The method usually comprises a screening procedure , brief feedback on the results, and personalized information about possible consequences. Five to 15 minutes is the average length of a BI . WrittenThe effectiveness of BI can be studied from different aspects. When measured by reduction of weekly alcohol consumption, BI delivered in primary and emergency care has been found to be effective at reducing hazardous drinking at 12 months or longer. In primMost studies on BI have been performed in primary care, in-hospital care, or emergency care settings,10,11. AHulse et al.,16 invesIn this study, we sought to investigate the effects of two variants of BI  in psychiatric outpatients. We hypothesized that patients who received the more intensive BI  would have lower AUDIT scores at 12-moThis study was part of a project aiming to determine the need for and suitability of BI in a psychiatric outpatient setting. The project was conducted at the Clinic of General Psychiatry at Uppsala University Hospital, Sweden . Study participants were adult outpatients aged ≥18 years who were treated at the clinic. Patients with psychosis or substance abuse disorders (SUDs) were treated at other clinics in the division of psychiatry and, therefore, were not included in this study.An important feature of the project was its naturalistic approach: interventions as well as the distribution of questionnaires were carried out by ordinary psychiatric clinical staff. Involving ordinary staff and using well-established questionnaires gives a realistic picture of effects and allow other psychiatric clinics to readily adopt the design. At the beginning of the project, all psychiatric treatment staff  received information and training for three hours. PhysiciThe AUDIT was developed in 1982 from a WHO collaborative project as a simple method to screen and identify individuals at risk of developing alcohol- and alcohol-related problems. The 10-The AUDIT-C is a condensed version of the full AUDIT. It consists of the three consumption items (items 1–3) from the full AUDIT. Scoring corresponds to that of the full AUDIT, ranging from 0–4. The AUDIT-C is frequently used in busy settings and for follow-up measurement-23.In a drinking diary, the number of standard drinks consumed each day per week is reported retrospectively.The RCQ is a 12-During a three-month period in the autumn of 2009, a questionnaire package that included the AUDIT as well as drug, tobacco and gambling items was administered to patients visiting seven outpatient units at the Uppsala University Hospital general psychiatric clinic. At each outpatient unit, receptionists were instructed to distribute a questionnaire package to each patient. After filling out the questionnaires, the patient handed them to his or her caregiver , who forwarded them to the first author of this paper to calculate AUDIT scores. The AUDIT score of every patient was communicated on paper to the patient’s caregiver. If the patient met the inclusion criterion for the study (AUDIT score of 6–19 for women or 8–19 for men), specific instructions to the caregiver were added. (Only three patients were enrolled by a physician).The only exclusion criterion was ongoing treatment for SUD. If patients fulfilled the inclusion criterion, caregivers were instructed to inform them that their drinking habits were considered hazardous and to invite them to participate in the study. The caregiver provided the patients with written information about the study. No incentives were offered. Patients who agreed to participate gave written consent. For every risky-drinking patient, the caregiver was instructed to return a form to the first author that included either the written consent or the information that the patient was not interested in participating. After randomization of consenting patients, the caregivers were handed written instructions on how to proceed at the forthcoming consultation. For patients indicating dependence (AUDIT score ≥20), the caregiver was advised to take further action.For the randomization process, a person from outside the research group, blinded from information of patient characteristics, created an allocation sequence by drawing pieces of paper out of a basket. Patients were assigned to intervention groups in the order the signed consent forms reached the principal author, following the allocation sequence.The interventions took place during the patient’s ordinary consulting time with their caregiver. Estimated time taken was 10 minutes for the minimal intervention and 15–20 minutes for the BI.Patients in the minimal intervention group were asked to complete the questionnaire package (RCQ and drinking diary) and were given an information leaflet. The leaflet included facts about risky drinking and practical advice on how to reduce alcohol consumption. Caregivers were instructed to avoid any further discussions on drinking habits.Patients in the BI group were asked to complete the same questionnaire as the patients in the minimal intervention group. The one difference from minimal intervention was that they also received a face-to-face intervention with their psychiatric caregiver. The intervention, for which a template was used, took the form of brief advice designed to meet the needs of a person with psychiatric problems, with the aim to enhance motivation to reduce drinking (see Appendix). The principles of MI were considered in the design of the intervention. Finally, BI patients were offered the information leaflet previously described.Staff training in how to perform interventions took place before baseline data collection and at follow-up two months later. During training sessions, intervention fidelity was stressed and discussed. The staff training course is described in detail elsewhere.Measurements were performed at baseline and at six and 12 months. The primary outcome was changes in AUDIT score at 12 months as measured by the full 10-item AUDIT. To measure changes in alcohol habits at six months, the AUDIT-C was used. Changes in AUDIT-C score were measured by extracting AUDIT items 1–3 from the baseline AUDIT and comparing them with the AUDIT-C score at six months. At both the six- and 12-month follow-ups, participants were asked to complete the RCQ and a drinking diary for the preceding week. Participants were contacted by the research team by mail and, if needed after one reminder, by telephone. See FigureInformation on the patient’s diagnosis and employment status at baseline was collected from medical records. To estimate psychiatric severity, the number of psychiatric consultations during the 12-month period after the initial screening was collected for each patient. The study was approved by the Research Ethics Committee at Uppsala University Hospital (DNR 2009/042).χ2 –test. Fisher’s test was used when applicable. Independent samples t-tests were used to test differences in AUDIT scores at baseline between dropouts and responders. All statistical analyses were performed using SPSS , version 19.A conservative intention-to-treat (ITT) analysis was applied in which all patients included had their last rank carried forward in the analysis in order to compensate for dropouts. Although data on alcohol habits may be defined as nonparametric, we chose to treat AUDIT scores as parametric data to make our results comparable with those of previous studies. For the outcome analysis, ANOVA was used . Effect sizes are denoted by d. Comparison of baseline characteristics between participants in each group were performed with Pearson’s Of the 559 eligible risky-drinking patients, 167 patients agreed to participate in the study. One hundred forty-one declined, and another 249 patients made no return visit during the specified period and were therefore not invited. Two patients were not invited because of ongoing SUD treatment elsewhere. After randomization, 150 of the 167 patients who agreed to participate were still willing and eligible. Of the 150 patients, 68 received a minimal intervention, and 82 received BI. Baseline demographic data are presented in TableThe mean AUDIT score at baseline in the enrolled group was 10.9 ± 3.5 points and was 10.6 ± 3.6 points in patients who declined to participate. Mean age in the enrolled group was 28.4 ± 9.9 years for women and 34.9 ± 13.6 for men; the mean age was 28.6 ± 11.0 years for women and 31.7 ± 11.8 years for men among patients who declined to participate. Gender proportions in the enrolled group were 65.3% women and 34.7% men. In the group that declined, the proportions were 73.8% women and 26.2% men. Interventions took place a mean of 21 ± 22.3 days from baseline AUDIT screening . The attrition rate was 15% (n = 22) at six months and 28% (n = 43) at 12 months. The results reported are based on the whole group of 150 participants.At 12 months, there was an overall reduction in AUDIT scores from 10.9 to 9.8 . Participants in the BI group reduced their score from 10.7 to 9.4 points. Participants in the minimal intervention group reduced their score from 11.2 to 10.3 points. There was no interaction effect between groups. The BI did not affect AUDIT scores more than the “minimal” intervention. In all, 21% of participants reduced their drinking to below hazardous level, regardless of intervention assignment. Another 8% reduced their drinking from the harmful to the hazardous level. There were no statistically significant differences in readiness to change stage over time in either group. No changes in number of drinks consumed per week could be discerned over time. At the six-month follow-up, there was a reduction in AUDIT-C scores from 5.0 ± 1.5 points to 4.7 ± 2.0 points  in the entire sample.χ2 = 9.4, p < 0.01). The improved group had a reduction in AUDIT-C scores already at the six-month follow-up .Further analysis was undertaken to compare participants who improved their drinking habits (the improved group) with those who did not improve their drinking habits (the nonimproved group). In the improved group, participants were significantly more motivated to change their drinking habits at baseline than participants in the nonimproved group. At baseline, 58.5% (n = 24) in the improved group were contemplating change versus 31.1% (n = 32) in the nonimproved group . No gender differences or differences in the number of psychiatric consultations were found between the improved and the nonimproved groups.In the improved group, the AUDIT consumption item with greatest reduction at 12-month follow-up was item 2: To our knowledge, this is the first study of BI in the psychiatric setting using clinical psychiatric staff. The major finding is that BI has a positive  12-month effect on alcohol habits in psychiatric outpatients. The minimal intervention produced similar results as the more intense BI: i.e., assessment, feedback, and a leaflet, with or without brief advice, reduced AUDIT score in risky-drinking patients. The reported major strategy among patients for reduced drinking was cutting down on number of drinks per drinking day. Gender or psychiatric severity as measured by number of psychiatric consultations had no impact on results.Investigations of BI in a psychiatric outpatient setting are rare. In one such study, by Eberhard et al., the desIn one other study conducted targeting inpatients in a psychiatric setting, Hulse et al. comparedPatients with psychiatric disorders were included in a recent review of BI for substance use. Except The results of our study are in line with those of Hulse et al. and Eberhard et al., suggesting that BI may be of some value also in the psychiatric setting. Our results imply that the intervention may be very brief and still be effective. Addressing the issue through assessment, feedback, and information leaflet may be sufficient. Intervention effects are relatively small, which indicates that regression to the mean as well as natural recovery processes cannot be ruled out.In this study we preferred the label “minimal intervention group” rather than control group. Patients in the minimal intervention group underwent assessment, were given feedback on drinking habits, gave consent, and received a leaflet. Intrinsic as they are to intervention studies, such factors are known to reduce drinking significantly over time,31.In general, the mechanisms that lead the risky-drinking person to change drinking habits after BI are unclear. It is plausible that BI works simply by pointing out a potential problem, which would stimulate risky-drinking persons to contemplate change. Most excessive drinkers who change, even those with more advanced alcohol problems or comorbid psychiatric disorders, change their drinking patterns without treatment,33. The Psychiatric outpatient units are busy; therefore, secondary alcohol assessment and prevention is not commonly given high priority. Consequently, such measures need to be short and easy to use but still efficient. In this study, BI was administered in a clinical psychiatric setting with caregivers of all categories performing both the screening and the intervention. In previous studies conducted in psychiatric settings, interventions were conducted by research staff. Physicians in primary care commonly are considered the most suitable professional group to perform BI, although nurses may assist,35. PsycMethodological limitations of this study need to be addressed. One limitation is the reliance on self-reported data. To engage all categories of psychiatric staff, we chose to use questionnaires only and not biological markers. However, biomedical markers have disadvantages: their use requires medical staff, and they are not reliable enough on their own. FurtherAnother limitation of our study is the small sample size. If a larger sample was used, conclusions could be more strongly supported. The dropout rate is another limitation. Women dropouts (n = 28) had higher AUDIT scores at baseline than women whom completed follow-up . No other systematic attrition could be identified. With the use of a conservative ITT analysis, we eliminated the risk of overestimating intervention effects. Also, follow-up data were collected by the research team only and not by the patient’s psychiatric caregiver, thus lowering the risk that the patient underestimated his or her drinking to please the caregiver.The major strength of this study is that it adds knowledge to the field of secondary alcohol prevention in a group of patients who are in urgent need of such strategies. By addressing alcohol habits in a simple, time-efficient manner, psychiatric staff of any profession may initiate small but measurable improvements in risky drinking behavior.Brief alcohol interventions in psychiatric care may result in a reduction of AUDIT score to a small extent in patients with hazardous or harmful alcohol use. Brief intervention can easily be performed by different psychiatric health professionals within their normal schedule and may be of some value in the psychiatric outpatient setting. Still, the modest effects of very brief interventions suggest that more profound forms of alcohol interventions with risky-drinking patients need to be elaborated in psychiatric care.Brief (approximately 10-minute) alcohol intervention for psychiatric patients with hazardous or harmful drinking (AUDIT score of 6–19 points for women and 8–19 points for men).Instructions:1. Inform the patient that her/his AUDIT score is above the limit usually considered low-risk; i.e., if she/he continues drinking at this level, there is a risk of harmful effects or dependence, even if that is not the case today.2. Alcohol often affects how one feels. What does the patient know about that? How does she/he use alcohol, and how does alcohol make her/him feel? Listen.3. Offer information; e.g., “Would you like some information about how alcohol affects mental health?”4. Describe why a person with psychiatric problems should be especially observant of drinking habits; for example:If you are having or have had psychiatric problems, you are more sensitive to the adverse effects of alcohol. This is the case even if you are a moderate drinker.Alcohol use can prolong periods when you feel more affected by your psychiatric symptoms.Alcohol may help you fall asleep more easily, but it also disturbs your sleep. It is common to wake up too early the morning after drinking with a feeling of anxiety.Alcohol may induce depression. Many research studies have shown this connection.If you’re on medication, alcohol interacts with medicines in an unpredictable way. Your medicine may fail to be effective, or you may get unexpected side effects.5. What are your patient’s thoughts when hearing this information? Listen.6. Ask if she/he would like some advice on how to cut down on drinking. If yes, introduce the tips presented in the leaflet, giving examples of how they can be used.7. Ask the patient to take the leaflet home to study it more thoroughly.The authors declare that they have no competing interests.CN carried out the collection of data, performed the statistical analysis, and drafted the manuscript. LG, AF, and LJ helped draft the manuscript. All authors took part in the design of the study and in interpreting the results. All authors have read and approved the final manuscript."}
{"text": "Oxygen tensions and gradients play essential roles in modulating biological systems in both physiologic and pathologic events. Thus, controlling oxygen tension is critical for mimicking physiologically relevant in vivo environments for cell, tissue and organ research. We present a new approach for on-demand generation of various oxygen tensions for in vitro hypoxia models. Proof-of-concept prototypes have been developed for conventional cell culture microplate by immobilizing a novel oxygen-consuming biomaterial on the 3D-printed insert. For the first time, rapid (~3.8 minutes to reach 0.5% O2 from 20.9% O2) and precisely controlled oxygen tensions/gradients (2.68 mmHg per 50 μm distance) were generated by exposing the biocompatible biomaterial to the different depth of cell culture media. In addition, changing the position of 3D-printed inserts with immobilized biomaterials relative to the cultured cells resulted in controllable and rapid changes in oxygen tensions (<130 seconds). Compared to the current technologies, our approach allows enhanced spatiotemporal resolution and accuracy of the oxygen tensions. Additionally, it does not interfere with the testing environment while maintaining ease of use. The elegance of oxygen tension manipulation introduced by our new approach will drastically improve control and lower the technological barrier of entry for hypoxia studies. Since the biomaterials can be immobilized in any devices, including microfluidic devices and 3D-printed tissues or organs, it will serve as the basis for a new generation of experimental models previously impossible or very difficult to implement.The development of  By innt years , 5. Oxygnt years , 7. Hypoin vivo or control at the microscale. Moreover, oxygen scavenging agents alter the cell growth environment and may affect cellular responses [Many technologies and devices have been developed for controlling oxygen in both macro and micro environments. Current techniques for global oxygen control of macro-environments include hypoxic chambers/workstations/perfusion chambers or directly adding oxygen scavenging agents. Critical challenges remain for these approaches including only providing a single condition of a hypoxic level at a time, slow equilibration time  and beinesponses . To fulfesponses –12. Theyesponses –20. Whilin vitro hypoxia models to overcome these challenges. Proof-of-concept prototypes have been developed for conventional cell culture microplates (96-well and 24-well cell culture microplates) by immobilizing a novel oxygen-consuming biomaterial on the 3D-printed insert. First, the effects of different biomaterial compositions were investigated. Oxygen tensions at different distance from the optimized biomaterial and the response time to reach each steady state oxygen tension were then measured. The dynamic modulation of oxygen tensions was further evaluated for the ischemia-reperfusion injury model. After that, the cytotoxicity tests of the biomaterials were performed. Finally, the developed prototypes were evaluated by examining the release of hypoxia-inducible factor 1α (HIF-1α) and tumor necrosis factor α (TNF-α) by macrophages under different hypoxic levels.In this study, we developed a new approach for on-demand generation of various oxygen tensions for 2 to 15 U/cm2 and from 1:0 to 1:50, respectively. The glucose oxidase and catalase enzymes were diluted with distilled water to have the density of 25 U/μl and 625 U/μl, respectively. A 1% chitosan solution was prepared by dissolving 1.0 g of chitosan in 100 ml of 1.0% acetic acid. For the biomaterial with 0 U/cm2, 10 μl glutaraldehyde solution (1.25%) was added into the 100 μl 1% chitosan solution. For the biomaterial with glucose density from 1.5 U/cm2 to15 U/cm2, 1–10 μl glucose oxidase solution and 2–20 μl catalase solution were dissolved in 100 μl 1% chitosan solution, and then 10 μl glutaraldehyde solution (1.25%) was added. The biomaterials were coated on the targeted surface and allowed to cure at 4°C for 24 hours prior to use.The biomaterial is a hydrogel comprising glucose oxidase  and catalase  enzymes cross-linked with chitosan (1% (w/v) chitosan acetic acid solution; 448877, Sigma) and glutaraldehyde . Glucose oxidase density and the ratio of glucose oxidase to catalase were adjusted from 0 U/cmGlucose oxidase (GOX) enzyme oxidizes D-glucose molecule, which is one of major components of the cell culture medium (most cell culture media have glucose concentrations between 5 and 25 mM), into gluconic acid and hydrogen peroxide. Catalase (CAT) enzyme decomposes hydrogen peroxide into water and oxygen. Based on the following chemical equations, two oxygen molecules are consumed and only one is produced, resulting in a net loss of oxygen .Net reaction:The insert device was designed to integrate with the multiwell cell culture microplates and fabricated through 3D printing technology. The insert was designed using CAD tools, and ordered by mail . The devOxygen tensions were measured using a precision temperature compensated fiber-optic oxygen measurement system . A 21 gauge hole was drilled on the bottom plate in order to insert the oxygen sensing probe. The tip of the oxygen microsensor was aligned to the bottom surface of the 96-well plate. The 3D-printed insert for 96-well was repositioned using a precision XYZ manipulator to control the distance between the biomaterial and oxygen microsensor.All experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the Feinstein Institute for Medical Research. Male Sprague-Dawley (SD) rats (250–350 g) purchased from Charles River Laboratories  were used. Rats were housed in a temperature-controlled room on a 12-h light/dark cycle and fed a standard Purina rat chow diet. At the day of harvesting peritoneal macrophages, rats were anesthetized by isoflurane inhalation, and the abdomen was shaved and washed with 10% povidone iodine. One hundred ml of HBSS were injected to the peritoneal cavity and the rats were gently shaken for 2 min. A small incision on the abdomen was performed to insert a 20-gauge needle to collect all the peritoneal fluid. Then, the rats were rapidly and painlessly euthanized using carbon dioxide exposure followed by exsanguination, as recommended by the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2013 Editions. All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.6 cells/ml RPMI 1640 media in 96-well and 24-well plate [2 incubator. After replacing the overnight media with fresh one, the sterilized inserts that can generate various oxygen tensions were placed into the pate.Extracted peritoneal macrophages were cultured at 1 X 10ll plate . The plaHydrogen peroxide assay kit  and glucose (GO) assay kit  were used to measure the levels of hydrogen peroxide and glucose. MTS viability assay  for mammalian cells was performed to test the biocompatibility of the biomaterials. TNF-α ELISA assay set  was used to evaluate the effect of hypoxia on peritoneal macrophages.The levels of HIF-1α protein from hypoxia induced peritoneal macrophage cells were determined by western blot analysis using HIF-1 alpha antibody . The β-actin western blot results from each individual line were used as the internal controls. The band densities from each line were digitalized using Bio-Rad densitometry image system. The ratio of HIF-1α / β-actin from each group were calculated, averaged and graphed in the histogram. The control group (cells only) and matrix group (no enzymes) were included as the background controls.All data were expressed as mean ± SD (n = 4–12) and compared by one-way ANOVA and the Student Newman-Keuls methods. P<0.05 was considered statistically significant.2 and the ratio of glucose oxidase to catalase was adjusted to 1:0, 1:1, 1:5, 1:10 and 1:50. As shown in 2, 3 U/ cm2, and 15 U/ cm2, the D-glucose concentration was decreased to 89.99±0.26%; 88.05±0.76% and 69.98±1.35% respectively after 24 hours incubation. With 48 hours incubation, the D-glucose concentration was further decreased to 86.82±0.31%; 85.89±1.27% and 54.77±2.18% respectively. Many classical media are supplemented with approximately 5.5 mM D-glucose which approximates normal blood sugar levels in vivo. RPMI 1640 media contains 11.1 mM D-glucose. With 15% glucose consumption, the glucose level decreases from 11.1 mM to 9.4 mM which is still much higher than normal glucose level (5.5 mM). Hence, there was acceptable glucose consumption for the coating density of 1.5 U/ cm2 and 3 U/ cm2. For the optimized biomaterial, glucose oxidase density and glucose oxidase to catalase ratio were fixed to 1.5 U/cm2 and 1:50, respectively.The working principle of the biomaterial indicates that it not only consumes oxygen but also consumes D-glucose and produces hydrogen peroxide and gluconic acid. First, hydrogen peroxide levels from different glucose oxidase and catalase combinations were measured. Biomaterials were coated on 96 well flat bottom plates and 100 μl of RPMI 1640 medium was incubated for 24 hours. Glucose oxidase density was fixed to 1.5 U/cm2 incubator with accurate pH meter . We found that the gluconic acid by-product accumulated in the medium did not affect the pH significantly . Most cell culture media contain a buffer system in their original formula to maintain the pH of the media at a certain range for a period of time. There are also supplemental buffer systems  available to minimize the pH changes for those pH sensitive cell lines. The RPMI 1640 Medium we used in this study contains a sodium bicarbonate buffer system (at 2.0 g/L). In addition, cell culture media is normally replaced by a fresh one twice a week. Therefore, the pH changes caused by the accumulation of gluconic acid will not be a factor for the cell growth in our hypoxia model.The production of gluconic acid may cause acidification of culture media. The pH of the cell culture media was measured after 48 hours incubation at 37°C in a humidified, 5% CO2 and glucose oxidase to catalase ratio of 1:50 were coated on the bottom plate of the 3D-printed insert for 96-well plate. After aligning the oxygen microsensor to the bottom surface of the well plate, the insert was repositioned using a precision XYZ manipulator. 100 μl of RPMI 1640 medium containing 11.1 mM D-glucose was added. First, the response time to reach the steady state oxygen tension was measured at the difference distance from the biomaterial. The response time was hereby defined as the time required reaching 90% of the steady-state value. As shown in Biomaterials with glucose oxidase density of 1.5 U/cm2 for this experiment), oxygen tensions were quickly recovered to almost baseline levels (<0.3% O2 deviation). 1.2% O2 level was generated when the distance between biomaterial and oxygen microsensor was 200 μm. The oxygen level was increased to 4.1%, 7.6%, 9.2% and 12.6% when the biomaterial was moved away from the oxygen microsensor 600 μm, 900 μm, 1000 um and 1200 μm distance, respectively.The dynamic changes of oxygen tensions were evaluated by modulating the distance from the biomaterial. Again, the oxygen microsensor was aligned to the bottom surface of the well plate and the insert with biomaterial was repositioned using a precision XYZ manipulator. As shown in 6 cells/ml) were incubated in supernatant for another 24 hours. 2. However, the biomaterial without catalase enzyme showed toxic. It indicates that the toxicity of biomaterials is mainly determined by the hydrogen peroxide levels and not other factors such as glucose, gluconic acid etc.The biocompatibility of the biomaterials with different glucose oxidase enzyme densities was evaluated. RPMI 1640 medium was exposed to the biomaterials for 24 hours and the supernatant was collected. Rat macrophages  to defend the host. In addition, macrophages also are capable of responding rapidly to hypoxia (low oxygen conditions) by altering their gene expression patterns. This way, macrophages are able to switch metabolism to anaerobic glycolysis, to produce more ATP and to increase their supply of oxygen and nutrients. It is well known that hypoxia and inflammatory signals share very similar transcriptional events in macrophages, including the activation of members of both the hypoxia-inducible factor (HIF-1α and HIF-2α) and nuclear factor κB  families . We, the6 cells/ml RPMI 1640 media in 24-well plates. Two hypoxic levels, 0.84% and 2.21%, were generated by placing two inserts with different pillar lengths  as shown in 2) and two different hypoxic conditions. Consequently, the experimental results demonstrate that oxygen tensions can be precisely controlled using the developed biomaterials.Primary peritoneal macrophages were cultured at 1 X 10In vivo physiological oxygen tension ranges from 1 to 11%, which is well below ambient atmospheric oxygen level of 20.9% [in vitro cell culture system is needed in which the oxygen level can be maintained at a low level. In this study, a novel device has been developed to generate rapid, highly accurate and controllable oxygen tensions by manipulating the distance between the immobilized biomaterial and cultured cells through 3D-printed inserts. Our experimental results have shown that it took around 3.8 minutes to reach 0.5% hypoxia from normoxia (20.9%). Compared to the conventional hypoxic chambers / incubators which require more than 3 hours for the medium inside the cell culture plate to equilibrate to the gas outside of it [Oxygen is critical in a wide variety of cellular signaling pathways. of 20.9% , 30. HypOxygen dissolves from air into the cell culture medium and oxygen consumption occurs on the surface of biomaterial. The oxygen equilibrium forms when oxygen consumption rate by biomaterial equals oxygen supply rate by air diffusion. By adjusting the distance between the cultured cells and biomaterial, various stable oxygen tensions can be achieved. The oxygen tension was controlled precisely with respect to the distance from the biomaterial, e.g., 2.68 mmHg per 50 μm increment in the range of 0 to 500 μm along the Z-axis. Our approach doesn’t require the cumbersome components of its forebears, such as syringe pumps or unwieldy interconnections. In addition, it can be reliably used into the cell incubators. 3D printing technology enables rapid, one-step fabrication of complex geometries not possible with planar lithography with high resolution , 32. It 2 deviation) hypoxic conditions (from 12.6% to 1.2% oxygen tension) can be induced by changing the distance between the cells and biomaterials from 200 μm to 1200 μm. The results highlighted the potential of the developed hypoxia insert to be used for in vitro hypoxia / reoxygenation model of ischemia-reperfusion injury [Dynamic test results further showed that rapid (<130 seconds) and reversible (<0.3% On injury , 34.In this study, we only used the biomaterial as an oxygen consuming material. However, the biomaterial also consumes the D-glucose in the cell culture media and generates hydrogen peroxide. All the chemical reactions happen on the biomaterial surface and diffuse into the media. It implies that hypoxic conditions with various D-glucose and hydrogen peroxide gradients can also be developed for different applications with different combinations of glucose oxidase and catalase enzymes –37. The in vivo environment not yet seen in tissue culture studies. This biomaterial will serve as the basis for a new generation of experimental models previously impossible or very difficult to implement.The developed biomaterial was applied to conventional cell culture microplates to control global oxygen of macro-environments as a proof of concept prototype. However, it can be applied to microfluidic devices to control local and complex oxygen of micro-environments that can closely mimic the microenvironment of living tissue . The elein vitro hypoxia models. Compared to the current technologies, this approach allows enhanced spatiotemporal resolution and accuracy of the oxygen tensions. Additionally, it does not interfere with the testing environment while maintaining ease of use. This biomaterial can be applied for both conventional cell culture microplates and microfluidic devices to control global oxygen of macro-environments and local oxygen of micro-environments. Furthermore, advanced disease models can be developed by coating the biomaterial on the 3D-printed tissues or organs.Taken together, we have developed a novel approach for on-demand generation of various oxygen tensions or gradients for"}
{"text": "Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for  Macrostomum lignano, as well as a new annotated genome sequence.Regeneration capable flatworms have emerged as powerful models for studying stem cell biology and patterning, however their study has been hindered by the lack of transgenesis methods. Here, the authors describe a transgenesis method for  Significant biological insights on stem cell biology and body patterning were obtained using free-living regeneration-capable flatworms (Platyhelminthes) as models4. The most often studied representatives are the planarian species Schmidtea mediterranea2 and Dugesia japonica5. Many important molecular biology techniques and resources are established in planarians, including fluorescence-activated cell sorting, gene knockdown by RNA interference, in situ hybridization, and genome and transcriptome assemblies4. One essential technique still lacking in planarians; however, is transgenesis, which is required for in-depth studies involving e.g., gene overexpression, dissection of gene regulatory elements, real-time imaging and lineage tracing. The reproductive properties of planarians, including asexual reproduction by fission and hard non-transparent cocoons containing multiple eggs in sexual strains, make development of transgenesis technically challenging in these animals.Animals that can regenerate missing body parts hold clues to advancing regenerative medicine and are attracting increased attentionMacrostomum lignano (Macrostomorpha) emerged as a model organism that is complementary to planarians9. The reproduction of M. lignano, a free-living marine flatworm, differs from planarians, as it reproduces by laying individual fertilized one-cell stage eggs. One animal lays ~1 egg per day when kept in standard laboratory conditions at 20 °C. The eggs are around 100 microns in diameter, and follow the archoophoran mode of development, having yolk-rich oocytes instead of supplying the yolk to a small oocyte via yolk cells10. The laid eggs have relatively hard shells and can easily be separated from each other with the use of a fine plastic picker. These features make M. lignano eggs easily amenable to various manipulations, including microinjection11. In addition, M. lignano has several convenient characteristics, such as ease of culture, transparency, small size, and a short generation time of three weeks7. It can regenerate all tissues posterior to the pharynx, and the rostrum12. This regeneration ability is driven by stem cells, which in flatworms are called neoblasts13. Recent research in planarians has shown that the neoblast population is heterogeneous and consists of progenitors and stem cells15. The true pluripotent stem cell population is, however, not identified yet.More recently, a basal flatworm M. lignano using microinjection of DNA into single-cell stage embryos and demonstrate its robustness by generating multiple transgenic tissue-specific reporter lines. We also present a significantly improved genome assembly of the M. lignano DV1 line and an accompanying transcriptome assembly and genome annotation. The developed transgenesis method, combined with the generated genomic resources, will enable new research avenues on stem cells and regeneration using M. lignano as a model organism, including in-depth studies of gene overexpression, dissection of gene regulatory elements, real-time imaging and lineage tracing.Here we present a method for transgenesis in M. lignano is an obligatorily non-self-fertilizing simultaneous hermaphrodite  within 3 h after injection  gene and made a transcriptional GFP fusion in the Minos transposon system  gene as described previously22. The injection of the transgene fragment followed by irradiation demonstrated 5% transgenesis efficiency  errors within contigs compared to 1871 in the ML2 assembly , compared to 94.88% of transcripts mapped on the ML2 genome assembly, and among the mapped transcripts more have intact open reading frames in the Mlig_3_7 assembly than in ML2 . The non-redundant transcriptome has TransRate scores of 0.4360 and 0.4797 for transcriptional units and gene sequences, respectively, positioning it among the highest quality transcriptome assemblies32. The transcriptome is 98.1% complete according to the Benchmarking Universal Single-Copy Orthologs33, with only 3 missing and 3 fragmented genes . For genes tested in this study, the regions up to 2 kb upstream of the transcription start sites are sufficient to faithfully reflect tissue-specific expression patterns of these genes , filled with nutrient enriched artificial sea water (Guillard’s f/2 medium). Worms were fed ad libitum on the unicellular diatom Nitzschia curvilineata  . Climate chamber conditions were set on 20 °C with constant aeration, a 14/10 h day/night cycle.The DV1 inbred M. lignano EFA promoter sequence was obtained by inverse PCR. Genomic DNA was isolated using a standard phenol-chloroform protocol; fully digested by XhoI and subsequently self-ligated overnight (1 ng/μl). Diluted self-ligated gDNA was used for inverse PCR using the EFA specific primers Efa_IvPCR_rv3 5′-TCTCGAACTTCCACAGAGCA-3′ and Efa_IvPCR_fw3 5′-CAAGAAGGAGGAGACCACCA-3′. Subsequently, nested PCR was performed using the second primer pair Efa_IvPCR_rv2 5′-AAGCTCCTGTGCCTCCTTCT-3′ and Efa_IvPCR_fw2 5′-AGGTCAAGTCCGTCGAAATG-3′. The obtained fragment was cloned into p-GEM-T and sequenced. Later on, the obtained sequence was confirmed with the available genome data. Finally, the obtained promoter sequence was cloned into two different plasmids: the MINOS plasmid (using EcoRI/NcoI) and the I-SceI plasmid (using PacI/AscI).The 8 and codon weight matrices were calculated using the 100 most abundantly expressed non-redundant genes. C. elegans Codon Adapter code48 was adapted for M. lignano (http://www.macgenome.org/codons) and used to design codon-optimized coding sequences (Supplementary Data ). Gene fragments  containing codon-optimized sequences, EFA 3′UTR and restriction cloning sites, were inserted into the pCS2+ vector to create optiMac plasmids used in the subsequent promoter cloning.Highly expressed transcripts were identified from RNA-seq dataM. lignano genome assemblies and MLRNA1509 transcriptome assembly8. RAMPAGE signal was used to identify the transcription start site and an upstream region of 1–2.5 kb was considered to contain the promoter sequence. An artificial ATG was introduced after the presumed transcription start site. This ATG was in-frame with the GFP of the target vector. The selected regions were cloned into optiMac vector using HindIII and BglII sites. Primers and cloned promoter sequences are provided in Supplementary Data Promoters were selected using Mlig_3_7, as well as several earlier Worms used for egg laying were kept in synchronized groups of roughly 500 per plate and transferred twice per week to prevent mixing with newly hatching offspring. The day before microinjections, around 1000 worms from 2 plates were combined (to increase the number of eggs laid per plate) and transferred to plates with fresh f/2 medium and no food . On the day of the injections, worms were once again transferred to fresh f/2 without food to remove any debris and eggs laid overnight. Worms were kept in the dark for 3 h and then transferred to light. After 30 min in the light, eggs were collected using plastic pickers made from microloader tips , placed on a glass slide in a drop of f/2 and aligned in a line for easier handling.Needles used in the microinjection procedure were freshly pulled using either borosilicate glass capillaries with filament  or aluminosilicate glass capillaries with filament  on a Sutter P-1000 micropipette puller  with the following settings: Heat = ramp-34, Pull = 50, Velocity = 70, Time = 200, Pressure = 460 for borosilicate glass and Heat = ramp, Pull = 60, Velocity = 60, Time = 250, Pressure = 500 for aluminosilicate glass. The tips of the needles were afterwards broken and sharpened using a MF-900 microforge . Needles were loaded using either capillary motion or microloader tips . Embryos were kept in position using glass holders pulled from borosilicate glass capillaries without a filament  using P-1000 puller with the following settings: Heat = ramp + 18, Pull = 0, Velocity = 150, Time = 115, Pressure = 190. The holders were broken afterwards using a MF-900 microforge to create a tip of ~140 µm outer diameter and 50 µm inner diameter. Tips were heat-polished to create smooth edges and bent to a ~20° angle.M. lignano embryos. An AxioVert A1 inverted microscope  equipped with a PatchMan NP2 for the holder and a TransferMan NK2 for the needle  was used to perform all of the micromanipulations. A FemtoJet express , with settings adjusted manually based on the amount of mucous and debris surrounding the embryos, was used as the pressure source for microinjections. A PiezoXpert  was used to facilitate the penetration of the eggshell and the cell membrane of the embryo.All microinjections were carried out on fresh one-cell stage γ-radiation within 1 h post injection.Irradiation was carried out using a IBL637 Caesium-137 source . Embryos were exposed to 2.5 Gy of 0) were selected based on the presence of fluorescence and transferred into single wells of a 24-well plate. They were then crossed with single-wild-type worms that were raised in the same conditions. The pairs were transferred to fresh food every 2 weeks. Positive F1 animals from the same P0 cross were put together on fresh food and allowed to generate F2 progeny. After the population of positive F2 progeny grew to over 200 hatchlings, transgenic worms were singled out and moved to a 24-well plate. The selected worms were then individually back-crossed with wild-type worms to distinguish F2 animals homozygous and heterozygous for the transgene. The transgenic F2 worms that gave only positive progeny in the back-cross (at least 10 progeny observed) were assumed to be homozygous, singled out, moved to fresh food and allowed to lay eggs for another month to purge whatever remaining wild-type sperm from the back-cross. After the homozygous F2 animals stopped producing new offspring, they were crossed to each other to establish a new transgenic line. The lines were named according to guidelines established at http://www.macgenome.org/nomenclature.html.Positive hatchlings  and 43HE (DsRed), an Axio Scope A1 with a MRc5 digital camera or an Axio Imager M2 with an MRm digital camera.2, overnight hybridization at 68 °C.Southern blots were done using the DIG-System (Roche), according to the manufacturer’s manual with the following parameters: vacuum transfer at 5 Hg onto positively charged nylon membrane for 2 h, UV cross-linking 0.14 J/cmThe Universal GenomeWalker 2.0 Kit  with restriction enzymes StuI and BamHI was used according to the manufacturer’s protocol. Sanger sequencing of PCR products was performed by GATC Biotech (Germany).49 (20 ng/µl), and stored at −80 °C. The final concentration of the probe and optimal hybridization temperature were optimized for every probe separately. Whole mount in situ hybridization was performed following a published protocol49. Pictures were taken using a standard light microscope with DIC optics and an AxioCam HRC  digital camera.cDNA synthesis was carried out using the SuperScript III First-Strand Synthesis System , following the protocol supplied by the manufacturer. Two micrograms of total RNA were used as a template for both reactions: one with oligo(dT) primers and one with hexamer random primers. Amplification of selected DNA templates for ISH probes was performed by standard PCR with GoTaq Flexi DNA Polymerase . Amplified fragments were cloned into pGEM-T vector system  and validated by Sanger sequencing. Primers used for amplification are listed in Supplementary Data 24. Head fragments were collected and treated with 0.2% colchicine in f/2  for 4 h at 20 °C to arrest cells in mitotic phase. Head fragments were then collected and treated with 0.2% KCl as hypotonic treatment for 1 h at room temperature. Fragments were then put on SuperfrostPlus slides  and macerated using glass pipettes while being in Fix 1 solution . The cells were then fixed by treatment with Fix 2 solution  followed by Fix 3 solution , before mounting by using Vectashield with Dapi . At least three karyotypes were observed per worm and 20 worms were analyzed per line.DV1 and NL10 worms were cut above the testes and left to regenerate for 48 h to increase the amount of dividing cells50. In order eliminate the residual diatoms present in the gut, animals were starved for 24 h. For each sample 100 worms were collected in an Eppendorf tube. Excess f/2 was aspirated and worms were macerated in 200 µl 1× Accutase  at room temperature for 30 min, followed by tissue homogenization through pipetting. 800 µl f/2 was added to the suspension and cells were pelleted by centrifugation at 4 °C, 1000 r.p.m., 5 min. The supernatant was aspirated and the cell pellet was resuspended in the nuclei isolation buffer . The cell suspension was passed through a 35 µm pore size filter  and treated with RNase A and 10 mg/ml PI for 15 min prior to measurement. Drosophila S2 cells (gift from O. Sibon lab) and chicken erythrocyte nuclei  were included as references. The S2 cells were treated in the same way as Macrostomum cells. The CEN were resuspended in PI staining buffer . Fluorescence was measured on a BD FacsCanto II Cell Analyzer first separately for all samples and then samples were combined based on the amount of cells to obtain an even distribution of different species. The combined samples were re-measured and genome sizes calculated using CEN as a reference and S2 as positive controls  according to manufacturer’s instructions. For the lysis step worms were kept in the supplied lysis buffer (with Proteinase K added) at 55 °C for 30–40 min and mixed by inverting the tube every 5 min. DNA was ethanol-precipitated once following the extraction and resuspended in TE buffer (for making 454 libraries Qiagen EB buffer was used instead). Concentration of DNA samples was measured with the Qubit dsDNA BR assay kit .454 shotgun DNA libraries were made with the GS FLX Titanium General Library Preparation Kit , and for paired-end libraries the set of GS FLX Titanium Library Paired-End Adaptors  was used additionally. All the libraries were made following the manufacturer’s protocol and sequenced on 454 FLX and Titanium systems.Illumina paired-end genomic libraries were made with the TruSeq DNA PCR-free Library Preparation Kit  following the manufacturer’s protocol. Long-range mate-pair libraries were prepared with the Nextera Mate Pair Sample Preparation Kit  according to manufacturer’s protocol. Libraries were sequenced on the Illumina HiSeq 2500 system.25 v. 1.4 with default parameters, except the errorRate was set to 0.04. The resulting assembly was polished with Pilon51 v. 1.20 using Illumina shotgun data mapped by Bowtie52 v. 2.2.9 and RNA-seq data mapped by STAR53 v. 2.5.2b. Next, scaffolding was performed by SSPACE26 v. 3.0 using paired-end and mate-pair Illumina and 454 data. Mitochondrial genome of M. lignano was assembled separately from raw Illumina reads using the MITObim software54 and the Dugesia japonica complete mitochondrial genome (acc. NC_016439.1) as a reference. The assembled mitochondrial genome differed from the recently published M. lignano mitochondrial genome55 (acc. no. MF078637) in just 1 nucleotide in an intergenic spacer region. The genome assembly scaffolds containing mitochondrial sequences were filtered out and replaced with the separately assembled mitochondrial genome sequence. The final assembly was named Mlig_3_7. Genome assembly evaluation was performed with REAPR27 and FRCbam28 software using HUB1_300 paired-end library and DV1-6kb-1, HUB1-3_6 kb, HUB1-3_7 kb, ML_8KB_1 and ML_8KB_2 mate-pair libraries (Supplementary Table PacBio data (acc. SRX1063031) were assembled with CanuM. lignano RNA-seq data31  and the de novo transcriptome assembly MLRNA150904 (ref. 8) were used to generate an improved genome-guided transcriptome assembly. First, trans-splicing and polyA-tail sequences were trimmed from MLRNA150904 and the trimmed transcriptome was mapped to the Mlig_3_7 genome assembly by BLAT56 v. 36 × 2 and hits were filtered using the pslCDnaFilter tool with the parameters “-ignoreNs -minId = 0.8 -globalNearBest = 0.01 -minCover = 0.95 –bestOverlap”. Next, RNA-seq data were mapped to genome by STAR53 v. 2.5.2b with parameters “--alignEndsType EndToEnd --twopassMode Basic --outFilterMultimapNmax 1000”. The resulting bam files were provided to StringTie29 v. 1.3.3 with the parameter “--rf”, and the output was filtered to exclude lowly expressed antisense transcripts by comparing transcripts originating from the opposite strands of the same genomic coordinates and discarding those from the lower-expressing strand (at least fivefold read count difference). The filtered StringTie transcripts were merged with the MLRNA150904 transcriptome mappings using meta-assembler TACO30 with parameters “--no-assemble-unstranded --gtf-expr-attr RPKM --filter-min-expr 0.01 --isoform-frac 0.75 --filter-min-length 100” and novel transcripts with RPKM <0.5 and not overlapping with MLRNA150904 mappings were discarded. The resulting assembled transcripts were termed ‘Transcriptional Units’ and the assembly named Mlig_RNA_3_7_DV1.v1.TU. To reflect closely related transcripts in their names, sequences were clustered using cd-hit-est from the CD-HIT v. 4.6.1 package57 with the parameters “-r 0 -c 0.95 -T 0 -M 0”, and clustered transcripts were given the same prefix name. Close examination of the transcriptional units revealed that they often represented precursor mRNA for trans-splicing and contained several genes. Therefore, further processing of the transcriptional units to identified boundaries of the encoded genes was required. For this, we developed computational pipeline TBONE , which utilizes exclusively experimental data to determine precise 5′ and 3′ ends of trans-spliced mRNAs. Raw RNA-seq data were parsed to identify reads containing trans-splicing sequences, which were trimmed, and the trimmed reads were mapped to the genome assembly using STAR53. The resulting wiggle files were used to identify signal peaks corresponding to sites of trans-splicing. Similarly, for the identification of polyadenylation sites we used data generated previously8 with CEL-seq library construction protocol and T-fill sequencing method. All reads originating from such an approach correspond to sequences immediately upstream of poly(A) tails and provide exact information on 3′UTR ends of mRNAs. The generated trans-splicing and poly(A) signals were overlapped with genomic coordinates of transcriptional units by TBONE, ‘cutting’ transcriptional units into processed mRNAs with exact gene boundaries, where such experimental evidence was available. Finally, coding potential of the resulting genes was estimated by TransDecoder58, and transcripts containing ORFs but missing a poly(A) signal and followed by transcripts without predicted ORF but with poly(A) signal were merged if the distance between the transcripts was not >10 kb and the spanning region was repetitive. The resulting assembly was named Mlig_RNA_3_7_DV1.v1.genes and includes alternatively spliced and non-coding transcripts. To comply with strict requirements for submission of genome annotations to DDBJ/ENA/GenBank, the transcriptome was further filtered to remove alternative transcripts with identical CDS, and to exclude non-coding transcripts and transcripts overlapping repeat annotations. This final transcriptome assembly was named Mlig_RNA_3_7_DV1.v1.coregenes and used in annotation of the Mlig_3_7 genome assembly for submission to DDBJ/ENA/GenBank.Previously published 59 v. 1.2.1 was used to assemble transposable element models directly from the repeated fraction of raw Illumina paired-end sequencing reads with the parameters “-k 31 -i 300 -m 200 -t 37 --big-graph = 1000”. To mine repeat models directly from the genome assembly, RepeatModeler package (http://www.repeatmasker.org) was used with the default settings. Identified repeats from both libraries were automatically annotated using RepeatClassifier perl script from the RepeatModeler package against annotated repeats represented in the Repbase Update – RepeatMasker edition database60 v. 20170127. Short (<200 bp) and unclassified elements were filtered out from both libraries. Additional specific de novo screening for full-length long terminal repeats (LTR) retrotransposons was performed using the LTRharvest tool61 with settings “-seed 100 -minlenltr 100 -maxlenltr 3000 -motif tgca -mindistltr 1000 -maxdistltr 20000 -similar 85.0 -mintsd 5 -maxtsd 20 -motifmis 0 -overlaps all”. Identified LTR retrotransposons were then classified using the RepeatClassifier perl script filtering unclassified elements. Generated repeat libraries were merged together with the RepeatMasker60 library v. 20170127. The resulted joint library was mapped on the genome assembly with RepeatMasker. Tandem repeats were annotated and masked with Tandem Repeat Finder62 with default settings. Finally, to estimate overall repeat fraction of the assembly, the Red de novo repeat annotation tool63 with default settings was applied.Two methods were applied to identify repetitive elements de novo both from the raw sequencing data and from the assembled scaffolds. Tedna software64, and aligned non-self blocks longer than 500 nt and at least 95% identical were calculated.To identify duplicated non-repetitive fraction of the genome, repeat-masked genome assembly was aligned against itself using LAST softwarehttp://gb.macgenome.org/downloads/Mlig_3_7.All raw data have been deposited in the NCBI Sequence Read Archive under accession codes SRX2866466 to SRX2866494. Annotated genome assembly has been deposited at DDBJ/ENA/GenBank under the accession NIVC00000000. The version described in this paper is version NIVC01000000. The genome and transcriptome assembly files are also available for download at Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Supplementary Movie 2"}
{"text": "Community pharmacy increasingly features in global strategies to modernise the delivery of primary healthcare. Medicine Use Reviews (MURs) form part of the English Government's medicines management strategy to improve adherence and reduce medicine waste. MURs provide space for patient–pharmacist dialogue to discuss the well-known problems patients experience with medicine taking. However, ‘underserved’ communities , who may benefit the most, may not receive this support. This study aims to develop, implement and evaluate an e-learning education intervention which is coproduced between patients from underserved communities and pharmacy teams to improve MUR provision.This mixed-methods evaluative study will involve a 2-stage design. Stage 1 involves coproduction of an e-learning resource through mixed patient–professional development (n=2) and review (n=2) workshops, alongside informative semistructured interviews with patients (n=10) and pharmacy staff (n=10). Stage 2 involves the implementation and evaluation of the intervention with community pharmacy staff within all community pharmacies within the Nottinghamshire geographical area (n=237). Online questionnaires will be completed at baseline and postintervention (3 months) to assess changes in engagement with underserved communities and changes in self-reported attitudes and behaviour. To triangulate findings, 10 pharmacies will record at baseline and postintervention, details of actual numbers of MURs performed and the proportion that are from underserved communities. Descriptive and inferential statistics will be used to analyse the data. The evaluation will also include a thematic analysis of one-to-one interviews with pharmacy teams to explore the impact on clinical practice (n=20). Interviews with patients belonging to underserved communities, and who received an MUR, will also be conducted (n=20).The study has received ethical approval from the NHS Research Ethics Committee (East Midlands–Derby) and governance clearance through the NHS Health Research Authority. Following the evaluation, the educational intervention will be freely accessible online. The involvement of underserved communities in the e-learning development process is central to the coproduction model. This provides a platform for their voices to be heard. It also incorporates the views of community pharmacy staff to ensure the intervention is sensitive to the context in which pharmacy teams deliver Medicine Use Reviews (MURs).There is a risk of social desirability bias through the implementation of self-reported questionnaires. We will seek to minimise this using self-completion online questionnaires and not through face-to-face completion.To triangulate findings, self-reported pharmacy staff behaviour change will be compared with the numbers of MURs performed with patients from underserved communities at baseline and postintervention. Pharmacy staff and patient interview accounts will further inform the interpretation of questionnaire findings and any impact on practice.Globally, community pharmacy increasingly features in delivering new services to modernise primary healthcare, often in response to the growing demand on general practitioners (GPs) and the shifting of care from resource impoverished hospital settings to comparatively cheaper care in the community.5To support medicine taking and to tackle problems of non-adherence and medicine waste, community pharmacies are increasingly being commissioned to deliver new services to support patient medicine taking.local needs and patient priorities’.Despite over 3 million MURs being conducted in 2015–2016,However, importantly, there is no obligation to target patients from underserved or ‘seldom heard voice’ communities . Although patients from these communities will have unique needs, they are all less inclined to participate in health or screening services and have poorer health outcomes.The coproduction philosophy, where health professionals and service users work in partnership to improve the patients' experience, is increasingly being used to improve services to patients.An e-learning intervention was identified as the most appropriate and cost-efficient method to deliver the training. E-learning tools have a track record in educating health professionals and are improving health-related behaviours.Coproduce, with patients from underserved communities and pharmacy teams, an e-learning resource designed to change the attitudes and behaviour of pharmacy staff to improve the provision of MURs to underserved communities.Evaluate the impact of the e-learning resource on pharmacy staff's reported behaviour scores on providing MURs to underserved communities.Characterise pharmacy staff's experience, perceived impact on practice and to investigate barriers and facilitators to successful implementation of the e-learning.This study aims to investigate whether an educational intervention coproduced with patients and professionals can change pharmacy staff attitudes and behaviour to improve the provision of MURs to underserved communities. The primary objectives are to:Explore underserved patient experiences of medicines and levels of support received from healthcare professionals.Investigate patients' experiences of community pharmacy service, their feelings of being offered and undertaking an MUR and how this has affected their knowledge of medicines and their use and perceptions of pharmacy services.The secondary objectives are to:This study will be conducted in two stages  at multiThe pharmacist e-learning educational intervention will take the form of a series (2–4) of reusable learning objects (RLOs). RLOs are small, pedagogically designed, ‘bite-sized’ chunks of e-learning that focus on a particular topic.Community pharmacy staff .Superintendent pharmacists, community pharmacy owners.Pharmacy representatives/professional leadership bodies/educational bodies , Royal Pharmaceutical Society, Pharmacy Voice, Centre for Pharmacy Postgraduate Education (CPPE)). EA is a CPPE tutor who has experience running pharmacy education workshops and will facilitate recruitment.Box 1Inclusion criteria1. Community pharmacy staff who have active involvement (on a day-to-day basis) in identifying, inviting or undertaking Medicine Use Reviews (MURs) within a community pharmacy2. Willing to provide consent to take part in the workshop activities/interviewExclusion criteria1. Pharmacy staff who are not actively involved with identifying, inviting or undertaking MURs within a community pharmacy2. Unwilling to provide consent to take part in the workshop activities/interviewPharmacy teams and patient groups from underserved communities will be recruited to workshops to coproduce the e-learning resources to inform the intervention. ‘Pharmacy teams’ will include participants from the following groups  identified as belonging to (or representing) an underserved community. For the purposes of this study, the term ‘underserved’ is used to describe patients who are eligible to receive the Medicines Use Review (MUR) service, but who, for whatever reason, not receive the service. We define patients from ‘underserved’ communities as including, but not limited to people with:People whose first language is not EnglishPeople with physical, visual, hearing or learning impairments, or any other disabilityPatients taking multiple medicines for more than one illness or conditionPeople who are housebound or their carersBlack and minority ethnic communitiesPeople with mental illnessPeople who are homeless or have no fixed addressPeople from refugee, asylum and traveller communitiesPeople from Lesbian, Gay, Bisexual and Transgender communitiesMen aged 16–252. Able to understand study information, willing and have capacity to consent to take part in the workshops/interviewExclusion criteria1. Not belonging to (or representing) an underserved community2. Unable to understand study information, unwilling or unable to provide informed consent to the workshops/interview Patients from underserved communities will be recruited via local organisations (n=15 participants) using the eligibility criteria detailed in In addition, one-to-one interviews will be undertaken with representatives from pharmacy teams (n=10) and patients from underserved communities (n=10) to further inform the e-learning intervention. The inclusion/exclusion criteria will be as above  and 2.Two patient and public involvement (PPI) representatives from undeserved communities, who are eligible themselves for an MUR, will be recruited to form an advisory panel for the study. PPI representatives will be recruited via the East Midlands Academic Health Science Network (AHSN) ‘Public Face’ newsletter. The AHSN was established by NHS England in 2013 to improve health and social care by bringing patients, carers and communities together with health and social care providers, industry and researchers.The PPI advisory panel will meet regularly to advise and challenge where necessary on the study design and information included in the e-learning educational intervention. Their involvement in the project will include attending research meetings, workshops, providing feedback on the development of the RLOs and study findings. They will bring their experience and expertise of the world outside academia and their time living with a long-term condition to ensure the patient voice is heard.The implementation and evaluation of the e-learning intervention will be assessed through three work streams.All community pharmacies (n=237) in Nottinghamshire will be approached to take part in the study. An information sheet and invitation letter will be sent to pharmacies which are currently providing the MUR service to patients. Frequently, pharmacy support staff are actively involved (on a day-to-day basis) in identifying and inviting patients to the MUR service. Pharmacists and their support staff will therefore be invited to participate in this study. The study is not powered to detect differences as there is no prior study on which to base a power calculation. It is assumed that 90% of the pharmacies in Nottinghamshire (n=237) are offering the MUR. With an estimated one pharmacist and one support-staff expressing interest to be involved, a response rate of 50% will give a minimum sample of 213 participants.Following completion of the postintervention questionnaire, a sample of pharmacists and support staff (n=20) who have completed the online questionnaire will be invited to take part in a semistructured interview to further explore their experience of using the e-learning educational resource and its impact on practice.Following their MUR, a purposive sample of patients from underserved communities that have had an MUR, will be provided with information about the study by the pharmacist. If the patient agrees to take part, a one-to-one, face-to-face or telephone interview (n=20) will be arranged. Interviews will explore their overall perceptions of MURs, in particular, their experience of how they were approached and engaged.This study will involve two stages that will be undertaken between August 2016 and March 2019 .The purpose of the workshops will be to help codevelop an e-learning training package for pharmacists to improve the provision and number of MURs to underserved communities. Initial development workshops (n=2) will focus on capturing themes through stories and experiences, including design aspects such as preferred media, on ‘storyboards’ to elicit the contents of the RLOs prior to production and enable an RLO specification to be written. The workshop will begin by providing participants with an outline of the day and reminder that there are no right/wrong answers. An ‘appreciative’ method will be used throughout the workshop to promote inclusivity. Examples of existing RLOs will be provided as well as an explanation of how they are developed. Small groups will work to explore (on flip-charts) what they think is important to include in a pharmacy staff e-learning intervention. Anticipated RLO topics may include: ‘identifying and learning about underserved communities’, ‘effective targeting and invitation strategies’ and ‘tailoring consultation skills to meet the needs of underserved communities’.Following initial concept ideas gathered from the development workshops and interviews, an iterative e-learning development process will be conducted with two ‘review’ workshops with patients from underserved communities and pharmacy teams. These workshops will be identical to the development sessions, but will be used to further develop and refine the e-learning specification. This will enable the refinement of initial ideas into a detailed specification  to be developed. Workshops will be held at the University of Nottingham and will be facilitated by AL (chief investigator (CI)), HW, other members of the University's HELM  team and PPI panel representatives will be present. A £200 inconvenience allowance will be made available to all participants.A review of the literature will ensure the contents are factually correct. The specification will be peer reviewed by experts in the field for clarity, factual content and appropriateness of any animations. For example, the peer review may identify errors in the content, and/or suggest content changes to improve the e-learning. These comments will be fed back to the author for revision of the specification and improvement.Software development and further peer review of the final RLOs will be undertaken before release.One-to-one semistructured interviews will be undertaken by the CI with patients and pharmacists to further inform the e-learning intervention. These interviews will take place at a time and place convenient to the participant and, with consent, will be audio recorded. Pharmacy staff will be invited to an interview to explore existing practice and experience of providing MURs to underserved groups and to identify knowledge gaps that will help create the contents for the e-learning materials. Patients from underserved communities who may be unable or unwilling to attend a workshop  will be invited. These interviews will explore medicine use, support from other health professionals , perceptions of the pharmacy and views on what would the patient like to see emphasised in an e-learning intervention.The e-learning resource and online questionnaires will be piloted on 10 undergraduate pharmacy students from the University of Nottingham to test the functionality of the RLOs and face validity of the data collection instruments. This will be performed by asking students to work through the e-learning and checking ease of use. Face validity of the questionnaire will also be assessed.Following permission from the pharmacy owner/manager, the pharmacist in charge of the pharmacy will be contacted and will inform their team and provide them with the study information. A poster will also be sent to each pharmacy to be displayed in staff areas to raise awareness. Assistance in recruiting pharmacies will be sought from LPCs, other pharmacy bodies and the NIHR Comprehensive Local Research Network (CLRN).All pharmacy staff will be invited to complete an online, baseline questionnaire which will be accessible over 3 weeks. Following baseline data collection (with a reminder after 2 weeks), the online e-learning educational intervention will be made available to participants for 30 days. A postintervention questionnaire will then be sent at 3 months to those who completed the baseline questionnaire (with one reminder). Consent to take part will be implied through the completion of the questionnaires.The questionnaire will collect the following data: (1) participant characteristics, that is, age, gender, educational achievement and details of any MUR training received, years qualified as a registered pharmacist; (2) pharmacy characteristics, for example, geographical area, ownership type  and economic deprivation  data for each pharmacy using the postcode as the lookup reference); (3) pharmacist and support-staff attitudes will be assessed through attitudinal statements developed from the ‘inequalities imagination’ frameworkWhereas self-reporting is a practical and efficient method of collecting data, there are limitations such as relying on respondents' honesty and accuracy of reporting. In order to triangulate and support questionnaire findings of self-reported behaviour with actual practice, ∼10 of the pharmacies taking part will be invited to record the number of MURs performed and how many were from underserved communities over a 2-week period. The sample size for the number of pharmacies included will be guided by the feasibility of data collection and resource constraints.This will occur at baseline and at 3 months following the e-learning.A purposeful sample of community pharmacy staff will be invited to a one-to-one interview about their experience of the e-learning, its usability (barriers and facilitators) of the RLOs and its effect on daily practice. Written consent will be taken before the start of the interview and permission sought for the interview to be audio recorded. It will be explained to the potential participant that entry into the study is entirely voluntary. The interviews will last for ∼30 min and will be conducted at the place of work or at any other convenient location according to participant preferences.A purposeful sample of at least 10 community pharmacies will be recruited to help identify patients from underserved groups. The pharmacist will sequentially invite every eligible patient at the end of their MUR to see if they are interested in taking part in the study. If the patient expresses interest, the pharmacist will hand them a study information sheet and seek consent for their details to be passed onto the CI. The patient will then be contacted by phone by the CI and the study will be fully explained. They will then be given time to decide whether to take part. If they agree, they will be invited to interview. The purpose of the interview will be to explore how the patient felt about being approached and engaged for the MUR, whether the strategies that were included in the e-learning were used by pharmacy staff and what benefit the MUR had to improve medicine understanding and use.2 test or Fisher's exact test as appropriate. Continuous data will be analysed using the within-group t-test or Wilcoxon signed rank-test as appropriate. Statistical significance will be assessed at the 5% (two-sided) level. All statistical analyses will be conducted using IBM Statistical Package for Social Sciences (SPSS) 22.Descriptive statistics will be used to describe participants' demographic and baseline characteristics. Continuous data will be presented using means and SDs if approximately normally distributed, and medians and IQR if non-normally distributed. Categorical data will be described using frequencies and percentages. To assess the effect of the e-learning, baseline and postintervention data scores will be compared. Categorical variables will be analysed using the χAll interviews will be transcribed verbatim and the data imported into qualitative analysis package NVivo; QSR International Pty for the purpose of coding and thematic analysis.24Initial reading and rereading of the transcribed data will be undertaken, with feedback and checking by several members of the qualitative research team, to identify common codes and categories. Actively searching for disconfirming data will be undertaken as well as regular detailed discussions among the qualitative researchers and PPI advisory panel. Consideration will then be given to how these issues group together in broader themes related to the research objectives. The principle of constant comparison will be used to test and refine the empirical conceptual consistency of codes and themes, which have been synthesised and narrated.27The study will be conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki, 1996; the principles of Good Clinical Practice and the Department of Health Research Governance Framework for Health and Social Care, 2005. All participants will be informed that participation (in any stage of the study) will be voluntary and that they may withdraw at any time.Central to the development of the e-learning educational intervention are the views of patients from underserved communities. A coproduction methodology will be used to enable patients and professionals to work in partnership to codesign the pharmacy e-learning intervention. By working alongside each other, a better understanding of the barriers to approaching people from underserved communities can be achieved. The collaborative approach can lead to long-lasting change that genuinely makes a difference to patients' experience.28However, patient participants from underserved communities may be more vulnerable and harder to reach or recruit than patients from the wider public who are not from these communities. We will ensure patients are fully informed about what is involved prior to the workshops. It is not anticipated that any participants will feel marginalised during the workshops as we will be guided by our PPI panel and use an ‘appreciative approach’ where all views will be valued. There will be sufficient facilitators during the workshops to accommodate participant needs. HW  has extensive experience in developing RLOs and will help facilitate the workshops.Face-to-face interviews will take place in the patient's home, pharmacy or any other location according to the patient's wishes. A telephone interview (or Skype/FaceTime) option will also be available. Patients will receive at least 24 hours or as long as is needed to decide whether to take part in an interview. Interviews are not expected to raise any distress or make the patient upset as they will be centred on the patient's medicines, the levels of support they receive and how services such as the MUR can help them.Should the researcher become aware that the patient requires urgent medical care, an intervention or is on the wrong medicines, they will be referred to their pharmacist, their GP or other health professional as appropriate.Following evaluation, the educational intervention will be made freely available online and accessible to pharmacy teams globally. The pharmacy workforce in the UK is made up of ∼50 000 registered pharmacists with 70% working within the community pharmacy sector.The Pharmaceutical Journal and Chemist & Druggist) and social media  to promote awareness.To support this study's contribution to wider knowledge, the research findings will be disseminated to regional, national and international audiences through conference and peer-reviewed publications targeted at service users, health professionals, academics, service commissioners and policymakers. We will also disseminate to pharmacy specific journals (eg, This paper is an important step in the dissemination process as it outlines the study's background, aims and details of methods that will be used. As well as providing pharmacy staff with knowledge and practical skills to engage patients from underserved communities, the study will also contribute to addressing the significant gap in the literature on how effective an intervention of this nature will be in improving pharmacy practice. In this respect, the study is novel and will provide information on the feasibility of the educational intervention, alongside the barriers and facilitators to implementation. Collectively, the findings from this study will act as the first stage in developing the coproduction methodology within community pharmacy and how this can be extended to address inequalities in other areas of pharmacy practice."}
{"text": "Abies faxoniana is the dominant plant species of the forest ecosystem on the eastern edge of Qinghai-Tibet Plateau, where the treeline is strongly defined by climate. The tree-ring chronologies and age structure of Abies faxoniana were developed in the treeline ecotones on the northwestern and southeastern aspects of the Min Mountains in the Wanglang Nature Reserve to examine the treeline dynamics of recent decades in response to climate change.On the northwestern aspect, correlation analysis showed that the radial growth was significantly and positively correlated with precipitation in current January and monthly mean temperature in current April, but significantly and negatively correlated with monthly mean temperature in previous August. On the southeastern aspect, the radial growth was significantly negatively correlated with monthly mean temperature in previous July and August.The different responses of radial growth to climatic variability on both the aspects might be mainly due to the micro-environmental conditions. The recruitment benefited from the warm temperature in current April, July and September on the northwestern aspect. The responses of radial growth and recruitment to climatic variability were similar on the northwestern slope. Recruitment was greatly restricted by competition with dense bamboos on the southeastern aspect.The online version of this article (doi:10.1186/1999-3110-54-15) contains supplementary material, which is available to authorized users. Upper treeline ecotones within alpine and arctic ecosystems are mainly controlled by climate  in the Min Mountains, on the eastern edge of QTP in western Sichuan Province, southwestern China   were established at the subalpine treeline ecotones on the northwestern  and southeastern  aspects of the Min Mountains in the Wanglang Nature Reserve  were obtained from the nearest meteorological station, Songpan , approximately 55 km southwest of the sampling sites. Monthly mean temperature and monthly total precipitation of the sampling sites were simulated by Mountain Climate Simulator  based on daily maximum and minimum temperature and precipitation of base station  and height  of the fir seedlings/saplings  on both northwestern and southeastern aspects are shown in Figure The age structures of On the southeastern aspect, the 1-10-year-old age class (fir trees that established in the 2000s) formed the largest age class (11%). The majority of fir trees (78%) were successfully recruited into the treeline ecotone from 1750 s to 1900s Figure b. HoweveThe short-time recruitment records in recent decades did not match with the available meteorological records (1951–2009) in the treeline on the southeastern aspect Figure b, so we During the last 60 years, significantly positive correlations existed between recruitment and monthly mean temperatures for the current April, July and September. However, monthly precipitation had no significant correlations with the recruitment Figure .Figure 7A. faxoniana was positively correlated with the temperature in current March at the treeline site in Wolong National Natural Reserve , which lies west of our study area. Warmer temperatures early in the growing season benefit A. faxoniana growth by inducing early snowmelt and increasing available soil water (Wang et al., Generally, the responses of radial growth to temperatures in previous summer were similar at the treeline ecotones on both the aspects. Significant negative correlations with previous July/August mean temperatures likely reflected the negative carry-over effects Figure . NegativA. faxoniana might reduce solar radiation and effective photosynthesis, thereby, shorten the growing season (Wang et al., The correlations between the precipitation and radial growth were different on both aspects Figure . There wThe differential responses of radial growth to climate factors on both sites might be mainly due to the difference in micro-environmental conditions between the contrasting slope aspects. The studied area is dominated by the southeast monsoon (Pu et al., A. faxoniana at the treeline of the Min Mountains on the eastern edge of QTP might be controlled mainly by temperature, wind, snowpack depth and winter drought.The age structure of a stand can provide a fairly accurate picture of temporal variations in the recruitment rate Kullman,  with theAbies spectabilis forest in the alpine timberline of the Mt. Everest in southern QTP, China (Lv and Zhang, Recruitment was mainly affected by the temperatures in the spring, summer and early autumn seasons, and it was found to have significantly positive correlations with mean temperatures in April, July and September Figure . TemperaThere was a large gap in recruitment during the middle of the 20th century on the southeastern aspect Figure . CompetiA. faxoniana had no obvious increasing trends with small fluctuations on both the aspects (Figure A. spectabilis on the southern QTP (Lv and Zhang, Picea schrenkiana and Abies fargesii in response to warm climate over several of the most recent decades. The different results suggest that the response of treeline ecotones to climate change varies with both local site conditions and the individual species (Luckman and Kavanagh, Over the most recent decades, the radial growth of s Figure . Howevers Figure a. Obvious Figure , Swedishs Figure  and U.S.s Figure . Our resA. faxoniana seedling recruitment were also similar to those that enhanced the radial growth of fir trees. For example, high temperatures in current April enhanced the recruitment of A. faxoniana and facilitated the radial growth (Figures At many upper treeline ecotones the climatic conditions that facilitate seedling recruitment are frequently similar to those conducive to radial growth of trees (Szeicz and Macdonald,  Figures  and 7; m Figures  and 7.A. faxoniana in the treeline ecotones on the eastern edge of QTP. For the geographical novelty, QTP is attracting more and more attention on conducting similar studies (Liang et al., In summary, we investigated the climatic response of radial growth and recruitment of"}
{"text": "The expression of CD40, CD80, CD83, and CD86 were upregulated in peripheral blood from the DC + TL + TRF group compared to other groups. In addition, there was higher expression of FasL in tumor-excised mice from the DC + TL + TRF group compared to other groups. FasL plays an important role in maintaining immune privilege and is required for cytotoxic T-lymphocyte (CTL) activity. Microarray analysis identified several genes involved in the regulation of cancer. In this study, we focused on the special AT rich binding protein 1 (SATB1) gene, which was reported to have dual functions, one of which was to induce aggressive growth in breast cancer cells. Tumors from DC + TL + TRF mice showed lower (p < 0.05) expression of SATB1 gene. Further study will be conducted to investigate the molecular functions of and the role of SATB1 in 4T1 mammary cancer cells and DC. In conclusion, TRF supplementation can potentiate the effectiveness of DC-vaccine immunotherapy.The aim of this study was to evaluate the effectiveness of immunotherapy using dendritic cells (DC) pulsed with tumor lysate (a DC vaccine) in combination with daily supplementation of tocotrienol-rich fraction (TRF) to potentiate anti-tumor immune responses. We had previously reported that DC-vaccine immunotherapy together with TRF supplementation induced protective immunity to tumor challenge. Breast cancer was induced in female BALB/c mice. The mice were randomly assigned into the treatment groups. At autopsy, peripheral blood was collected in heparinized tube and the expression of cell surface molecules  that are crucial for T-cell activation and survival were analyzed by flow cytometry. Tumor was excised from each animal and snap-frozen. Total RNA was extracted from each tumor tissue for microarray and gene expression analysis. Total protein was extracted from tumor tissue for protein expression studies using Western blotting. The results show that systemic administration of 1 mg TRF daily in combination with DC-vaccine immunotherapy (DC + TL + TRF) caused a marked reduction ( Efforts to find a cure for cancer have been ongoing for some time. Researchers have used several approaches, including developing vaccines against some tumors such as breast, lung, colon, and prostate cancers ,4,5,6,7.Tocotrienol-rich fraction (TRF) is the vitamin E fraction extracted from palm oil, which contains 70% mixed tocotrienols and 30% alpha-tocopherol ,23. TocoThe aim of the present study was to investigate the effectiveness of TRF as an adjuvant in enhancing immune response to prevent tumor growth using a syngeneic mouse model of breast cancer to elucidate the mechanism(s) of action using microarray and quantitative PCR approach.Five to eight-week-old inbred female BALB/C mice were purchased from the Experimental Animal Facility of the Institute for Medical Research (IMR), Kuala Lumpur. The animals were housed at the same facility and were given a commercial pellet diet and water ad libitum. The cage bedding was changed every three days. The mice were allowed to acclimatize in their environment and housed under a 12 h light/12 h dark cycle in an air-conditioned room with temperature set at 25 °C. This study was conducted in accordance with international animal ethics guidelines and was approved by the Research and Ethics Committee of the International Medical University (IMU), Kuala Lumpur (Ref: 4.9/55/2007 dated 26 June 2007).2 . At day 5–7, 20 ηg/mL of tumor necrosis factor-alpha  were added to the culture to induce maturation in cells. The DC were usually harvested on day 7–9, depending on their maturation and differentiation, which were monitored using an inverted microscope. The DC were characterized by analyzing the expression of CD11c using flow cytometry  Biosciences, San Jose, CA, USA)The bone marrow (BM) was harvested under aseptic conditions. The tibia and femur bones were obtained from euthanized BALB/c mice and the BM cells were obtained by flushing the cut tibia and femur bones with complete medium using a 22 cc needle syringe as described previously ,24. CompThe tumor cell line used in this study was the 4T1 murine mammary cancer cells, purchased from the American Type Culture Collection (ATCC) . The 4T1 murine mammary cancer cells are spontaneously metastatic tumor cells from mammary gland tumor of BALB/C mice which are reported to be comparable to human stage IV breast cancer . The 4T12 . The next day the 4T1 cells were collected into a 15-mL tube  and harvested by centrifugation (1000 rpm for 5 min). The supernatant was discarded and the cells were resuspended in 1 mL culture medium. Tumor lysate (TL) from the 4T1 cells was prepared by subjecting these cells to several freeze–thaw cycles. The cells were first frozen in liquid nitrogen and then rapidly thawed at 65 °C. The freeze–thaw cycle was repeated 3–5 times. The cell lysates was centrifuged (2000 rpm for 5 min) and the supernatant was passed through a 30-μm nylon filter-column before it was aliquoted in vials and stored at −80 °C until use.The 4T1 cells were cultured in the presence of 8 μg/mL TRF in T25 flasks overnight at 37 °C in a humidified incubator with 5% CO2 for 24 h. Following this, culture supernatant was removed and the cells were washed thrice in Hanks’ Balanced Salt Solution (HBSS). The tumor-pulsed DC were recovered by centrifugation (2000 rpm for 3 min) and resuspended in 1 mL complete medium and left on ice prior to injection into mice.The spent culture medium from DC in a T25 flask was replaced with fresh medium and TL was added. The amount of TL added was based on a 3:1 ratio of confluent DC:TL. The flask was incubated at 37 °C in a humidified incubator with 5% COThe six-week-old BALB/c mice were randomly assigned in to one of the five groups  used in this study . Each grg for 5 min at 4 °C). The supernatant was discarded and the PBS wash step was repeated twice. Following this, the cells were stained with fluorochrome-conjugated antibodies against some mouse antigens, such as CD40-FITC , CD80-PE , CD83-FITC , and CD86-PE , for 30 min on ice. Then, the cells were washed with buffer ) and recovered by centrifugation (1800 rpm for 5 min). The cells were fixed by addition of 500 µL of wash buffer, followed by 500 µL of fixing solution . Each sample was prepared with unstained cells for comparison with stained cells. The tubes were thoroughly mixed before they were analyzed using flow cytometry using the Cell-Quest software provided by the manufacturer  Biosciences, San Jose, CA, USA). The population of interest was gated using the forward scatter (FSC) and side scatter (SSC) dot plot. For each acquisition, 10,000 events were acquired for data analysis. The acquired data was analyzed using the Cell-Quest software. For each sample, the percentages of cells stained with the fluorochrome-conjugated antibodies (FL1 (FITC/green fluorescence) versus FL2 (PE/red fluorescence)) in the gated population were obtained. The changes in the FSC and SSC channel were adjusted and compensated so that the populations were centralized on the dot plot.Whole blood (500–800 µL) was collected into heparin tubes. The red blood cells (RBC) were lysed using a commercial RBC lysis buffer according to the manufacturer-recommended protocol . Briefly, two drops of RBC lysis buffer were added to each tube containing the mice blood. The tubes were incubated at room temperature for 5 min. The lysis reaction was stopped by addition of PBS pH 8.0 solution. The tubes were centrifuged (1000× n = 3) from three groups . Total RNA was also extracted from 4T1 cells for this analysis. Total RNA was extracted using the TRI-reagent solution according to the manufacturer’s instructions . The concentrations of the extracted RNA and ratio of absorbance at 260 nm to 280 nm (A260/A280 ratio) were measured using the NanoDrop ND-1000 spectrophotometer . The integrity of the extracted RNA samples was evaluated with the RNA integrity number (RIN) for each sample using the Total RNA 6000 Nano Kit with the Agilent 2100 Bioanalyzer . The RIN describes a gradual scale of RNA integrity from 1 (RNA completely degraded) to 10 (RNA without degradation). In general, a RIN that is higher than seven is accepted to be optimal in most of experiments. For the microarray analysis, total RNA was extracted from tumors of mice  database , supplemented with probes derived from the Mouse Exonic Evidence Based Oligonucleotide (MEEBO) set as well as exemplar protein-coding sequences described in the RIKEN FANTOM2 database. The microarray facilities were provided by Malaysian Genome Institute (MGI), University Kebangsaan Malaysia (UKM), Bangi, Malaysia. The complementary RNA (cRNA) synthesis and purification were performed using IlluminaTotalPrep RNA Amplification . All the RNAs were subject to cleanup using RNA cleanup  and hybridization steps followed with the manufacturer’s protocol.SATB1) at the mRNA level. For this experiment, RNA from tumors of three animal groups  and 4T1 cells were used. The RNA samples treated with DNase treatment  were used for the RT-PCR. The reaction mix consisted of 5 µL purified RNA (5 ng), 0.5 µL of enzyme mix, and 12.5 µL of 2 × reaction mix ); 10 µM of reverse and forward primers were added to the microwell plate. Ultra-pure water  was added to each microwell to make the final volume 25 µL. The reaction was run at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 55 °C for 30 s, and an extension phase for 1 cycle at 95 °C for 60 s, 60 °C for 60 s, and 95 °C for 60 s  in a thermocycler . Triplicate samples were run for each RNA. The primers for murine beta-actin (β-actin) and SATB1 genes were purchased from Invitrogen  and R&D Research , respectively. The primer sequences are as follows: (a) β-actin  and (b) SATB1 . Data were normalized relative to the housekeeping gene.In this study, we used quantitative PCR (RT-PCR) to confirm the expression of special AT-rich binding protein 1  for Western blot analysis. Protein concentration of each sample was determined using the Bradford method . Protein extracted (30 ng) from each sample was resolved on 10% SDS-PAGE gels and electro-blotted onto nitrocellulose membrane using a semi-dry transfer system . The membrane was incubated overnight at 4 °C with 5% blocking agent  in wash buffer (0.1% (v/v) Tween 20 in PBS). After the blocking step, membrane was probed with primary antibody (SATB1 or FasL)  for 2 h at room temperature. Subsequently, the membranes were incubated for another two hours at room temperature with a secondary antibody, horseradish peroxidase (HRP) anti-rabbit . Detection was performed using an ECL detection kit according to the manufacturer’s instructions . Following this, the membranes were stripped off from the probed antibodies, blocked, and re-incubated with mouse monoclonal GAPDH  followed by HRP-labeled anti-mouse secondary antibody . Bands on autoradiography films  were quantified using ImageJ software and normalized against GAPDH. The list of primary and the corresponding secondary antibodies used for the Western blotting assay is listed in t-test and SPSS version 20 for the most of data. For the tumor incidence data, statistical analysis was done using the one-way ANOVA with post hoc Tukey test for multiple comparisons (SPSS version 20). The IMAGE J software (http:rsb.info.nih.gov/ij/index.html) was used for the densitometry analysis of protein bands whilst the Bead Studio 2.1 and GENE SPRING 7.0 were used for microarray analysis. Most of the data represent the ± SD of triplicate measurements. The quantitative PCR data represent mean value with ± SEM of the triplicate measurements.Statistical analysis was done using the Student The anti-tumor effect was assessed using a syngeneic mouse model of breast cancer. Previously, we had reported that the combination of DC pulsed with TL (DC + TL) and TRF supplementation showed marked anti-tumor effects, as a marked inhibition of tumor growth and metastasis was observed in the mice that received this treatment ,24. In tp > 0.05) was observed in mice in the DC + TL + TRF groups  in the number of PBL that expressed CD40, CD80, CD83, or CD86 in PBL from the DC + TL + TRF groups compared to the other groups  of mice was determined as these molecules are reported to be important surface markers that provide co-stimulatory signals required for T-cell activation and survival ,25,26. Gr groups . The expr groups . For CD8r groups . For CD8r groups . For CD8r groups .Fas by FasL expressed on the activated CTL [The Fas/Fas ligand (FasL) is reported to be one of the process required for the maintenance of immune privilege ,28,29. Ip < 0.05) or 99% (p < 0.01). For this analysis, we focused on studying the difference in gene expression in tumors from the DC + TL and DC + TL + TRF groups as these treatment groups showed very similar genes expression pattern in the dendogram (p < 0.05) for, there were four genes that were differentially regulated  were down-regulated (p < 0.01), only one gene was differentially regulated  down-regulated in tumors from the DC + TL + TRF group when compared to tumors from the DC + TL groups . In order to reduce non-specific binding errors, the normalization intensity was conducted on all samples and groups. A dendogram a was creendogram a and Conendogram b analysiegulated c. The exegulated . When thegulated c. This gL groups .SATB1 gene in the tumor tissues extracted from the three DC-treated groups  was analyzed using quantitative PCR (qPCR). Total RNA extracted from cultured 4T1 cells was also included in the analysis. A marked (p < 0.05) reduction in the expression of SATB1 gene was observed in tumors extracted from mice treated with DC + TL + TRF (SATB1 was only 0.14-fold (p < 0.05) lower expression of the SATB1 gene when compared to DC alone (0.59) or cultured 4T1 cells (1.0)  .SATB1 protein expression in tumors from the DC + TL + TRF and DC + TL groups when compared to tumors from the control or vehicle groups (SATB1 expression to be reduced in DC + TL + TRF (23.3% of relative density) group compared to other groups  was upregulated, whilst three genes  were down-regulated (p < 0.01) was used, only one gene (SATB1) was found in this category  in tumors from mice treated with DC + TL + TRF , which are implicated in the loop domain organization of chromatin or chromatin remodeling [SATB1 gene [p < 0.0001) with prognostic outcomes of cancer patients [SATB1 gene was able to reprogram chromatin organization and the transcription profiles of breast tumors, which promoted cancer growth and metastasis [Microarray analysis performed to compare gene expression in tumors from DC + TL and DC + TL + TRF flagged four differentially expressed genes. One gene (egulated  when thecategory c. The SATL + TRF . This SAi et al.  reportedi et al. ,39. In ty in TH2 ,39,40. Tmodeling ,39,40. ISATB1 gene correlated well with the protein expression in tumors excised from the experimental animals. As observed with the gene expression studies, there was marked suppression of SATB1 protein expression in tumors from mice treated with DC + TL + TRF (The expression pattern of the TL + TRF .SATB1 was identified to be one of the potential targets that could have produced some of the anti-tumors effects observed. Further studies are being planned to elucidate the role of the SATB1 gene in tumor growth and spread as well as to understand how TRF is able to modulate the expression of this gene.In conclusion, this study has shown that TRF can be used to supplement DC-vaccine immunotherapy using an immunocompetent experimental model. The DC + TL + TRF approach appears to induce activation and promote survival of T-cells, as judged by the increased expression of CD40, CD80, CD83, and CD86 in PBL as well as FasL in the tumors. Increased expression of FasL protein in tumor tissue from the DC + TL + TRF-treated mice suggest that CTL activation may have taken place and this could have contributed to inhibition of tumor growth. One of the limitations of this study is the small number of mice used for the study. Also, the scope of the present study is rather narrow as we were not able to include more parameters to investigate. However, despite this the results are significant and we have managed to identify a key target protein that we could do more work on."}
{"text": "Skull fractures are common injuries in young children, typically caused by accidental falls and child abuse. The paucity of detailed biomechanical data from real-world trauma in children has hampered development of biomechanical thresholds for skull fracture in infants. The objectives of this study were to identify biomechanical metrics to predict skull fracture, determine threshold values associated with fracture, and develop skull fracture risk curves for low-height falls in infants. To achieve these objectives, we utilized an integrated approach consisting of case evaluation, anthropomorphic reconstruction, and finite element simulation. Four biomechanical candidates for predicting skull fracture were identified  and evaluated against well-witnessed falls in infants (0–6 months). Among the predictor candidates, first principal stress and strain correlated best with the occurrence of parietal skull fracture. The principal stress and strain thresholds associated with 50 and 95% probability of parietal skull fracture were 25.229 and 36.015 MPa and 0.0464 and 0.0699, respectively. Risk curves using these predictors determined that infant falls from 0.3 m had a low probability (0–54%) to result in parietal skull fracture, particularly with carpet impact (0–1%). Head-first falls from 0.9 m had a high probability of fracture (86–100%) for concrete impact and a moderate probability (34–81%) for carpet impact. Probabilities of fracture in 0.6 m falls were dependent on impact surface. Occipital impacts from 0.9 m onto the concrete also had the potential (27–90% probability) to generate parietal skull fracture. These data represent a multi-faceted biomechanical assessment of infant skull fracture risk and can assist in the differential diagnosis for head trauma in children.The online version of this article (10.1007/s00414-018-1918-1) contains supplementary material, which is available to authorized users. Falls are the leading causes of nonfatal, unintentional injuries in infants ≤ 1 year of age . HistoriGiven ethical restrictions for conducting controlled falls in children, pediatric biomechanics of falls are investigated using case reports , 5, retrTo improve on the limitations of the individual study types, laboratory-based studies are often paired with FE model simulations to identify fracture thresholds. Roth et al. , 20 and In this study, we utilized an integrated approach combining case-study evaluation, full-body anthropomorphic infant fall reconstruction, and FE simulation to identify the skull fracture risk associated with low-height falls in infants. The infant head FE model incorporates material properties of the human infant skull and suture from children over the age range of 19 days to 4.5 months . Using mTo identify biomechanical response metrics and thresholds that are predictive of parietal skull fracture, data from simple well-witnessed falls with detailed event descriptions and radiological reports were examined. The study was approved by the Institutional Review Board of the University of Pennsylvania and Children’s Hospital of Philadelphia (CHOP) and carried out in accordance with the IRB approved guidelines and regulations. All infants less than 6 months of age with a history of a fall  admitted to the CHOP between June 2006 and September 2009 were evaluated as potential subjects. To be considered for the study, the medical history had to state that the fall event had been witnessed by two adults, one adult and one child old enough to communicate, or one adult whose statement had been designated as without suspicion of abuse by the CHOP child protection team. When the incident was witnessed by two or more persons, the incident description was reviewed carefully to ensure that there was no inconsistency in the details of the incident history. Inclusion criteria also specified that CT and/or MRI scans were obtained within the first week of hospitalization and evaluated by a radiologist. Exclusion criteria were a history of child abuse, mental retardation, drug dependency, birth complications, hemophilia, and pre-existing skull or brain abnormalities . Cases with a history of a complex fall that would be difficult to reconstruct , and those with ambiguous event descriptions were also excluded. Cases with fall heights greater than 5 ft were excluded due to limits of the test facility. Of 263 cases screened, 44 cases met the inclusion/exclusion criteria. Cases were further evaluated to determine whether the details of the incidents required for reconstruction were explained clearly in the medical records. If needed, care providers and/or parents were contacted following approved consent procedures to obtain additional event information. The required details were the position of child immediately prior to and after the fall, an estimation of the height of the fall, type of flooring that child landed on, the first part of the child’s body to come into contact with the ground, anything else the child came into contact with during or after the fall, the height of the person involved in the fall, and the age of the witnesses. Additional details about the sustained injuries and health conditions of the child prior to the accident were also gathered. Twenty-six of the 44 cases had unambiguous, well-described fall histories. These 26 patient records and images were reviewed carefully by a radiologist to document; if possible, the exact location of skull fracture or other physical signs of head impact, such as soft-tissue swelling or subgaleal hematoma. Cases were excluded from further consideration if images were of poor quality or if no physical signs of head impact were observed. Cases were excluded if there was a lapse of more than 1 day between accident and hospital visit, thus decreasing the efficacy of using the CT/MRI data to determine exact impact location. Based on the medical record, witness and/or interview report, and radiology report, 11 of the 26 subjects were enrolled in the study: 7 with diagnosed parietal skull fracture  and 4 with no skull fracture. Details of each case, including a brief description of the event, age, sex, estimated fall height, impact surface, area of impact, and skull fracture are provided (Table E = 535 ± 139 MPa) attached together with a silicone material (E = 2.1 ± 0.2 MPa) and covered with a 1-mm-thick latex cap that duplicate the mechanical response of the infant skull, suture, and scalp reported in the literature [G = 765 ± 44 Pa) similar to infant brain. There was no air space in the surrogate head. The mass of the body was also adjusted so the overall surrogate mass matched the overall child’s body mass. Additional details on the biofidelity and validation of the surrogate are reported in [To estimate the head impact force of each child, the 11 cases were reconstructed using a whole-body biofidelic anthropomorphic infant surrogate . A six dterature . The surterature . For eacterature , and theorted in .The contact surfaces for the 11 cases were concrete, tile, linoleum, laminate, or carpet with carpet pad. The rigid metal surface of the heavy force plate was used as the closest approximation to concrete and tile. Examples of the other three impact surface materials as stated in the history report were obtained and clamped to the force plate. For each case, the primary impact location was determined from the fall description, medical history, and the radiological reports of fracture and soft-tissue swelling. For fall reconstructions, the fall height, flooring, identified head impact location, and the fall description from the medical and witness reports for each case were matched and verified via a high-speed digital video . Carbon paper was used to mark impact location on the surrogate head. The nature of accident reconstruction entails some uncertainties, and there is trial-to-trial variability, so it is impossible to determine with absolute certainty the forces applied to the head during the impacts. Creating a corridor of possible responses by performing multiple trials is a way to take these uncertainties into consideration in real-world accident reconstruction studies , 34. Theμ0; strain-sensitive nonlinear characteristic parameter, α; Poisson’s ratio, υ; the relaxation moduli, C1 and C2; and time constants, τ1 and τ2, in the Prony series  . The infbase1/3) . Becausebase1/3) . Specifientclass1pt{minimabase1/3) . The braentclass1pt{minima−3 m/s to 2224 N. Depending on available surface area, 5–24 locations on each surface were tested. Tests near boundaries of the flooring sample were excluded. An average stress-strain curve for each material was implemented in the material evaluator of ABAQUS . Linoleum and laminate were represented with second-order-reduced polynomial models. Carpet and carpet pad were modeled, respectively, with second-order and first-order forms of ABAQUS’s hyperfoam material model. The concrete and tile impact surfaces were represented as rigid bodies. Table ρ) and coefficients for each flooring surface.To model the impact surfaces in the simulations, the linoleum, laminate, carpet, and carpet pad were indented with a 0.076-m platen at 0.02 × 10N = 33) in ABAQUS explicit. For each FE simulation, the initial velocity of the head was adjusted until the simulated impact force-time history matched the peak and duration of experimental impact force trace within 5% error tolerance  is the probability of parietal skull fracture for the given value x of the predictor candidate. Variables a and b are the regression coefficients. The quality of fit for the risk curves was then evaluated for the four candidate parameters using the Nagelkerke R2 and Cox and Snell R2 statistics to determine the parameter with the highest correlation with skull fracture. Receiver operating characteristic (ROC) curves were also generated, and the area under the ROC curves (AUROC) was used as another measure of how well each parameter distinguished between parietal bones with and without skull fracture. For each candidate fracture parameter, the threshold values corresponding to the 50 and 95% likelihood of skull fracture were extracted from the logistic regression, and the optimal ROC threshold (optimizing specificity and sensitivity) extracted from the ROC curve. For each candidate, the Q2 (or median) risk curve and associated skull fracture threshold value were used for further analysis in this study. The Q1 and Q3 risk curves, and associated threshold values, were used to demonstrate the range of potential uncertainties or errors in injury risk curve development using our real-world accident reconstruction approach.Each of the 33 simulations generated two peak values for the parietal skull plate  for all 4 candidate predictors. For each of the 4 candidate skull fracture parameters, the 66 generated data points were categorized into one of the three simulation groups representing the first, second, and third peak head impact force quartiles (Q1-Q2-Q3) measured in the reconstruction experiments. For each simulation group, a binary classifier was assigned to each response data point to designate the presence (1) or absence (0) of fracture, based on the radiological report. Binary logistic regression analyses were then performed, and Q1, Q2, and Q3 parietal skull fracture risk curves were created  with distribution functions of the following form:N = 10 drops/condition). The trials producing the minimum, median, and maximum head impact forces for each height-surface-location combination were selected for FE analysis in this study , and Cox and Snell R2 (0.519 and 0.516). The threshold values corresponding to the 50 and 95% probabilities of parietal skull fracture, as determined from the developed injury risk curves for the four predictors are provided in Table For each potential skull fracture predictor, 66 data points were extracted from the FE simulations . Each data point was assigned a designation of presence of parietal fracture or absence and is displayed in Fig. SThe peak first principal stress and strain associated with the minimum, median, and maximum head impact force from low-height falls is given in Table SClinical differentiation of accidental and abusive head injury etiologies has proven to be a persistent problem for physicians. Given the paucity of documented pediatric accidental cases in the literature, the 11 well-witnessed, real-world infant falls in our clinical dataset, along with kinematics estimated from anthropomorphic surrogate event reconstructions and biomechanical parameters from FE simulations, provide the biomechanics community with valuable data that can facilitate validation of future computational models of infant head injuries. In addition, kinematics measured in our controlled surrogate drops and biomechanical parameters from corresponding FE simulations representing fall scenarios involving three heights, two surfaces, and two head impact locations, can serve as a data set for future studies investigating the likelihood of head injury in low-height falls.To develop skull fracture risk curves, we used anthropomorphic surrogate studies and multiple biomechanical response parameters from FE simulations. Potential of using these biomechanical parameters to be used as skull fracture predictors in infants were assessed not only in terms of quality of fit of the corresponding developed risk curves and their prediction accuracy rates but also by comparing the distribution of these parameters with the actual location and pattern of skull fracture in real-world falls. An additional large set of controlled head-first fall drop tests  were useUsing surrogate and FE reconstructions of real-world fall accidents, we found skull fracture thresholds similar to failure stress and strain values reported for infant parietal skull . Our 50 When placed in context with ultimate stresses reported via three- and four-point bending of pediatric crania of varying ages, our first principal stress fracture threshold (25.229–36.015 MPa) and Coats and Margulies’ ultimate stress 30.95 MPa) underscore the order of magnitude range in skull fracture thresholds for ultimate stress over the age spectrum from infant to adults, with ultimate stress increasing dramatically during the first year, then asymptoting to adulthood Fig. . Marguli.95 MPa u−1, respectively. Of note, only Motherway et al. [Several studies have tested the adult skull in tension or compression. Evans and Lissner  reportedy et al.  performey et al.  and Daviy et al. , respectIn both the pediatric and adult literature, ultimate strain measurements are surprisingly sparse. Wang et al.  reportedIn addition, in all simulated fall scenarios, we identify consistent predictions for parietal fracture likelihood and fracture pattern using first principal stress and strain as skull fracture metrics. Coats et al.  used firTo date, the present study represents the first FE model to assess likelihood of infant skull fracture in a variety of low-height fall scenarios with the aid of injury criteria developed based on real-world fall cases and validated against ultimate stress and strain values derived from mechanical tests on infant skull cadavers. Previously, we reported that peak head impact forces for 0.9 m falls onto carpet approach those for concrete, but peak head impact forces for 0.6 m falls onto carpet were significantly lower than those for concrete . In the Examining published clinical studies on pediatric head injuries following low-height falls, we focus on those limited to young children and infants. Among the children ≤ 2 years old who experienced non-inflicted head injuries in Duhaime et al.’s  prospectBoth first principal stress and strain predictors in our study indicate probabilities of parietal fracture for 0.9 m falls onto concrete with occipital impact. The bone-suture junction is less structurally rigid than the skull plate in infants  that makStudies published nearly 70 years ago reported fractures on the external skull surface in regions of outward bending and on the internal surface in regions of inward bending. While inward bending always occurred under the site of impact, outward bending sometimes occurred at considerable distances away from the site of impact , 47. GivAlthough this study significantly advances pediatric head trauma literature by proposing new biomechanical fracture criteria and predicting parietal fracture in low-height fall scenarios, the limitation of our study is the lack of any occipital fractures in our clinical data set. As a consequence, we were unable to develop injury criteria for occipital fracture or to predict occipital fracture in fall scenarios. However, making the assumption that principal stress and strain fracture thresholds are similar for parietal and occipital skull plates, we would predict a low probability of fracture for the three occipital impact cases in Table One additional important consideration with our parietal skull fracture risk curves is that a sideways fall often concludes with shoulder or limb contact before the head. Our whole-body surrogate reconstructions of short falls simulated worst-case risk scenarios with head-first contact. Thus, the likelihood of skull fracture risk might differ from those reported here if the fall were “broken” by contact of another body part during the fall.We developed parietal skull fracture risk curves for infants under 5.5 months old through reconstruction of real-world accidental falls in infants. To minimize the uncertainties accompanied by accident reconstruction procedure, rigorous inclusion criteria were employed during data collection process, impact kinematics were estimated through whole-body anthropomorphic surrogate reconstructions, and precise and comprehensive approaches were used to verify the reliability and precision of surrogate and FE reconstructions. FE simulations were validated against not only the peak impact forces but also the entire impact force-time histories measured in surrogate reconstruction drops. As an improvement over the existing accident reconstruction studies, FE simulations were also verified by considering stress and strain distribution in terms of the location and pattern of skull fracture. Among all four potential predictors, maximal first principal stress and strain best correlated with the occurrence of parietal skull fracture, and their corresponding proposed tolerances are in agreement with published in vitro material failure tests. Finally, the resulting risk curves were used to evaluate the worst-case likelihood of parietal skull fracture in head-first, low-height infant falls. We conclude that the likelihood of parietal skull fracture in head-first falls from 0.3 m is very low, regardless of fall condition. Falling from 0.9 m onto the concrete on either occipital or parietal site can result in parietal skull fracture. An improvement over anecdotal clinical reports and heuristic evidence, the outcomes of the present study can be used to more accurately assess likelihood of skull fracture in infant falls for a variety of fall heights, impact locations, and contact surfaces.ESM 1(DOCX 15 kb)ESM 2(PNG 518 kb)High resolution image (EPS 205668 kb)"}
{"text": "Modern mass spectrometry, including electrospray and MALDI, is applied for analysis and structure elucidation of carbohydrates. Cyclic oligosaccharides isolated from different sources (bacteria and plants) have been known for decades and some of them (cyclodextrins and their derivatives) are widely used in drug design, as food additives, in the construction of nanomaterials, etc. The peculiarities of the first- and second-order mass spectra of cyclic oligosaccharides  are discussed in this minireview. The most studied of them, cyclodextrins  by mass spectrometry alone is impossible (because of m/z = nm/nz), but they are separable by ion mobility spectrometry (IMS) due to their difference in size and shape + in a positive ion mode.In MALDI MS, CDs give only singly charged ions. For example, for permethylated β-CD in DHB matrix, only [M + Na]ion mode . Howeverion mode . It was 4), heptakis-β-(2→1)-fructofuranose , octakis-β-(2→1)-fructofuranose   2+ “fragment by glycosidic cleavages giving B-series of ions...”  in the table of the MS2m/z data and presented a description of them below the table. Briefly, A-series is formed by doubly charged ions differed in 81 Th. B-series arises due to charge separation reaction due to loss of [Glcn + H]+ (difference in 162 Da). Low abundant C-series is formed by singly charged ions smaller than B-peaks on 264 Da; this cleavage is accompanied by C-H hydrogen transfer (demonstrated by H/D isotopic exchange in OH groups). Low abundant D-series may be regarded as a result of loss of H2O from B-series ions . For a representative MS2 spectrum, see It is impossible to find out “reducing” and “non-reducing” ends for a cyclic oligosaccharide molecule , so an attempt to apply the generally accepted Domon-Costello nomenclature of oligosaccharide fragmentation  leads toaimed in ), thoughaimed in : “...we aimed in  (p. 1568aimed in  was refeaimed in  introduc2O (ca. 18 Da), for permethylated OSs, C2H6O (ca. 46 Da), for peracetylated OSs, C4H6O3 (ca. 102 Da), etc. Note, that the MS of acyclic OS having an anhydro unit in its composition looks like that of cyclic one due to their isomerism. Some structural studies based on MS  are presented below. A previously unknown glucan  was isolated from a recombinant strain of a Rhizobium meliloti TY7, an ndvB mutant carrying a locus specifying β- glucan synthesis from Bradyrhizobium japonicum USDA110, using reversed phase chromatography +  and [M + Na]+ , respectively (M = Glc12). Acyclic dodecahexaose should be heavier by 18 Da, so, this one had likely a cyclic structure. 1H- and 13C-NMR spectra were rather complex, but they demonstrated the presence of mainly β-(1→3)-linked (definitely not of α-configuration) glucose residues along with a minor β-(1→6)-glycosidic bond. The first Smith degradation afforded the product consisting of eleven hexose units . The second Smith degradation gave the product consisting of only β-(1→3)-Glcp units according to 1H- and 13C-NMR spectra -β-glucosyl), which was resistant to the next Smith degradation. Surprisingly, the MS of this carbohydrate expected to be cyclodecalaminarinose exhibited the main peak at m/z 1735, which the authors assigned it to [M + 5Na]+, which is definitely unbelievable. A more reasonable explanation of this peak is complexation of the analyte with glycerol usually used as a FAB matrix , for [Hex10 + Gro + Na]+, calculation gives m/z 1735.56, so the structure of the starting material was assigned to β-(1→6)-laminaro(cyclodekakis-β-(1→3)-glucosyl). Later, the same authors isolated the both cyclic OSs  from different mutants of Bradyrhizobium japonicum + were mentioned with no data. Heterogeneity of these cycles (the presence of one β-(1→6)-Glcp units inside each cycle) was approved by NMR spectrometry  and molecular dynamics calculations. Cyclic OPGs usually bear side chains, e.g., phosphatidylglycerols giving a +C3H7O5P increment (ca. 154 Da each) +, calcd. m/z 2792.8 , m/z 2969.1, [M2 – 2H + 3Na]+, calcd. m/z 2968.80. In ESI MS, the main triply and doubly charged ions were observed: m/z 915.9, [M1 – 3H]3− ; m/z 967.7 [M2 − 3H]3−, ; m/z 1374.6, [M1 − 2H]2− , m/z 1451.6, [M2 − 2H]2− . There is no explanation why the exp./calcd. data coincidence for ESI MS was not so good as for MALDI TOF MS, possibly, this due to low resolution of the triple quadrupole analyzer used in this work. No MS2 data were reported.Similar studies were carried out for OPGs from several agriculturally important microorganisms  . In this3 in DMF, and the purified amine was acylated with biotinamidohexanoic acid N-hydroxysuccinimide ester. MALDI TOF MS (DHB matrix) of monoaminated and monobiotinylated cyclooligosophoraoses displayed prominent [M + Na]+ peaks for d.p. 17 to 22 which showed good coincidence with the calculated values. As expected, the difference between the corresponding peaks with the same d.p. is equal to ca. 339 Da .The appropriate side chain  can be introduced artificially. Biotinylation through amidocaproyl spacer was done by Cho et al. . A mixtu+, K+, and Cs+ (not H+) in MALDI MS were much higher that of corresponding ionized maltohexaose and maltoheptaose, respectively; differences showed an increasing trend with the radii of metal cations, and β-CD had higher ionization efficiency than α-CD. However, no proofs of general character of this regularity were demonstrated and even if such proofs will be found, it seems problematic to use this phenomenon in assignment of isomeric acyclic and cyclic OSs without authentic or reference samples  between linear and cyclic oligosaccharides. In fact it was shown by Choi et al.  that theles (cf. ).To use fragmentation techniques to distinguish linear and cyclic OSs is promising in order to avoid isolation of individual compounds. The first attempt to realize this approach was done by P.J. Derrick and colleagues . Dextran+ and [M + H + K]2+ ions were fragmented for each compound. It was shown that three methods of activation are complementary. For CDs, all of the fragments were formed by glycoside bonds rupture, no cleavages of residues were observed. Comparison of CE50 demonstrated that slightly higher collision energy is needed to cleave CD than the related linear maltooligosaccharide . Though the authors claim that CID/HCD combination opens a way for deciphering of structures of complex carbohydrates “alone or in mixture”, we do not share their optimism because the observed effects seem very tiny.Later, a similar regularity was demonstrated for ESI MS in more sophisticated experiment using CID, PQD, and HCD activation procedures for three pairs of α-CD/maltohexaose, β-CD/maltoheptaose and γ-CD/maltooctaose . A LTQ oThus, the choice between acyclic or cyclic structure can be supported by ESI or MALDI MS if a small amount (enough for an NMR study) of rather pure compound is isolated, but such a choice for a component in a complex mixture  remains problematic despite the great progress of tandem techniques during the last thirty years.2 [n + H]+, n = 2–5 by activation of the protonated complex .At present time, there are various derivatives of cyclic OSs, especially CDs prepared for different purposes. First-order MS  can obviously applied for profiling and determination of d.s. ,37. SincO-6-tosylation of Glcp unit + and [M + Na]+ were observed in MALDI and ESI MS. LID and CID of the protonated molecule resulted in similar secondary-order mass spectra with predominant formation of aminohexose-containing fragments, so, a proton is definitely localized on the amino group and the first cleavage of glycosidic bond occurs near the HexN residue  and bis(6-O-tosyl)-β-CD (12–14). One can assume that mass spectra of decay for regioisomers of modified CDs (as well as for any cyclic oligosaccharides) may differ from each other since a double rupture of glycosidic bonds can result in different combinations of primarily bonded units. A critical consideration of this approach may be formulated in two questions:(1)Are the bonds between substituents and the carbohydrate units strong enough in comparison to glycosidic bonds, and(2)Do transitions of substituents between carbohydrate residues  take place?The influence of the positions of regioisomeric, disubstituted β-cyclodextrins on their ESI MSudied in  for two 9a, in AC, they are separated with one and four unsubstituted units, 10a, and in AD, 11a, substituted units are separated with two and three unsubstituted units, 2 for all three [M + H]+ ions , but it is absent in MC2 of AD and AC. The fragment consisting of two substituted and one unsubstituted unit (m/z 963) is absent в MS2 of AD. The peak of the ion resulting from elimination of one substituent (m/z 1732.5) is present in MS2 of all three regioisomers, nevertheless, this pathway does not dominate. For bis-6-O-tosylates, the fragmentation differs drastically: CID MS2 of [M + H]+ for ditosylates AC and AD are practically the same, and for AB, the same pattern of ions was observed though peaks assigned to ditosylated maltose (m/z 633) and ditosylated maltotriose (m/z 795) have higher relative abundances than those of AC and AD  followed by NHMe2 loss. The [M − (NMe2R)]2+ ion eliminated one singly charged Glc residue from β-CD thus opened the cycle. Two pathways of further fragmentation were realized. Path 1: Glc neutrals are sequentially lost. Path 2: the second alkyl and NHMe2 were lost followed by sequential loss of Glc units. Cleavage of the carbohydrate residues also occurred simultaneously. This complicated scheme of thoroughly investigated fragmentation was supported using MS3 to reveal fragment ion transitions . All studied compounds had similar fragmentations, no drastic differences between O- and N-succinates were reported.Cyclic OSs, especially CDs, are used as starting compounds for the construction of complex organic and hybrid molecular systems. For example, CDs are applied for preparation of drug delivery agents in which a CD core is tethered by ether or ester bonds with linkers bearing side ionogenic groups. A scheme of CID fragmentation of a series of surfactants based on 6-ted β-CD  was pres+ were also identified. It was stressed that “LC with on-line ESI MS detection has problems with the analysis of polymer species related to formation of multicharged ions thus creating difficulties in spectra interpretation, especially in the case of polydisperse oligomer mixtures” + and [M + Na]+ were present, and their MS2 spectra differed drastically. For MS2 of [M + H]+ (c.e. 20 eV), stepwise losses of HOAc and Glc units were observed: after elimination of four HOAc molecules, elimination of one Glc unit occurred until all co-monomer units were depleted. This process was readily illustrated in the table of all possible m/z values, where really observed m/z ones were highlighted forming a kind of ladder. For MS2 of [M + Na]+ (c.e. 120 eV), only consecutive losses of four molecules of HOAc  were observed. In a small m/z range, abundant peaks corresponding to acetylated Glc unit fragments were present. As it was demonstrated previously by NMR and MS [2 alone that there were no oligomeric hydroxybutyryl substituents (which may occur due to esterification of a hydroxyl to give a hydroxybutyryl moiety). Abundant [M + Na]+ ions along with quite visible [M + H]+ ones were observed for each component in ESI MS. For MS2 of [M + H]+ of 5HB-substituted β-CD (c.e. 15 eV), a subsequent loss of Glc units (162 Da) accompanied by losses of HB substituents (104 Da) and their anhydro forms (86 Da) occurred. A similar \"ladder\", but with smaller steps was pictured in the table created analogously to MS2 of TABCD. MS2 of the [M + Na]+ ion (c.e. 110 eV) contained peaks corresponding to different fragmentation pathways. Ions m/z 1483 and m/z 1379 are formed due to losses of one and two HB molecules. The ion m/z 1425 resulted in loss of one Glc unit, and the ion m/z 1339 was formed due to (Glc-HB) loss (104 + 86 Da). Because of the ion m/z 1397  was not observed, one can conclude that there were likely no di-HB substituent attached to β-CD. The origin of the quite abundant m/z 1543 ion (loss of 44 Da) was not definitely explained by the authors; due to low resolution, it was impossible to find out what neutral (C2H4O or CO2) was eliminated. The above fragmentation pathways classified by Peptu et al. are presented in For the last two decades, CDs were used as scaffolds for star polymers, the best known being the oligolactide-tethered CDs produced by ring-opening polymerization of lactide (the cyclic dimer of lactic acid) initiated by CD, where oligolactide forms a chain attached to the CD ,62,63,64ixtures” ,66. Totaixtures” , and β-CR and MS , the app2 and MALDI LID) were thoroughly described for the [β-CD-LA4 + Na]+ ion with total assignment of composition of fragment ions. In contrast to the above examples, multiply repeated units in substituents capable of fragmentation were definitely present, so three other pathways were observed designated as G (loss of substituted Glc unit) and E1 and E2 (fragmentation of side lactide chains), + and Li+ adducts; heavier ions having more lactate units gave homologous fragmentation patterns.Recently, a mixture of derivatized β-CDs partially substituted with oligolactide chains was studied by GPC, NMR, and MS . From thO-benzylated (with only two free OH groups) β-CD was transferred to a homologous mixture of oligopolylactides, which was characterized by MALDI TOF profiling along with NMR spectrometry +, [M + 2H]2+, [M + Na]+, [M + K]+, and a moderate peak of an [M − H]− anion. The ions [M + NH4]+ are formed only for cyclic N-acetylglucosamines 19b–24b, not for cyclooligoglucosamines 19a–24a.First-order, electrospray ionization (ESI) mass spectra of compounds 2 spectra of cyclooligo-β-(1→6)-d-glucosamines 19a–24a and cyclooligo-β-(1→6)-d-N-acetylglucosamines 19b–24b, cleavages of the glycosidic bonds were the principal processes, they are accompanied by eliminations of molecules of water (and ammonia for cyclooligo-β-(1→6)-d-glucosamines) [19a produced the [M + H]+ and [M + 2H]2+ ions. The main fragment of activation (c.e. 25 eV) of the singly charged ion, [GlcN + H]+  was described as a result of rearrangement. Peak at m/z 203.1023 . The peaks at m/z 185.0919, [GlcN2 + H − C4H8O4 − H2O]+ and m/z 167.0814 [GlcN2 + H − C4H8O4 − 2H2O]+ was explained analogously . The peak at m/z 180.0861 was assigned to the [GlcN + H + H2O]+ ion. The origin of peaks at m/z 174.0756 and m/z 186.0761  was difficult to understand, possibly they occurred due to unknown skeletal rearrangements different from those known before  [m/z 186 and m/z 168 were formed due to the transfer of the CH2CO fragment from one unit to the amino group of the other one followed by cleavage of the glycosidic bond and the loss of one or two molecules of water . Activation of the [M + 2H]2+ ion (c.e 10 eV) resulted in appearance of the main peak at the same m/z, but differed from the residual signal by the position of isotopic peaks (see above) along with smaller peaks of the [GlcN + H − H2O]+ and [GlcN + H − 2H2O]+ ions  and the small peak at m/z 204.0871 . The CID MS2 of the [M + Na]+ ion was not described due to its low intensity.In the positive ion mode ESI CID MSsamines) . For exacN + H]+  had the residue) . In thisO]+ ions . The pea−. As an example, the CID MS2 (c.e. 20 eV) of the ion [M − H] − of 20a was considered. The decay of this anion differed from that of protonated molecule because the main fission occurred not at glycosidic bonds. The small peak at m/z 160.0612 can be considered as a deprotonated link of glucosamine [GlcN − H]−, i.e., the product of cleavage of two glycosidic bonds, nevertheless, the ion [GlcN2 − H]− was not observed. The ions of lower masses occurred due to elimination of molecules of ammonia  and water , and the ion at m/z 101.0247 (C4H5O3−) was possibly formed by elimination of the neutral C2H5NO moiety that may correspond to the O(1)-C(1)-C(2)-N(2) fragment. The main fragment peak at m/z 220.0831 (C8H14NO6−) can be formed only as a result of a cleavage of the C—C bond (presumably C(4)-C(5) and C(5)-O(5) of one of the residues with the retention of one (1→6)-glycosidic bond and the rupture of another one). Small peaks at m/z 464.1906 and m/z 446.1797 corresponded to elimination of one or two molecules of water. The peak at m/z 423.1628 (C16H27N2O11–) may be a result of elimination of the O(1)-C(1)-C(2)-N(2) fragment of one unit. The further elimination of the C2H4O2 fragment resulted in formation of m/z 363.1435 (C14H23N2O9−) anion. The ion at m/z 280.1053 (C10H18NO8−) corresponded to the formal addition of the C4H8O4 fragment to the deprotonated glucosamine residue. Formation of this ion was proposed through the rupture of C-C and C-O bonds in two glucosamine residues with the retention of the third one as a core. It was concluded that primary fragmentation of the negatively charged ions (deprotonated molecules) proceeds in a different way than that of positively charged ones [Mass spectra of the negatively charged ions of the studied compounds demonstrated low abundant deprotonated molecules [M − H]ged ones .-N-acetylglucosamines were similar to those of their acyclic analogs. For example, MS of cyclic dimer of N-acetylglucosamine 19b possessed peaks of [M + H]+, [M + NH4]+, [M + Na]+, and [M + K]+ ions. CID MS2 of [M + H]+ ion resulted in a cleavage of glycosidic bonds and formation of the ion at m/z 204.0865 ([GlcNAc + H]+); under c.e. of 10, 15, and 20 eV, the peak of this ion was the most intense. Elimination of one, two, and three molecules of water resulted in formation of ions at m/z 186.0761, m/z 168.0655, and m/z 150.0550, respectively. At c.e. 40 eV, the peak at m/z 138.0549  became the main one. Two other intense peaks (c.e. 40 eV) at m/z 144.0654 (C6H10NO3) and m/z 126.0549 (C6H8NO2) were assigned to the fragment ions [GlcNAc + H − C2H4O2]+ and [GlcNAc + H − C2H4O2 − H2O]+, respectively. Fragmentation of the ion [M + NH4]+ (c.e. 20 eV) was similar to that of [M + H]+. In the range of higher m/z, the products of elimination of one ant two molecules of water, m/z 389.1544 and m/z 371.1451 were found. In CID MS2 (c.e 50 eV) of metallated molecules of 19b, only cleavages of two glycosidic bonds were revealed , and elimination of H2O from these fragments resulted in [GlcNAc + Na − H2O]+, m/z 208.0575 and [GlcNAc + K − H2O]+, m/z 224.0247. For the [M + Na]+ ion, elimination of one and two molecules of water was observed. For cyclic oligomers having from three to seven GlcN units, the same losses were observed.Mass spectra of cyclic oligo2 of protonated molecules of isomers 25 and 26 was claimed in [2 of their [M + H]+ ions  contained the same signals with slight difference in relative abundance. A small peak at m/z 323 related to the disaccharide fragment [GlcGlcN + H]+ was observed for both oligosaccharides .The aim to reveal characteristic differences in the CID MSaimed in . The pos2 of the doubly charged ions [M + 2H]2+  were much more prominent : for 25, its relative abundance is 0.6 %, whereas for 26 it is equal to 33 %, so, in more than 50 times higher 2+ of 25 and 26 contained only peaks of glucosamine unit and its fragmentation products.At the same time, the differences in CID MSrominent a,b. The n of 21a c gave anshown in  for aminA critical review of traditional methods and recent achievements in the analysis of cyclodextrins and their derivatives is presented in . This paN-succinylamido linker was done by high-resolution positive and negative ESI MS + was presented and described. Successive losses of HBr (80/82 Da) and C6H5CO2H (122 Da) occurred. In the low-mass region, abundant ions m/z 311/313, 293/295, 189/191 corresponding to the fragmentation of a single 2,3-di-O-benzoyl-6-deoxy-6-bromo hexose unit along with m/z 105 (C6H5CO+) were observed.ESI MS was used for the characterization of regioselectively derivatized maltooligosaccharides obtained starting from natural β-cyclodextrin . For two2 inclusion complex was reported [2 + 2H]2+ and [C60:(γ-CD)2 + 2Na]2+, only [(γ-CD) + H]+ or [γ-CD + Na]+, respectively, were registered along with ionized fragments of γ-CD ([Glcn + H/Na]+), whereas for negatively charged ion [C60:(γ-CD)2 − 2H]2−, the major fragments gave superposed C60− and [C60 + H]− peak clusters along with minor superposed clusters of deprotonated cyclodextrin [γ-CD − H]−/[(γ-CD)2 − 2H]2−.An interesting observation concerning charge differentiation in tandem ESI CID mass spectra of C60:(γ-CD)reported . For posO-sulfated, polyethylene glycol α,ω-dimethylacrylate-tethered, dipyrenyl-terminated rotaxanes were profiled using Orbitrap ESI MS. Positive ion mode ESI MS2 was successfully applied for structure characterization of individual components in a mixture of rotaxanes.A mass spectrometric study of complex, supramolecular object was performed quite recently . PolydisO-isopropylidene and 3-O-dodecyl, were subjected to ESI MS2 fragmentation in triple quadrupole instrument using Ar as a collision gas. Schemes of low-energy CID fragmentation were proposed; the main losses were acetone (from isopropylidene) and dodecanol/dodecene (from dodecyl). Ether cycle cleavage also occurred; the authors included this fission as a central part in both schemes.A study of unusual, separately classified compounds was published in . These c2 fragmentation is now in progress; we believe that discoveries will happen concerning these unusual compounds in nearest future.For the last three decades, mass spectrometry of cyclic oligosaccharides has achieved great success, especially in the field of gas-phase chemistry of non-covalent, host-guest complexes. Nevertheless, some problems remain unsolved. First, there is no convenient and universal nomenclature of cleavages of cyclic oligosaccharides free of the reducing/nonreducing end formalism. No easy-to-use and reliable methods to differentiate between isomeric cyclic and acyclic oligosaccharides have been proposed. For induced decay of host-guest cyclodextrin complexes, no direct proof of guest-host proton transfer is presented. The boundaries of application of tandem mass spectrometry for determination of positions of substituents in cyclic oligosaccharides are not yet determined. A few works were done concerning fragmentation of negative ions of cyclic oligosaccharides. The methods of activation are now restricted by CID (ESI MS) and LID (MALDI MS), for example, no studies in ECD of multiply charged positive ions of cyclic CDs were carried out. A search of structure effects in complex, mixed cyclic oligosaccharides on their MS"}
{"text": "Night shift work has become highly prevalent in our 24/7 societies, with up to 18% of the US work force working alternate shift schedules. However, studies indicate that there may be adverse health effects of chronic night work across diverse populations. These effects are likely due to misalignment of the circadian system with work schedules, mediated by the system’s primary marker melatonin as well as other downstream molecules.Melatonin has multiple biologic actions that are relevant to cardiometabolic disease, including modulation of oxidative stress, inflammation, and (via the melatonin receptor) vasoconstriction. Behavioral traits, such as chronotype and meal timing, have recently been shown to interact with the effects of night work on cardiometabolic health.Together with recent findings suggesting a role for circadian genes in cardiometabolic risk, the interactions of night shift work and behavioral traits are likely to facilitate novel treatment and prevention approaches for cardiovascular disease and type 2 diabetes, incorporating aspects of clock and timing. Involvement of the circadian system in the pathogenesis of cardiovascular disease (CVD) has been suspected since older anecdotal evidence indicated a higher frequency of myocardial infarction in the early morning hours. More recent reports have continued to support this notion . In the A number of epidemiologic studies have explored the association between shift work and diabetes in recent decades. The majority of these studies were conducted since 2000 across the United States (US), Europe, and Asia. A recent meta-analysis identified 12 such studies involving more than 200,000 participants, and reported that, overall, shift work history was related to an increased risk of diabetes . AlthougPtrend < 0.001). Specifically, compared to participants without such a history, participants with 1–2, 3–9, 10–19, and ≥ 20 years of rotating night shift work history had the following elevated risks of diabetes after adjustment for important confounding factors: hazard ratios (HRs) 1.05 (95% CI = 1.00–1.11), 1.20 (1.14–1.26), 1.40 (1.30–1.51), and 1.58 (1.43–1.74), respectively. These results were consistent across the two cohorts. Additional analyses adjusting for body mass index attenuated these estimates, although the overall trend remained highly significant; still, this result suggests that body weight might mediate the observed association.A landmark study of rotating night shift work and diabetes  was conducted by our group within the Nurses’ Health Study (NHS) and Nurses’ Health Study II (NHS II) in 2011 . This st•]. In particular, a mismatch in sleep and work timing appeared to increase the risk of diabetes slightly among early chronotypes who worked ≥ 10 years of rotating night shifts, and to increase the risk strongly among late chronotypes who worked no rotating night shifts. This finding is consistent with recent evidence suggesting a role of meal timing  have been identified in all nucleated cells –30 that The circadian system is controlled by the “master pacemaker,” which regulates the sleep/wake cycle and pineal melatonin biosynthesis . It alsoBehaviors that induce misalignment between internal clock and external timing  have the potential to upset the inherent, roughly regular circadian rhythm in humans. Several studies have demonstrated negative effects of circadian misalignment on measurable biomarkers, including melatonin. Levels of melatonin’s major urinary metabolite, 6-sulfatoxymelatonin (aMT6s), are closely correlated with nightly peak plasma melatonin levels in blood and saliva, and therefore allow determination of nightly peak levels of melatonin in human studies . In addi•]). Additional evidence for the protective effect of circulating melatonin on CVD/T2DM risk comes mostly from cross-sectional studies reporting decreased melatonin levels  in patients with versus without diabetes [Human observational studies have shown higher melatonin levels to be inversely associated with higher melatonin levels to be inversity associated with hypertesion, T2DM and incident myocardial infarction (MI). For example, in prospective, nested case-control studies of the NHS, after adjusting for an extensive set of confounding variables, the odds ratio for a unit decrease in log-transformed sulfatoxymelatonin:creatinine ratio was 1.48 (95% CI = 1.11–1.98) for T2DM and 1.40 (95% CI = 1.02–1.93) for incident MI •). Additdiabetes , 43, anddiabetes , 45.Several lines of evidence from experimental studies support that melatonin may have a beneficial effect on glucose metabolism  and cardMTNR1B (type 2 melatonin receptor) and T2DM, with odds ratios ranging from 1.09 to 1.20. Such findings provide further support for an association between the circadian system—and hence night shift work, which adversely affects this system—and cardiometabolic health [CRY2 gene has been associated with increased insulin resistance and T2DM at genome-wide levels of significance [Several associations between circadian gene variants and health outcomes have previously been described (e.g., sleep disorders  and obesc health –73. Addiificance .In summary, several lines of evidence, both from mechanistic studies as well as observational data in humans, provide support for a causal association between circadian disruption as encountered by night shift workers, and the primary circadian marker melatonin, with risk of T2DM and CVD. While night workers have been found to experience a higher risk of cardiometabolic diseases including T2DM and CVD, the mechanistic studies suggest that these effects are mediated by alterations in melatonin levels and melatonin’s function on melatonin receptors throughout the body, as well as through clock-genetic variants, and inflammatory and endothelial pathways common to CVD and T2DM.There is ample evidence for a variety of important pathways that are, at least partially, under clock control and suggest close involvement of the circadian system in cardiometabolic pathophysiology. Recent studies of behavioral traits, such as chronotype and corresponding meal timing, also suggest an interaction with night shift work , and fut"}
{"text": "The pharmacokinetics of four upadacitinib extended-release formulations with different in vitro release characteristics and an immediate-release capsule formulation of upadacitinib were evaluated in 20 healthy subjects in a single-dose, randomized, crossover study. In vivo pharmacokinetic data and in vitro dissolution data  were used to establish a level A IVIVC. Three formulations were used to establish the IVIVC, and the fourth formulation was used for external validation. A non-linear IVIVC best described the relationship between upadacitinib in vitro dissolution and in vivo absorption profiles. The absolute percent prediction errors (%PE) for upadacitinib Cmax and AUC were less than 10% for all three formulations used to establish the IVIVC, as well as for the %PE for the external validation formulation and the overall mean internal validation. Model was cross-validated using the leave-one-out approach; all evaluated cross-validation runs met the regulatory acceptance criteria. A level A IVIVC was successfully developed and validated for upadacitinib ER formulation, which meets the FDA and EMA regulatory validation criteria and can be used as surrogate for in vivo bioequivalence.Upadacitinib is a selective Janus Kinase 1 inhibitor which is being developed for the treatment of several inflammatory diseases including rheumatoid arthritis. Upadacitinib was evaluated in Phase 3 studies as an oral extended-release (ER) formulation administered once daily. The purpose of this study was to develop a level A The online version of this article (10.1208/s12248-019-0378-y) contains supplementary material, which is available to authorized users. Upadacitinib ABT-494) is a novel, selective Janus kinase (JAK) 1 inhibitor that potently inhibits JAK 1, but is less potent against other JAK isoforms –4. Upada94 is a nCmax and Cmin to 6 mg and 12 mg BID using the IR formulation under fasting conditions [in vitro assessments, upadacitinib has been shown to be highly permeable and highly soluble at clinically relevant doses across the pH range of 1 to 7.5 (data on file at AbbVie). Based on in vitro assessments, upadacitinib is considered to be a class I drug according to the Biopharmaceutics Classification System [Upadacitinib pharmacokinetics was characterized in healthy subjects following the administration of the immediate-release (IR) and the extended-release (ER) formulations ,12. The n System . The relin vivo clinical study that is adequately powered to demonstrate bioequivalence [in vivo–in vitro correlation (IVIVC) can allow for the prediction of the plasma concentration time profile of a formulation without having to conduct in vivo bioavailability studies, thus saving time and costs and avoiding the need to administer a drug to healthy volunteers [in vivo bioavailability studies which may be needed to approve and maintain a drug product on the market [Throughout the life-cycle of a drug product, the need often arises to change some aspects of the formulation, the manufacturing process, or the manufacturing site. Some post-approval changes require the need to demonstrate bioequivalence between the modified and the marketed formulation through an ivalence ,18. Furtlunteers . The avae market ,18.in vitro dissolution and in vivo pharmacokinetics for four ER formulations (30 mg strength) with various in vitro release profiles, including the proposed commercial formulation, were evaluated relative to a 24-mg dose of the immediate-release (IR) capsule formulation. The methodology for the evaluation and establishment of a non-linear level A IVIVC for upadacitinib ER formulations are described.In the present study, the in vivo clinical study.Four ER formulations  were developed with identical amounts of upadacitinib (30 mg) but varying amounts of the release-controlling polymer HPMC. Three formulations  contained 10%, 15%, and 35% HPMC, respectively. The fourth formulation evaluated (Formulation C) contained 20% HPMC as a prototype for the planned commercial formulation. A single 24-mg dose of upadacitinib (2 × 12 mg IR upadacitinib capsules) was used as the reference formulation in the in vitro dissolution profiles. The dissolution medium was 900 mL of 0.05 M phosphate buffer (pH 6.8). An automatic sampler collected 1.5 mL of each of the sample at multiple time points , and the samples were then analyzed with high-performance liquid chromatography (HPLC). Several additional dissolution methods were also evaluated .A USP Dissolution Apparatus 1 operating at a rotating speed of 100 rpm was used to generate the N = 20) in general good health were selected to participate in the study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee  and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individuals included in the study prior to any study-related procedure. Enrolled subjects were randomly assigned in equal numbers to one of five sequences of regimens A, B, C, D, and E consisting of five periods. In each of the five periods, a single dose of upadacitinib was taken orally with approximately 240 mL of water after a fasting for a minimum of 10 h and at least 4 h before lunch. Each dose was separated by a washout interval of 4 days. Blood samples for upadacitinib assay were collected into dipotassium ethylenediaminetetraacetic acid-containing collection tubes prior to dosing (0 h) and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 36, 48, and 72 h after dosing in each period. Plasma concentrations of upadacitinib were determined using a validated liquid chromatography method with tandem mass spectrometry [A Phase 1, single-dose, open-label study conducted according to a five-period, randomized, crossover design was used to characterize the bioavailability of four upadacitinib ER tablet formulations with different dissolution release rates relative to upadacitinib immediate-release capsules under fasting conditions. The study was conducted at PPD Development , and subjects were confined to the study site and supervised for approximately 21 consecutive days. Adult male subjects (trometry . The lowCmax) of upadacitinib and time to Cmax (tmax) values were determined directly from the plasma concentration versus time data for each subject. The apparent terminal phase elimination rate constant  was obtained from the slope of the least squares linear regression of the logarithms of the plasma concentration versus time data from the terminal log-linear phase of the profile. The terminal phase elimination half-life (t1/2) was calculated as ln (2)/β. The area under the plasma concentration versus time curve from zero to the last measurable concentration (AUCt) and area under the plasma concentration versus time curve from zero to infinity (AUCinf) were calculated by the linear trapezoidal method.Pharmacokinetic parameters were estimated using non-compartmental methods in Phoenix® Version 7.0 . The maximum observed plasma concentration , and b is the slope factor.in vitro dissolution data and the Akaike Information Criteria (AIC) from each equation [in vivo data. The individual UIR parameters were estimated by fitting a maximum of three polyexponential function with a time lag to each individual profile from the IR formulation and choosing the one which best fits the profile based on the AIC with a uniform weighting scheme. Since the reference formulation was an IR formulation, the “strip Ka” approach was followed to ensure that the UIR is decoupled from the absorption process as previously described [Tlag) was incorporated into the model to allow for a time lag in absorption. The adequacy of the selected model was determined through evaluating the agreement between model-predicted and observed upadacitinib plasma concentration versus time profiles.Individual unit impulse response (UIR) parameters were generated using the IR formulation escribed . This asin vivo (Fa) from each of the four upadacitinib ER formulations relative to the IR formulation was estimated by numerical deconvolution of the observed plasma concentration versus time profiles against the UIR from the reference IR formulation of upadacitinib. Exploratory plots were generated to evaluate the relationship between Fa and Fdiss, vitro as well as between the in vitro dissolution time (Tvitro) and in vivo absorption time (Tvivo).The fraction of upadacitinib dose absorbed Cmax and a linear IVIVC could not be established. A user-specified non-linear IVIVC was evaluated by fitting a non-linear Emax model to scale the Tvitro to the Tvivo, as shown in Eq. Fa and Fdiss, vitro.Several linear and non-linear models were evaluated for correlation between the fraction dissolved versus time and fraction absorbed versus time profiles of upadacitinib. Three formulations  were used to establish the IVIVC, and the fourth formulation (formulation B) was used for external validation. Default linear models in Phoenix IVIVC toolkit which employ absorption scale, time scale, and/or time shift for different dissolution conditions were initially evaluated. However, all the evaluated linear models demonstrated under-prediction for upadacitinib The equation to use was selected based on adequacy of fitting the equation .IndividuA1, A2, and B2 are constants characterizing the Emax relationship between Tvitro and Tvivo.Fa  corresponding to Fdiss, vitro at different time points for each formulation. The Fa, pred for each of the four ER formulations and the individual UIRs from the IR capsule formulation were used as the input function for convolution to generate individual predicted plasma concentration versus time profiles for the different ER formulations. The predicted pharmacokinetic parameters, Cmax, AUClast, and AUCinf were estimated using non-compartmental analysis within the Phoenix IVIVC toolkit.The developed IVIVC relationship was used to predict inf and Cmax for each ER formulation as:The prediction error, %PE, was calculated for AUCAdditionally, a cross-validation was conducted using the leave-one-out approach. The final IVIVC model was re-run using each of the four extended-release formulations  as an external validation formulation and the remaining three formulations for model building and internal validation. The %PE was calculated for internal and external validation formulations for each of the cross-validation runs.Cmax and AUCinf was required. In addition, the absolute % PE for each individual formulation was required not to exceed 15%. For external validation, the absolute %PE was required not to exceed 10% for Cmax and AUCinf.The predictive ability of the IVIVC was assessed for the numerical IVIVC through evaluating each method’s internal and external predictability per FDA and EMA guidance documents ,18. To ein vitro drug-release profiles for the four upadacitinib ER formulations using the selected method (USP App 1 at pH 6.8) are shown in Fig. in vitro dissolution data as it provided better fit compared to a Hill function  and ER tablets (30 mg) are described in Table Tmax of upadacitinib was 2.0, 2.5, 3.0, and 3.0 h for ER formulations A, B, C, and D, respectively, as compared to 1.0 h for the IR formulation. Upadacitinib terminal half-life was similar across the ER formulations and comparable to that of the IR formulation (approximately 10 to 13 h).The mean plasma concentration versus time profiles from the ent Fig. . The ratFa and Fdiss, vitro as well as between Tvivo and Tvitro are presented in Fig. Fa, obs and Fd, obs is linear  as well as for the external validation formulation (formulation B) are presented in Table inf and Cmax for the ER formulations . The %PE for each of the individual ER formulations A, C, and D were also well below 15% for both AUCinf and Cmax. In addition, the external %PE for ER formulation B was less than 10%  of 10 to 35%. This range encompasses the target to be marketed formulation (formulation C) which contains 20% HPMC. The established IVIVC meets both the internal and external predictability per the FDA and EMA criteria and can potentially be used as surrogate for in vivo study if there is a need for formulation change (within the design space of the IVIVC) or manufacturing process change. Additionally, the established IVIVC enables setting the release specifications based on clinical relevance using the predicted range of upadacitinib exposures for formulations that fall within the proposed specifications. These analyses highlight the importance of evaluating non-linear models when linear IVIVC relationships cannot be established.A predictive level A non-linear IVIVC was established for upadacitinib ER formulation using Cmax, (data not shown) suggesting slower dissolution in vitro than in vivo dissolution that was not adequately accounted for using a linear time scaling factor. This was also the case for various other in vitro test conditions evaluated to mimic the in vivo conditions . Given the high permeability and high solubility of upadacitinib, intestinal transporters are not expected to have clinically relevant role in upadacitinib disposition Tvitro and Tvivo appeared linear up to approximately 8 h in vivo. An approach that is alternative to the use of non-linear model would be to establish a linear IVIVC with in vivo cutoff time (tcutoff). However, use of tcutoff implies ignoring collected in vivo and in vitro data after the selected tcutoff. Additionally, Phoenix software that does not allow estimating the optimal tcutoff value and tcutoff will need to be selected as an arbitrary value based on observed data. Both of the aforementioned points can potentially introduce bias in the analysis when tcutoff is used. We opted to use a non-linear IVIVC approach rather than use of linear IVIVC with tcutoff to be able to use the totality of the data thus to avoid ignoring any in vitro or in vivo data and to allow the software to estimate the best-fitting parameters for the non-linear relationship based on the data.It is worth noting that the relationship between The IVIVC model was developed while a Phase 3 study which evaluated the safety and efficacy of upadacitinib doses of 15 mg and 30 mg QD using the ER formulation was ongoing. The upadacitinib ER formulation is considered proportionally similar between the 15 and 30 mg strengths, and upadacitinib plasma exposures are linear over a wide range of IR and ER doses ,15. Therin vitro test can potentially serve as a surrogate for bioavailability testing as well as a tool to screen formulations and set the dissolution and drug-release acceptance criteria.The robustness of the model was evaluated through cross-validation using the leave-one-out approach; all cross-validation runs meet the acceptance criteria . This assessment, although not a requirement per regulatory guidance documents, demonstrate robustness of the IVIVC and that it is not sensitive to data from a specific formulation. With a validated IVIVC, an A robust non-linear level A correlation that meets the FDA and EMA validation criteria for both internal and external predictability was established for upadacitinib ER formulation. This IVIVC can be used as surrogate for bioequivalence studies in case of future formulation changes that are covered by the IVIVC release rates tested. This correlation will enable the setting of clinically meaningful dissolution specifications based on acceptable differences in plasma concentrations corresponding to the upper and lower limit of the dissolution specifications.ESM 1(DOCX 14 kb)"}
{"text": "We present SlicerDMRI, an open-source software suite that enables research using diffusion magnetic resonance imaging (dMRI), the only modality that can map the white matter connections of the living human brain. SlicerDMRI enables analysis and visualization of dMRI data and is aimed at the needs of clinical research users. SlicerDMRI is built upon and deeply integrated with 3D Slicer, a National Institutes of Health–supported open-source platform for medical image informatics, image processing, and three-dimensional visualization. Integration with 3D Slicer provides many features of interest to cancer researchers, such as real-time integration with neuronavigation equipment, intraoperative imaging modalities, and multimodal data fusion. One key application of SlicerDMRI is in neurosurgery research, where brain mapping using dMRI can provide patient-specific maps of critical brain connections as well as insight into the tissue microstructure that surrounds brain tumors.In this article, we focus on a demonstration of SlicerDMRI as an informatics tool to enable end-to-end dMRI analyses in two retrospective imaging data sets from patients with high-grade glioma. Analyses demonstrated here include conventional diffusion tensor analysis, advanced multifiber tractography, automated identification of critical fiber tracts, and integration of multimodal imagery with dMRI.We illustrate the ability of SlicerDMRI to perform both conventional and advanced dMRI analyses as well as to enable multimodal image analysis and visualization. We provide an overview of the clinical rationale for each analysis along with pointers to the SlicerDMRI tools used in each.SlicerDMRI provides open-source and clinician-accessible research software tools for dMRI analysis. SlicerDMRI is available for easy automated installation through the 3D Slicer Extension Manager. We provide the clinical rationale for each analysis along with pointers to the workflow of the SlicerDMRI tools used. We begin by demonstrating a conventional diffusion tensor imaging analysis followed by advanced multifiber and automated tractography analyses. We give two examples of how SlicerDMRI, used within the platform of 3D Slicer, enables multimodal image analysis for neurosurgical planning and guidance research.RelevanceOne key application of SlicerDMRI is in neurosurgery research, where brain mapping using dMRI can provide patient-specific maps of critical brain connections and insight into the tissue microstructure that surrounds brain tumors. SlicerDMRI is available for easy automated installation through the 3D Slicer Extension Manager.18 The most popular measures used in neurosurgical patients are derived from modeling water diffusion using tensor mathematics (diffusion tensor imaging [DTI]19). These measures include fractional anisotropy (FA) and mean diffusivity (MD). In neurosurgical patients, alterations in FA and MD can reflect combinations of increased water content from edema and/or tumor infiltration.20dMRI also provides quantitative microstructure measures. These measures allow the assessment of changes of tissue microstructure caused by tumors and the effects of treatment.Brain mapping using dMRI requires sophisticated computational processing to enable visualization and quantification, and multiple software packages exist for this purpose. In fact, several packages are US Food and Drug Administration (FDA) approved and purpose-built for neurosurgical brain mapping, neuronavigation, and radiation therapy planning. However, these dedicated packages do not make cutting-edge developments in dMRI analysis available to clinicians or are specifically designed for research needs. Rather, they are designed to provide robust results within the constraints imposed by routine clinical settings and FDA approval, where there is relatively little time to analyze the data of an individual patient and where the operators of the navigation systems are typically residents with limited training in dMRI.21) includes dMRI visualization, interactive and automated tractography, tissue microstructure modeling, and quantitative analyses. SlicerDMRI can import dMRI data, including clinical Digital Imaging and Communications in Medicine (DICOM) images, research formats of Neuroimaging Informatics Technology and Nearly Raw Raster Data, and the new DICOM tractography format compatible with multiple software platforms.22 The open-source codebase of SlicerDMRI can be extended as needed to fit the needs of a specific research project.SlicerDMRI is a suite of clinician-accessible software tools with a strong focus on the needs of clinical researchers. SlicerDMRI has an average of > 200 downloads per month. The main functionality of SlicerDMRI .27 The data were acquired at Brigham and Women’s Hospital. The use of the data was approved by the Partners Healthcare institutional review board, and informed consent was obtained from the patients before scanning. All data analyses in the current work were performed retrospectively for research purposes only.Retrospective imaging data sets from two neurosurgical patients were selected to illustrate two different clinical scenarios: a high-grade glioma displacing the corticospinal tracts (CSTs), with significant surrounding edema, and a high-grade glioma abutting the arcuate fasciculus (AF). Preservation of these tracts is critical for voluntary motor movements (CSTs)2, six baselines, and a matrix of 112 × 112 with 66 axial slices. Functional MRI (fMRI) was acquired as the patient performed motor and language tasks as clinically indicated, and data were processed using FDA-approved software . Ultrasound data were acquired with the BK5000 system  with a slice thickness of 0.5 mm coregistered to the T1-weighted anatomic volume using the Curve navigation system  and extracted using OpenIGTLink28 and 3D Slicer.All magnetic resonance data were acquired on a 3T MAGNETOM Prisma MRI scanner  with a 20-channel head coil. Anatomic imaging included volumetric T2 weighted and gadolinium-enhanced T1 weighted. dMRI was acquired with a repetition time of 3,500 ms and echo time of 58 ms with a flip angle of 90°, a voxel size of 2 × 2 × 2 mm, a b value of 1,000 seconds/mmA 69-year-old, right-hand-dominant man with a recurrent left-sided frontoparietal glioma initially presented with a diffuse large fronto-insular infiltrative pattern lesion. A biopsy sample was found to have a WHO grade 2 astrocytoma IDH1 wild type, which was treated with chemoradiation. Follow-up imaging at 3 years revealed progression in the left-side perirolandic area, which did not respond to salvage systemic therapy. Radiographic progression and clinical deterioration (motor weakness) led to the decision to pursue surgical resection for debulking and tissue diagnosis. Image-guided left frontal craniotomy was performed with intraoperative motor- and somatosensory-evoked potentials and cortical and subcortical mapping. Gross total resection of enhancing tumor (now grade 4) was accomplished with stable motor function immediately postoperatively and improved motor function relative to baseline at 1-week follow-up.A 49-year-old, right-hand-dominant man who presented with secondarily generalized seizures and word-finding difficulties was found to have a left-sided temporoparietal tumor immediately adjacent to posterior language areas by fMRI. He underwent image-guided craniotomy and resection of tumor with gross total resection of the lesion. Pathology demonstrated high-grade glioma, WHO grade 4.1 DTI enables scalar measurements such as FA and MD19 and single-fiber tractography that accounts for one fiber tract per voxel.5 In this section, we give two clinically relevant example applications using DTI analysis in SlicerDMRI for neurosurgical planning research.DTI, which models water diffusion using a tensor model, is the most widely used method for analyzing dMRI data.The analysis of scalar measurements from DTI can give insight into tissue microstructure differences between the tumor and surrounding tissues. SlicerDMRI provides the DiffusionTensorEstimation module to calculate DTIs from diffusion-weighted image sequences. The 3D Slicer Volumes module allows visualization of diffusion tensors with multiple options, including both two-dimensional (2D) and three-dimensional (3D) displays.SlicerDMRI supports computation of quantitative diffusion measures, such as FA and MD, on the basis of the estimated diffusion tensors using the DiffusionTensorScalarMaps module.SlicerDMRI, together with other modules in the 3D Slicer core, enables quantification of region-specific diffusion measures. This can be done using 3D fiducials to define regions of interest (ROIs) and Data Probe to view diffusion measures from an ROI. This process can also be performed with the SegmentEditor module that enables manual drawing of an ROI and the LabelStatistics module that calculates statistics for different ROIs.DTI tractography can identify patient-specific fiber tracts for visualization. This can inform surgical planning to minimize morbidity associated with disruption of critical white matter connections. 5 Tractography seeding using two clinically relevant approaches is provided: seeding from an interactively visualized 3D object that can be manipulated by the user and seeding from an ROI that is stored as a label map, or mask. These two seeding strategies are provided in the TractographySeeding and TractographyROISeeding modules. Tractography visualization is performed using the TractographyDisplay module, where multiple tract visualization modes are provided .SlicerDMRI supports single-fiber DTI tractography that performs fiber tracking by following the principal direction of the estimated diffusion tensors.SlicerDMRI provides multiple tools to enable quantification of tract diffusion and geometric measures. For example, the TractographyMeasurement module allows for computation of multiple statistics  of diffusion-derived values of the fiber points along a tract. The TractographyToMask and LabelStatistics modules together enable the measurement of tract volume, and the WhiteMatterAnalysis package enables computation of the fiber length distribution of a tract.Advanced analyses in SlicerDMRI include more sophisticated mathematical modeling of the white matter tissue, which enables depiction of crossing fiber tracts, and automated identification of critical fiber tracts, which enables rapid, standardized assessment of patient-specific neuroanatomy.29 because of the effects of edema and infiltration. DTI tractography is also limited to model only one fiber tract per voxel, despite that there are tens to hundreds of thousands of axons packed per square millimeter.30 DTI tractography cannot reconstruct fiber tracts that cross one another,33 such as the CST and AF. Modern multifiber tractography approaches can map a more-complex fiber architecture  to better trace important anatomic fiber tracts.34Conventional DTI tractography is often unable to fully trace fiber tracts in the peritumoral region35 which provides an expanded range of multifiber models, including multitensor and multicompartment models. UKF tractography is more sensitive than standard single-tensor tractography, particularly in the presence of crossing fibers and peritumoral edema.38 Thus, UKF can better match the established understanding of surgical neuroanatomy than traditional DTI tractography. However, it should be noted that such comparisons are difficult because of the lack of ground truth, and tractography validation and standardization remain an open problem.40SlicerDMRI supports advanced multifiber unscented Kalman filter (UKF) tractography,41 and neurite orientation dispersion and density imaging,42 are available during UKF tractography fiber tracking.SlicerDMRI provides the InteractiveUKF and UKFTractography modules for fiber seeding from an interactively modifiable 3D object and an ROI mask, respectively. Advanced tissue modeling options, such as free water modeling43 Using a combination of machine learning and expert anatomic annotation, we created an anatomically curated white matter atlas.44 The atlas contains 58 deep white matter tracts, including major long-range association and projection tracts, commissural tracts, and tracts related to the brainstem and cerebellar connections. This atlas is released as an open source as part of SlicerDMRI.45 Using this atlas and our accompanying open-source software, we have demonstrated automatic identification of fiber tracts in patients with brain tumors, despite the challenges of edema and tract displacement as a result of mass effect.46Conventional identification of critical fiber tracts requires expert manual interpretation, which is time consuming and has variable results across experts.48 The 3D Slicer Ruler function can be used to measure the distance of the tracts to other image data  displayed in the 3D Viewer.SlicerDMRI provides the WhiteMatterAnalysis Python package to perform automated tract identification using a data-driven fiber clustering pipeline. A one-step batch script is provided to run the pipeline for identification of fiber tracts according to the anatomically curated white matter atlas.v 1.5 hours on a data set containing 1 million fibers).49 This fast computation can motivate the use of automated analyses for intraoperative mapping of white matter connections.In addition to WhiteMatterAnalysis, in the near future, SlicerDMRI will also support fast tractography segmentation using deep learning techniques. This will be provided in the DeepWMA package. While achieving visually similar tract identification results to WhiteMatterAnalysis, DeepWMA performs much faster by leveraging deep learning and graphics processing unit computation , is already being used for surgical planning57 and may be ideal for children or patients who are unable to perform a task. A prospective multi-institutional study to analyze fMRI, rsfMRI, and multifiber DTI techniques is needed.Preserving function during brain tumor operations is associated with increased quality of life and overall survival.Anatomic fiber tracts identified using the SlicerDMRI WhiteMatterAnalysis tool are displayed together with functional activations computed from the fMRI data . Visualization of the multimodal data can be performed in either 2D mode (to show the intersection of 3D objects such as tracts and activations on a 2D image) or 3D mode through the 3D graphical user interface .58 The only commercially available option for intraoperative reregistration of a brain is intraoperative MRI, which is expensive, requires nonferromagnetic instruments, and adds up to 1 hour of operative time per scan. Because ultrasound resolution has improved, intraoperative ultrasound is increasingly used for updated real-time imaging.59 This allows the surgeon to better estimate the effect of brain deformation while also obtaining a real-time navigated view. Ongoing work is addressing quantification of this brain deformation such that the preprocedure magnetic resonance data, including tractography, can be updated to match the current brain configuration, thus improving the use of tractographic information to augment intraoperative surgical decision making.61Another consideration during intraoperative identification of function is brain shift, which is the deformation of the brain that occurs during craniotomy and resection. As brain shift worsens during an operation, preoperative imaging becomes less reliable.Anatomic tracts from presurgical dMRI data are computed using the SlicerDMRI WhiteMatterAnalysis tool and are registered to the presurgical anatomic T1-weighted image. Intraoperative ultrasound data are overlaid on the presurgical MRI data for an integrated visualization.62 SlicerDMRI is open source, so all code is available.63 User and software development support for SlicerDMRI is available through the 3D Slicer forums.64 SlicerDMRI has been used successfully for research with dozens of dMRI protocols from various vendors, with appropriate approvals from institutional review boards for each intended research use.SlicerDMRI is available for easy automated installation through the 3D Slicer Extension Manager, an app store for distribution of software extensions to 3D Slicer, with installation instructions and tutorials provided.In conclusion, SlicerDMRI provides open-source and clinician-accessible software tools for dMRI research. In this work, we demonstrate SlicerDMRI functionality as an informatics tool to enable end-to-end dMRI analysis. We provide examples that demonstrate the use of SlicerDMRI to retrospectively analyze data from brain tumor resection."}
{"text": "Virtual care, the use of videoconferencing technology to connect with patients, has become critical in providing continuing care for patients during the current COVID-19 pandemic. Virtual care has now been adopted by health care providers across the spectrum, including physicians, residents, nurse practitioners, nurses, and allied health care professionals. Virtual care is novel and nuanced compared to in-person care. Most of the health care providers who are delivering or expected to deliver virtual care have little to no prior experience with it. The nuances of virtual care involve regulatory standards, platforms, technology and troubleshooting, patient selection, etiquette, and workflow, all of which comprise critical points in the provision of health care. It is important to consistently deliver high-quality, equitable, and professional virtual care to inspire patients with the trust they need to continue follow-up of their care in these difficult times. We have been adopting virtual care in our clinical practice for over two years. In partnership with Canada Health Infoway, we have assembled a primer for virtual care that can serve as a guide for any health care provider in Canada and globally, with the goal of providing seamless transitions between in-person and virtual care. As part of the response to the COVID-19 pandemic, health care organizations across Canada have cancelled elective and nonurgent clinics as a measure to reduce the risk of exposing patients to COVID-19 -7. VirtuVirtual care is novel and nuanced compared to in-person care. Virtual Care has significant benefits to patients, health care systems and society at large by offering patient-centered care -16. VirtThe Division of Neurology at Queen's University/Kingston Health Sciences Center has been an early adopter of virtual care for ambulatory services since 2016 for stroke, epilepsy, and sleep medicine, and it has an active virtual care research program ,26. LeveAt this time, no rigorous standalone virtual care guidelines have been established by licensing bodies in Canada . Some guFurther information on medicolegal liability can be obtained from the Canadian Medical Protective Association (CMPA) ,38. NonpSecure messaging, secure email, and secure video conferencing are some of the most used virtual care modalities. These services can be delivered on multiple platforms and devices. The platforms are broadly divided into regulated and unregulated categories. Regulated platforms are those that comply with Canada's federal Personal Information Protection and Electronics Document Act (PIPEDA), as well as provincial and territorial privacy laws that apply to health or medical records, such as Ontario's Personal Health Information Protection Act (PHIPA). A comprehensive list of regulated platforms in Canada is available ,36. UnreFor the virtual care provider, the choice of platform may depend on the features offered, such as secure messaging, video conferencing, patient portals, integration into electronic health records (EHRs), and integration with other remote monitoring devices and applications. Before finalizing a decision on a platform, we recommend that practitioners seek clearance from their own organization's privacy and security officer if available. Each organization and each individual provider will have a unique set of circumstances, resources, and abilities. It is essential for each provider to develop familiarity with the platform selected by their organization. The platforms currently used at Queen's University are the Ontario Telemedicine Network and Reacts .It is important to ensure that the appropriate precautions are taken by the physician and the patient to ensure the privacy of health care information . For addThe equipment (or hardware) required for delivering virtual care is widely available and includes smartphones, tablets, laptop computers, and desktop computers. Ideally, all these devices should have built-in or peripheral hardware for video conferencing . The network (bandwidth) requirements must be confirmed before use, although most of these platforms enable the quality of the video conference to be adjusted to low bandwidth. In general, the faster and more reliable the internet connection, the better the experience will be for the provider and the patient.The choice of the device used by the provider depends on multiple factors, including the physical location of the provider at the time of the virtual video visit. Although virtual visits can be conducted from any internet-enabled device, we suggest using a laptop or a desktop computer with a built-in camera and speaker. These devices typically provide the highest quality internet connection, a large screen, and a better speaker and microphone setup. However, this is highly dependent on the technology available to the patient, as modern-day tablets and smartphones have high-quality built-in cameras and speakers. Perhaps most importantly, the patient and provider should be familiar with their respective devices in case troubleshooting is required.It is essential to identify when virtual care is most appropriate for patients. Virtual care is an effective alternative to in-person care for many but not all medical conditions and clinical scenarios. Some general guidelines on patient selection and screening are provided in table 1 . Given tIt is mandatory in all jurisdictions to document all health care encounters as part of the patient's health care record. Provincial and territorial medical regulatory authorities and the CMPA consider appropriate documentation to be an essential communication skill and a core component of physicians' professional best practice.Although some virtual care platforms integrate the video visit into the provider's EHR, patient portal, or hospital information system, this practice is not yet commonplace. Thus, two parallel workflows often coexist for the provider: the first for the virtual care platform, and the second for the provider's medical record system.This “double workflow” ensures that the virtual care encounter details  are captured in the patient's health record.It is necessary practice to discuss consent with the patient at the time they sign up for virtual care, covering subjects such as the inherent risks related to privacy and security as well as the limitations of virtual care . There aThe limitations of virtual care should be made clear to the patient. It should be clear that when the clinician cannot see the patient physically, there is inherent risk of missing physical findings. This is only exacerbated by the potential for unstable network connections or poor quality/malfunctioning equipment. It should also be made clear to patients that with this in mind, should there be a requirement for in-person assessment, there may a delay relative to the traditional method of seeing the patient in-person. The option of seeing a clinician in person should always be given to the patient and should always be suggested by the clinician if they feel that the virtual visit was insufficient for the patient’s care or may even have caused harm.Generic consent forms are readily available on the web through many of the resources referenced in this paper ,37,39, aIt is crucial to gauge the degree of a patient's or caregiver's comfort level with using technology before proposing the use of virtual care. Some health conditions can affect motor strength, coordination, language, vision, and cognition. All these challenges can adversely affect a patient’s ability to use personal internet-enabled devices and participate in virtual care, even if the patient is proficient with technology. In addition, it is important to recognize that not all patients will have easy access to the ideal technology for virtual care, including video capability and a sufficiently fast internet connection. We have found that modern mobile phones are capable and ubiquitous but may have poor video and/or audio quality depending on the internet connection. Some patients have chosen to use audio-only telephone visits instead. As we transition to virtual care, we need to be keenly aware of patient-readiness for this transition. Although audio-only visits are shown to be useful, an unexpected change to an audio-based visit from a planned video-based visit may result in slight inconvenience at best and a delay in essential visual assessment and therefore care at worst. In cases of communication breakdown, an in-person visit is often suggested.With the patient's consent, providers should explore the option of having a patient's family member or friend assist with facilitating a virtual visit . PatientIt is also helpful to provide some tips for the patient to prepare for the virtual care visit. The patient’s clothing should allow a reasonable range of motion if the provider requests physical maneuvers during the virtual visit. If the patient requires a hearing or visual aid, these should be readily available. For patients with hearing, visual, or cognitive challenges, it is often more challenging to see and hear via a screen and speakers, in contrast to an in-person visit. Patients requiring mobility aids should keep these devices close by and may benefit from the presence of a nondisabled individual to assist them as necessary.Many health care providers are not familiar with how their patients will perceive them in a virtual visit. With this in mind, it is useful to be aware of the etiquette that is unique to virtual care ,43. The Due to technical limitations, auditory and visual information can be lost during a virtual consultation. At the beginning of the session, both cameras should be directed so that the faces and shoulders of both the patient and the health care provider are framed. Remember that slower, broader movements decrease visual blurring, and obscuring the face and mouth should be avoided. The physician should be mindful that gazing at the patient's image on the screen can make it seem as though the physician is looking down at them. When possible, the physician should look into the camera.During the session, the interviewer must recognize the importance of clear communication. Virtual visits require communication adjustments. Physicians should speak slowly and use nonmedical terms for maximum clarity. They should be prepared for lags in communication for technical reasons. Recognizing these technical limitations reinforces the need for speaking clearly. It may be helpful for the interviewer to check in with the patient to see if they can hear and understand their messaging. Reiterating and summarizing what a patient has said may also be helpful. In addition, clear signposting by the interviewer can indicate the progress and direction of the session. For example, the interviewer could say, “I am now going to summarize what I understand of your history, then proceed to the physical exam.”The primary sensory modalities available in a virtual visit are limited to auditory and visual. However, touch is a standard method of communication during an in-person visit, especially during the physical examination. When requesting that patients perform physical examination maneuvers during a virtual visit, adjustments are required. The clinician should clearly state the movement that the patient is asked to perform. They should consider documenting the absent physical examination maneuvers that would have typically been included but were not feasible due to the virtual nature of the consultation. This suggests, at a minimum, that these steps have been considered. Also, the clinician should inform the patient before they navigate to another screen to look at laboratory or imaging data. When possible, screen-sharing modes should be used to show patients their images or laboratory results.Last but certainly not least, kindness and compassion are paramount in a virtual visit. Listening carefully to a patient's concerns, taking time to clarify their statements, and following up on their questions are vital skills, regardless of what form of medical care is being offered.Preparing one's virtual visit environment is vital for both patients and providers. Clinical administrative staff play a key role. Prior to the appointment, the staff should send a message to the patient with recommendations on setting up for a visit. Some recommendations are relevant to patients and virtual visit providers alike:The room should be reasonably private, with minimal potential for distractions. It may be helpful to notify other members of the household or building that space will be used privately for the duration of the appointment. If possible, adequate lighting, presenting oneself in an uncluttered room, and ensuring the visibility of one's face and shoulders are ideal. Background should be reduced to a minimum. This may include distancing from a louder area . We recommend posting a notice at the entrance to the room indicating that a confidential meeting is occurring. Personal communication devices  should be switched to silent mode to avoid distraction.If other parties are involved in the meeting, such as trainees, family members, and friends, they should be introduced prior the beginning of the meeting. As with standard in-office visits, the people who are involved in the meeting should be transparent and within the control of the patient and physician.Other recommendations are more relevant to patients and can be tailored to their medical conditions and clinical scenarios :For some clinical specialties, it may be helpful for the patient's room to have enough space to allow them to stand and move away from the camera, so their entire body is visible.It is helpful if the camera is high-definition (720p or higher) and wide-angle. The room should have a comfortable yet easily movable chair available for the patient to rest.If the consultation requires equipment, the patient should have an easily accessible area to place these materials.A high-quality internet connection within the selected room is also essential. A wired connection is ideal, but if only Wi-Fi is available, the room should not be too far from the wireless router to ensure a reliable connection.During the presession preparation, attention must be paid to the equipment, environment, and external appearance. In addition to checking the equipment and network, it should be ensured that the general guidelines laid out in the above section with regard to etiquette and the environment are followed. The patient's information or EHR should be available within reach of where the video visit is happening.A good habit is to check the platform appointment software to confirm that the patient or patients have been booked as expected. If there is an issue with connecting to the patient, it is always useful to have an alternative means of communication. Ideally, this will be a telephone number; however, email or instant messaging can be used. Most platforms allow the patient to join and wait in a virtual “room.” It is recommended to mute the microphone before the patient enters the appointment.The first thing to do when the patient logs in is to confirm that both parties’ cameras and microphones are working correctly and troubleshoot as necessary. In all first visits, it should be expected that there will be delays due to technological issues. Technical factors play a significant role in one’s ability to communicate effectively with a patient. It is vital for the medical professional to be familiar with the platform they are using, as it is not uncommon to have to walk through teaching a patient how to modify camera or audio settings. It is essential to explain the steps in a simple and organized fashion.Formal introductions can be made next, in which it is essential to obtain consent for the virtual visit and establish the identity of the patient. The ID verification step may not be necessary for clinical scenarios in which the patients are well known to the interviewer . Announcing who is involved in the meeting for both parties is useful, as some participants can be virtual and others can be physical but not visible (off-camera).The history will likely feel very similar to an in-person visit. It is useful to be clear and intentional with questions, as information can easily be lost through the virtual connection. The physical examination will be a different experience, as traditional medical training in conducting examinations anticipates that the patient will be physically present. When the correct group of patient, diagnosis, and visit purpose are selected, a virtual physical examination is equivalent to an in-person visit examination. Investigations can be reviewed with the patient, including sharing the actual results and images directly via screen sharing. If discussions with colleagues or other staff are necessary, the patient may be notified and muted. At the end of the appointment, as with most appointments, the clinician should discuss and establish a plan. Most relevant in this context is whether a follow-up appointment will be a virtual visit or in-person visit. Because the patient will not be taking any items  with them, it is crucial to verify details with the patient .If orders and prescriptions are integrated into the EHR, clinicians can follow their routine workflow. Otherwise, orders and prescriptions must be sent post-encounter . It should be ensured that follow-up arrangements have been made. Finally, the encounter can be documented by clinicians as per typical workflow , clearly identifying the visit as an eVisit or virtual visit.Before the virtual video visit (presession):Check that the device, video conferencing equipment, and network are working properlyEnsure that the camera and microphone are positioned properlyEnsure that the room is well lit and does not contain any distractions and that only professional objects are visibleEnsure privacy during a virtual video visit:Mute your mobile phone or place it on vibrateClose the office door and place a “Do Not Disturb” or “Doing Video Visits” signEnsure beforehand that you have access to the patient charts, results, and any other health informationMute the microphone until the videoconference startsEnsure that the patient’s telephone number is available should it be necessaryDuring the virtual video visit:Confirm that both parties’ videoconferencing technology is working and optimized for sound and videoIntroduce yourself and bring your photo ID close to the camera for the patient to seeAsk the patient to show a valid photo ID Obtain verbal consent for the virtual visitAsk the patient to ensure their room is well lit and has enough space for movementAsk the patient to ensure privacy during the virtual visit:Mobile phone(s) are placed on mute or vibrateDoors are closed and indicate “Do Not Disturb”Ask the patient to adjust their position so they are framed adequately within the camera viewEnsure that the patient has all objects and tools necessary for the full assessmentIntroduce any other health care providers or learners present in the roomAfter finishing the virtual video visit:Ensure that all reports and clinical documentation are completeEnsure that the planned investigations and medication prescriptions are orderedEnsure that follow-up plans have been madeVirtual care will become a reality in many future medical practices, and the COVID-19 pandemic is serving as a catalyst for this impending change. Virtual care is effective at removing barriers to access, especially to specialists who are typically located in larger centers, for the remarkably distributed rural and remote patients across Canada. As we have outlined, there are important nuances in the application of virtual care that help ensure patients receive at least equivalent care, or in some ways, more effective care than that received in the traditional in-person format. Many clinical organizations are working toward establishing baseline principles, guidelines, and protocols to guide Canadian physicians. This paper is intended to contribute to an emerging Canadian body of knowledge and to support optimal professional practice in an emerging and rapidly evolving field. Most importantly, virtual care should not fragment care. It should support both high-quality care, the foundation of which is an established trust-based physician-patient relationship, and continuity of care."}
{"text": "Despite widespread interest in the use of virtual  visits for ambulatory patient care during the COVID-19 pandemic, studies examining their adoption during the pandemic by race, sex, age, or insurance are lacking. Moreover, there have been limited evaluations to date of the impact of these sociodemographic factors on the use of telephone versus video visits. Such assessments are crucial to identify, understand, and address differences in care delivery across patient populations, particularly those that could affect access to or quality of care.The aim of this study was to examine changes in ambulatory visit volume and type  by patient sociodemographics during the COVID-19 pandemic at one urban academic medical center.We compared volumes and patient sociodemographics  for visits during the first 11 weeks following the COVID-19 national emergency declaration  to visits in the corresponding weeks in 2019. Additionally, for visits during the COVID-19 study period, we examined differences in visit type  by sociodemographics using multivariate logistic regression.Total visit volumes in the COVID-19 study period comprised 51.4% of the corresponding weeks in 2019 . Although patient sociodemographics between the COVID-19 study period in 2020 and the corresponding weeks in 2019 were similar, 60.5%  of the visits were virtual, compared to 0% in 2019. Of the virtual visits, 61.2%  were video based, and 38.8%  were telephone based. In the COVID-19 study period, virtual (vs in-person) visits were more likely among patients with race categorized as other (vs White) and patients with Medicare  insurance and less likely for men, patients aged 0-17 years, 65-74 years, or ≥75 years (compared to patients aged 18-45 years), and patients with Medicaid insurance or insurance categorized as other. Among virtual visits, compared to telephone visits, video visits were more likely to be adopted by patients aged 0-17 years (vs 18-45 years), but less likely for all other age groups, men, Black (vs White) patients, and patients with Medicare or Medicaid  insurance.Virtual visits comprised the majority of ambulatory visits during the COVID-19 study period, of which a majority were by video. Sociodemographic differences existed in the use of virtual versus in-person and video versus telephone visits. To ensure equitable care delivery, we present five policy recommendations to inform the further development of virtual visit programs and their reimbursement. The COVID-19 pandemic has significantly altered the landscape of health care delivery. One of the major changes resulting from the pandemic has been the rapid adoption of virtual  visits and other telemedicine programs that facilitate health care services via health care information technologies to accommodate necessary reductions in in-person care . A majorUnfortunately, access to virtual visits may not be equitable in the United States. Differential access to the internet and devices and differences in health literacy may leave patients without the ability to attend video visits. Thus, those patients may only be able to participate in telephone visits if they are unable to attend in-person visits. Surveys by the Pew Research Center in 2019 found lower rates of internet usage and smartphone ownership among people ages ≥65 years compared to younger adults ,5. When There is already existing evidence that other recent innovations in health care technology may exacerbate differences in health care access. For example, patient portal use, which has the potential to improve the quality and efficiency of health care delivery, differs with respect to race, insurance, and neighborhood broadband internet access . One stuDespite widespread interest in the use of virtual visits for ambulatory patient care during the COVID-19 pandemic, few studies to date have evaluated the adoption of ambulatory virtual visits during the pandemic by age, race, sex, or insurance . The stuIn this study, we aimed to (1) assess changes in visit volume, type, and patient sociodemographics from the start of the COVID-19 national emergency to the end of May 2020, compared to the same weeks in 2019; and (2) elucidate differences in the use of ambulatory virtual visits (as compared to in-person visits) and, for those using virtual visits, the use of video visits (compared to telephone visits) by age, sex, race, and insurance. We hypothesize that (1) total visit volumes decreased and virtual visits increased during the COVID-19 pandemic, while patient sociodemographics remained similar between the two time periods; and (2) patients who utilized in-person visits during the COVID-19 study period were more likely to be younger than patients who utilized virtual visits, and of those using virtual visits, patients utilizing video visits were more likely to be younger, White, and have commercial insurance than patients utilizing telephone visits -23.The University of Chicago Medical Center (UCMC) is the flagship institution of University of Chicago Medicine, and includes 5 multispecialty faculty ambulatory practice sites in Chicago, IL, and the surrounding area, with over 600,000 encounters per year. UCMC began offering virtual visits in March 2020 in response to the widespread shelter-in-place orders at the city, state, and regional level due to the COVID-19 pandemic. Telephone visits began during the week of March 15, 2020. Video visits began with a pilot program in the hematology/oncology, pediatrics, psychiatry, gastroenterology, and obstetrics/gynecology practices on March 26, 2020, followed by a broad roll-out to all ambulatory faculty clinics on April 6, 2020. All practices used a HIPAA -compliant Zoom platform to enable video visits, which was not integrated into the institution’s electronic health record system (Epic) during the evaluated time period.Immediately after the City of Chicago and State of Illinois shelter-in-place orders were enacted, patients with previously scheduled in-person office visits were contacted and given the option to either reschedule or convert their appointment to a virtual visit. If a patient agreed to a virtual visit, a video visit was encouraged. Patients scheduled for video visits were sent the following through the patient portal or email: a Zoom link for the video visit; a brief prevideo visit checklist followed by more detailed instructions describing the technical requirements to participate in the video visit; and a link to a video highlighting methods to best prepare for the video visit and a demonstration of what to expect. If the patient was unable or unwilling to participate in a video visit, a telephone visit was scheduled, and they were told to expect a call from their provider at the scheduled appointment time. Patients reaching out to schedule new virtual visits were also preferentially offered video visits but were given the opportunity to schedule a telephone visit as well in accordance with their preferences. The availability of virtual visits was marketed widely to our patient population through our patient portal, marketing emails, and our health system’s internet home page. Beginning on May 1, 2020, patients were given the option to begin self-scheduling video visits (but not telephone visits) through the patient portal.All adult and pediatric outpatient clinic visits occurring in UCMC faculty practice locations from March 15 to May 31, 2019, and March 15 to May 31, 2020, were included. The type of outpatient clinic visit was classified as in-person or virtual, and virtual visits were further classified as telephone or video, based on the scheduled visit type for all completed visits. Patient sociodemographic data were examined for each visit, including age, sex, race, and insurance. Age was categorized into 5 groups: 0-17 years, 18-45 years, 46-64 years, 65-74 years, and ≥75 years. Patients were grouped into 3 racial categories: White, Black, and other . Insurance was categorized as Medicare (including Medicare-Medicaid Alignment Initiative), Medicaid, commercial, or other. The data were extracted from the institution’s electronic health record data warehouse. This project received a formal determination of Quality Improvement according to institutional policy. As such, this initiative was not reviewed by the Institutional Review Board.First, we used descriptive statistics to examine weekly and overall visit volumes during the study period, which were the 11 weeks following the COVID-19 national emergency declaration , compared to visit volumes in the corresponding weeks of the 2019 calendar year. Next, we examined visit type  and patient sociodemographics  associated with the visit and compared these characteristics to those visits occurring during the same date range in 2019. Last, we examined differences in ambulatory visit type  by patient sociodemographics  for visits occurring during the COVID-19 study period.P≤.001. To estimate the association between patient sociodemographics and visit type , we performed logistic regression. Results were similar between unadjusted and adjusted analyses; only adjusted analyses are presented. Data were analyzed using RStudio, version 3.6.3 .Data were summarized with chi-square tests where appropriate. Because of the large sample size, statistical significance was set at In the week of March 15-21, 2020, the ambulatory visit volume dropped to 34% of visit volumes when compared to the same week in 2019  and reached a nadir of 20.8% of 2019 levels  in the following week. By the week of May 24-30, 2020, the ambulatory visit volume had rebounded to 81.8% of the volume of the same week in 2019 . Total visit volumes from March 15 to May 31, 2020, were 51.4% of 2019 volumes .Virtual ambulatory visits increased from 0 to 48,475 visits between March 15 to May 31, 2020, and comprised 60.5% of total ambulatory visit volume, with the remaining 39.5%  conducted in person . Among vIn unadjusted analyses, there were statistically significant differences between those who received in-person and virtual visits for all sociodemographics examined . In adjuIn unadjusted analyses, there were statistically significant differences across all sociodemographics examined except sex between those using telephone versus video visits . In adjuTotal visit volumes in the COVID-19 study period were approximately half of that in 2019, although patient sociodemographics were similar. Recovery of clinic volumes after the escalation of the pandemic was largely driven by virtual ambulatory care, which comprised over 60%  of total ambulatory clinic volumes from March 15 through May 31, 2020, a majority of which were video visits. Children, adults ≥65 years, men, and patients with Medicaid coverage were less likely to have virtual visits, whereas patients with Medicare coverage were more likely to have virtual visits compared to patients with commercial insurance coverage. For those who attended virtual visits, children were more likely to have video visits, while adults ≥46 years, men, Black patients, and patients with Medicare or Medicaid coverage were less likely to have video visits.The sociodemographic differences in virtual visits we identified are in line with prior research. For example, prior research found that women were more likely than men to shelter in place due to concerns about the risk of COVID-19 infection for themselves and their family; this would make virtual visits a more appealing visit type for women . AdditioThe sociodemographic differences in virtual (vs in-person) visits and video (vs telephone) visits illustrate the digital divide . The patThe shift in the delivery of ambulatory care through virtual visits was incentivized by the new virtual reimbursement policies from CMS and private insurance companies. The significant contribution of virtual visits to overall ambulatory visit volumes is likely to continue once the COVID-19 pandemic has ended. The volume of virtual ambulatory visits at UCMC has continued to grow even after the end of the study period, indicating sustained interest in virtual visits likely due to continued safety concerns related to the pandemic, ongoing reimbursement for these services, and physician and patient satisfaction with this new option for care delivery ,28. Givemaintaining reimbursement parity between video and telephone visits. Second, given the rapid growth and early success of virtual visits, and the role they will likely play in blended models of care, legislation that makes virtual visit reimbursement permanent is essential to allow for the long-term investment by health care systems and providers needed to improve the virtual visit infrastructure and experience. Third, government insurers and specialty societies should collaborate to establish guidance to help distinguish ambulatory care best suited for virtual versus in-person care. Fourth, quality improvement initiatives should be undertaken at medical institutions to support and improve access to and usability of video visits in populations encountering the greatest barriers to its use. Last, advocacy for policy changes and more universal broadband access are essential to help close the digital divide experienced by our most vulnerable patient populations, which would help address the differential access to virtual visits described in this study.The results of this study and our review of the virtual visit landscape has prompted us to offer five recommendations . First, Maintain reimbursement parity between video and telephone visitsPass legislation making virtual visit reimbursement permanentEstablish guidance to distinguish ambulatory care best suited for virtual versus in-person carePerform quality improvement initiatives to improve access to and usability of video visits in vulnerable populationsAdvocate for policy changes and universal broadband access to close the digital divideOur study has limitations. First, this study only examined a single medical center and was a retrospective analysis; despite this, the diversity of the patient population examined in our study enabled our analysis of ambulatory virtual visit use. Second, our study only examined a limited set of variables, which were used as surrogates for the social determinants of health described in this paper, such as access to broadband internet, health literacy, tech literacy, education, and income, and did not examine virtual and video visit use by ethnicity due to limited data availability. Third, this area of clinical practice and study is rapidly changing and will likely continue to change rapidly over the next few months to years. Further studies at other medical institutions should be conducted to confirm our findings and examine additional sociodemographic variables. Future analyses of ambulatory virtual visits should also investigate patient satisfaction and outcomes by patient visit type , given the differences in reimbursement by visit type category, and whether ambulatory virtual visits increase the geographic area served by academic medical centers or medical institutions with subspecialty care, as already suggested by limited data .The COVID-19 pandemic has drastically changed the health care delivery landscape largely due to the growth of ambulatory virtual visits, which have rapidly become a vital component of health care delivery. Given the differential use of these technologies by age, sex, race, and insurance, these changes also risk perpetuating and even exacerbating existing disparities in health care access and quality, especially if reimbursement policies do not sufficiently account for these differences and the digital divide remains unaddressed."}
{"text": "Hints and Kinks, we discussed the role of causal inference in tasks of health services research (HSR) using examples from health system interventions  as a health system intervention.In a previous would not have been implemented. Note that we never observe one of the two situations, because either the HM is implemented or not, but not both. We now introduce a formal notation for causal inference which allows us to mathematically define a causal effect.We introduced counterfactuals as hypothetical outcomes which are actually not observed in a real-world setting Hernán . We usedYnoHM) together with the outcome under the HM (denoted as YHM). The superscripts denote the counterfactual outcomes we can formalize, but which are actually not observed: Only YHM can be observed if the HM is implemented. An average causal effect in the study population can then be defined by the risk difference Probability(YHM  = 1)–Probability(YnoHM  = 1), abbreviated as RDCausal. Note that we could also use other risk measures, for example a relative risk, for the definition of a causal effect. The choice of the used effect measure depends on the research question because the underlying scale  influences its final interpretation  if the HM had not been implemented . For example, the patient with ID 5 was treated in the HM region with no observed hospital readmission (YObserved = 0). The observed outcome is equal to the counterfactual outcome in the HM region (YObserved = YHM = YHM = 0). Note that if this patient would have been treated in the control region, he or she would have had a readmission (YnoHM  = 1). Because this patient is actually only observed in the HM region, one will never observe the outcome of the control region (YnoHM is missing). The mathematical notation for counterfactuals might be initially confusing, yet it is a necessary component for a causal inference framework.An important question remains: How can we assess an effect measure based on outcomes which are actually not observed? One could compare the outcomes in the region with HM to outcomes in a 'control' region with no HM. Table Causal is zero, because Probability(YHM = 1) = 3/5 and Probability(YnoHM  = 1) = 3/5. Thus, the HM does not reduce hospital readmissions.What is the average causal effect in the study population from Table associational effect measure generally compares risks in subsets of a study population by conditioning on certain study characteristics  and the second expression 1/3 . Thus, RDAssociational is equal to 0–1/3 = –1/3, i.e., the risk of hospital readmissions in the HM region is lower compared to the risk in the control region.An see Fig.  (Hernán see Fig. . In the mentclass2pt{minimCausal and the associational effect RDAssociational leads to the famous ‘association is not causation’ adage. Likely because of this adage, many researchers in HSR avoid any causal terminology, especially when they use ‘only’ observational data (Hernán The difference between the derived causal effect RDa Hernán . They arHow can researchers integrate ‘causality’ in HSR? Our above introduced components of a framework for causal inference is the backbone for modern causal inference. Modern causal inference allows for inference which mimics a situation as if patients would have been assigned by random allocation, despite using an observational study design. Topics for recent calls of causal inference approaches in HSR include, for example, comparative effectiveness research, payment scheme evaluations, health care utilization or the use of simulation studies (see Table Hints and Kinks, we introduced components for a principled framework for causal inference in HSR. Because ‘causal inference’ is conceptually different from ‘description’ or ‘modeling’, HSR needs the integration of a causal inference framework which includes a specific notation, definitions and analysis techniques to extend the traditional tasks of ‘description’ and ‘modeling’. Public health decision-making which solely relies on associational effect measures might lead to inappropriate decisions because questions about optimal decision-making are inherently causal. We plea that students and researchers in the field of HSR are aware of the different available frameworks to successfully address ‘description’, ‘modeling’ and ‘causal inference’, depending on the intended research question.In the present"}
{"text": "Hints and Kinks, we explain why a solidly principled causal inference framework should be integrated into the tasks of HSR.In a recent issue of the American Journal of Public Health, Hernán and other colleagues strongly plea for causal thinking in scientific research where the research question investigates consequences of decisions and interventions Ahern . Hernán Table The first example in Table The second example in Table would not have been implemented, i.e., outcomes which are actually never observed. Counterfactuals allow to mathematically define a causal effect, which is conceptually different from an associational effect. In a follow-up Hint and Kinks, we continue on the introduction of a principled framework for causal inference in HSR (Moser et al. Unfortunately, public health decisions on interventions or policies are often only based on ‘descriptive’ and ‘modeled’ results, without the integration of a solidly principled causal inference framework. Despite both, ‘traditional’ and ‘causal inference’, approaches have their proper legitimation in the investigation of specific research questions, many researchers and students in the field of HSR are not trained to the notions of causal inference. Modern causal inference identifies effects of interventions by using the concept of counterfactuals, which are a key component of a causal inference framework Hernán . A countAs Hernán and others, we believe that causal reasoning plays an important role in HSR to support public health decision making. This can only be done by using an explicit ‘causal inference’ framework, besides the traditional tasks of ‘description’ and ‘modeling’. None of these tasks should be viewed superior to another, but each should be adequately chosen, related to the intended research question (Hernán et al."}
{"text": "Coronavirus disease 2019 (COVID-19), the most hectic pandemic of the era, is increasing exponentially and taking thousands of lives worldwide. This study aimed to assess the prevalence of pre-existing comorbidities among COVID-19 patients and their mortality risks with each category of pre-existing comorbidity.To conduct this systematic review and meta-analysis, Medline, Web of Science, Scopus, and CINAHL databases were searched using pre-specified search strategies. Further searches were conducted using the reference list of the selected studies, renowned preprint servers , and relevant journals’ websites. Studies written in the English language included if those were conducted among COVID-19 patients with and without comorbidities and presented survivor vs non-survivor counts or hazard/odds of deaths or survivors with types of pre-existing comorbidities. Comorbidities reported in the selected studies were grouped into eight categories. The pooled likelihoods of deaths in each category were estimated using a fixed or random-effect model, based on the heterogeneity assessment. Publication bias was assessed by visual inspection of the funnel plot asymmetry and Egger’s regression test. Trim and Fill method was used if there any publication bias was found.A total of 41 studies included in this study comprised of 27 670 samples. The most common pre-existing comorbidities in COVID-19 patients were hypertension (39.5%), cardiovascular disease (12.4%), and diabetes (25.2%). The higher likelihood of deaths was found among COVID-19 patients who had pre-existing cardiovascular diseases (odds ratio (OR) = 3.42, 95% confidence interval (CI) = 2.86-4.09), immune and metabolic disorders , respiratory diseases , cerebrovascular diseases , any types of cancers , renal , and liver diseases .This study provides evidence that COVID-19 patients with pre-existing comorbidities had a higher likelihood of death. These findings could potentially help health care providers to sort out the most susceptible COVID-19 patients by comorbidities, take precautionary measures during hospitalization, assess susceptibility to death, and prioritize their treatment, which could potentially reduce the number of fatalities in COVID-19. The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus from the large coronavirus family, was first detected on December 31st, 2019 in Wuhan, China, and has now been deemed a global pandemic by the World Health Organization (WHO). The virus is highly transmissible  [The virus is equally transmissible in all ages; however, people who are now in critical conditions or who have died were more likely to be in the elderly population and had one or more morbidities -11. CommThis systematic review and meta-analysis was conducted by following the Preferred Reporting for Systematic Review and Meta-Analysis (PRISMA) consensus statement . StudiesFour databases: Medline, Web of Science, Scopus, and CINAHL, were searched using pre-specified search strategies for each database and concluded on May 01, 2020. The search strategy consists of keywords on COVID-19 disease , pre-existing morbidity , and patients’ survival status  combined using the Boolean operators . Details of the search strategies are presented in Table S1-S4 in the All peer-reviewed and preprint (not-peer-reviewed) studies met the pre-specified inclusion criteria were included in this study.Studies were included if they met the following inclusion criteria: (i) conducted for the hospitalized patients infected with COVID-19 with or without pre-existing comorbidities, (ii) presented survivor and non-survivor counts following COVID-19 among patients with or without pre-existing morbidity or presented hazard/risk/odds ratio of deaths or survival following COVID-19 with the types of morbidities, and (iii) published in the English language. Studies without complete information but met our inclusion criteria were included in the narrative review.Studies were excluded if COVID-19 was reported among pregnant women or children (aged <18 years) because there are additional conditions or morbidities existing in these groups, such as pregnancy complications. We also excluded papers written in languages other than English. Additionally, we excluded review papers, correspondence, viewpoints, editorials, commentaries, and studies where no information related to previous morbidities were reported.All articles identified through the specified search strategy that were compiled and screened in Endnote version X9 . Two investigators  independently screened the included articles based on inclusion and exclusion criteria. Initially, the titles and abstracts of the included studies were screened for a full-text review. Finally, the full-text of each of the selected studies were reviewed by the same investigators. Any conflict of interest faced by the investigators was reported to the senior authors (MNK and MIK) and solve based on the group discussion.A data extraction form was designed, trialed, and modified to extract information from the selected studies. Two authors (MMAK and MGM) used the pre-designed form to extract information independently. The following information was extracted: study location, design, sample size, study population characteristics , and survivor vs non-survivor counts among COVID-19 patients with or without specific comorbidities. If available, the odds/risk/hazard ratio of deaths among COVID-19 patients with comorbidities were extracted against the types of morbidity. Disagreements reported in data extraction were reviewed and solved by the corresponding and senior authors (MNK and MIK). The Modified Newcastle-Ottawa scale, as part of the data extraction strategy, was used to assess the quality of selected studies . The sca2I statistics) was moderate (50%-74%) or high (≥75%), the pooled estimates of ORs were computed using the random-effects model [One or more pre-existing comorbidities among COVID-19 patients reported in the selected studies were grouped into eight broad categories based on the type of comorbidities. These were cardiovascular system diseases , immune and metabolic disorders , respiratory system diseases , acute respiratory distress syndrome, tuberculosis, etc.), cancer , cerebrovascular diseases , renal system diseases , liver system diseases , and gastrointestinal system diseases . The case definition of these comorbidities is presented in ts model . Subgrouts model . When evts model . Stata sA total of 247 articles were identified from the databases searched, and the additional 15 articles were identified by checking the reference list of the selected articles and the selected journals’ websites (A summary of the 41 selected articles is represented in Distribution of the type of morbidity presented in 2I = 81.2%) than the patients who had no cardiovascular diseases. The odds of death among COVID-19 patients with immune and metabolic disorders were also found to be 246% higher  than among COVID-19 patients without such disorders. The incidence of COVID-19 among people with respiratory diseases increases mortality risk around two times  more than COVID-19 patients without respiratory system diseases. Similarly, we found higher mortality risk among COVID-19 patients who had pre-existing any types of cancers  and cerebrovascular system diseases  more than their counterparts. Moreover, the incidence of COVID-19 among patients with pre-existing renal disease and chronic liver disease increased mortality risk by about three times  more and one and half times  more, respectively compared to the COVID-19 patients who did not have such comorbidities.The pooled ORs of deaths for each category of pre-existing comorbidities among COVID-19 patients, publication bias, and Trim and Fill estimates are presented in We found evidence of publication bias for the four categories of pre-existing comorbidities: cardiovascular diseases, cerebrovascular diseases, renal system diseases, and liver diseases  for cardiovascular diseases (81.2%) and respiratory diseases (75.7%). To examine the sources of heterogeneity, we conducted stratified analysis across types of comorbidities, study design , sample size , age of the total sample (divided based on mean age and classified as ≤60 years and >60 years), and age at death (divided based on mean age and classified as ≤70 vs >70) , diabetes (25.2%), and cardiovascular disease (12.4%). The likelihood of death was higher among COVID-19 patients who had comorbidities like cardiovascular diseases, cerebrovascular diseases, respiratory diseases, renal diseases, immune and metabolic disorders, hepatic diseases, and cancer. This evidence will guide physicians to take precautionary measures, which could reduce the number of fatalities following a secondary infection with COVID-19.Among the total positive COVID-19 cases included in this systematic review, around 43% had one or more pre-existing comorbidities, mostly cardiovascular diseases, and immune and metabolic disorders. These underlying diseases are increasing the risk of death among COVID-19 patients. Recent reviews have confirmed that COVID-19 infection among patients with cardiovascular diseases, and immune and metabolic disorders, particularly, having hypertension and diabetes elevate the risk of infection severity, ICU admission, and the risk of death . The undPooled likelihoods in this study provide evidence of higher deaths among COVID-19 patients who had pre-existing chronic respiratory diseases or any type of cancer- as found in other reviews . The chrThis study also suggests that patients with cerebrovascular, liver, and renal diseases are more vulnerable to mortality following the secondary incidence of COVID-19 than patients who do not have such diseases. The results are comparable to deaths among previously reported SARS patients . DiseaseThis study has several strengths and limitations that should be reported. To our knowledge, this is the first study of its kind that summarizes all morbidities among COVID-19 patients that lead to death using data from globally representative studies. Moreover, comorbidities reported among COVID-19 patients were classified into board groups based on their characteristics, and the likelihood of death was estimated separately for each group. This evidence will inform health care providers about the risk of death among COVID-19 patients with different groups of pre-existing comorbidities. Thus, they will be able to take precautionary measures early targeting to prevent deaths. However, this study reported the odds of death for COVID-19 patients with one pre-existing comorbidity only. Many COVID-19 patients may have multi-morbidities (COVID-19 with pre-existing two or more comorbidities), and they may have a higher risk of death. The studies included in this review considered each morbidity separately; for instance, if COVID-19 patients had both hypertension and diabetes, they were included in both groups. None of the included studies considered COVID-19 patients with two or more comorbidities together; therefore, we failed to provide the likelihood of deaths for higher risk patients. Moreover, the percentage distribution of morbidity among COVID-19 patients estimated in this study could be different from the actual situation depending upon the counts in the included studies. However, as we included all available studies conducted worldwide, therefore, any distortion is likely to be random. In addition, the likelihoods presented in this study were mostly unadjusted (31 of the 35 articles included) as they were calculated from the extracted raw data. This may overestimate or underestimate the actual likelihood of deaths in COVID-19 patients because age and other socio-demographic characteristics are potential confounders of their deaths, which should be adjusted for unbiased estimates. Despite these limitations, this study is unique and beneficial for health care providers to handle COVID-19 patients with pre-existing comorbidities.About 43% of the sample included in this systematic review had one or more pre-existing comorbidities and had COVID-19 as a secondary infection. The most common pre-existing comorbidities were hypertension, diabetes, and cardiovascular disease. The likelihood of death was higher among COVID-19 patients who had pre-existing cardiovascular and cerebrovascular diseases, respiratory diseases, renal diseases, immune and metabolic disorders, liver diseases, and any types of cancer. These findings will help health care providers to sort COVID-19 patients by comorbidities, take precautionary measures during hospitalization, assess susceptibility to death, and prioritize their treatment. These could potentially reduce the number of fatalities from secondary infection with COVID-19 disease."}
{"text": "The method was selective and linear over a concentration range of 0.5–50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of  Streptomyces hygroscopicus in Easter Island + at m/z 931.5 and 809.5, respectively, which are frequently observed in ammonia-mediated ionization mass spectra of compounds with oxygen groups  in their structures [m/z 864.4 for sirolimus and m/z 756.5 for IS, which is in accordance with previous findings [m/z 864.4 of sirolimus was likely formed due to the separation of an -OCH3 and two H2O, whereas the product ion at m/z 756.5 of the IS was potentially the result of three H2O separation. The declustering potential, collision energy, and collision cell exit potential were optimized to obtain maximum responses of sirolimus and the IS (m/z 931.5 → 864.4 for sirolimus and m/z 809.5 → 756.5 for the IS. An ammonium formate solution (5 mM) was employed as a mobile phase component to induce the ammonium adducts of sirolimus and the IS. Several mobile phase compositions and reverse phase columns were tested to produce peaks of sirolimus and the IS with adequate retention time and separation. Peaks of sirolimus and IS were not appropriately observed with isocratic modes; thus, gradient modes were used. Finally, a mixture of ammonium formate buffer (5 mM) with 0.1% formic acid (A) and acetonitrile (B) in gradient mode  and a SynergiTM 4 μm polar-RP 80A column were used to obtain optimized peaks of sirolimus and the IS.The chemical structures of sirolimus and ascomycin  are depicted in ructures . In the findings ,34. The d the IS . ThereafTypical chromatograms of blank porcine whole blood/lung tissue, zero calibrators, a sirolimus standard, and a porcine sample from the pharmacokinetic studies are shown in 2 = 0.99317 ± 0.00150  least squares regression analysis), where y, x, and R refer to peak area ratio (sirolimus/IS), sirolimus concentration in whole blood, and the correlation coefficient, respectively. The calibration curves (peak area ratio versus concentration) for sirolimus in porcine lung tissue were linear over a range of 0.5–50 ng/mL with the developed analytical conditions. Using the weighted (1/x) least squares regression analysis, the calibration equation for sirolimus was y = (0.05056 ± 0.00356) x + (0.00003 ± 0.00553), with R2 = 0.99792 ± 0.00162 , where y, x, and R refer to peak area ratio (sirolimus/IS), sirolimus concentration in lung tissue, and the correlation coefficient, respectively. The weighted (1/x) least squares regression analysis demonstrated the acceptable precision and accuracy of the corresponding calculated concentrations at each level for both whole blood and lung tissue. The precision was 4.33–13.26% for whole blood and 1.66–10.52% for lung tissue, whereas the accuracy was 90.42–107.63% for whole blood and 94.06–102.29% for lung tissue. Thus, the response (sirolimus/IS peak ratio) was directly proportional to the whole blood/lung tissue concentration of sirolimus, indicating the linearity of the LC-MS/MS assay in the range of 0.5–50 ng/mL.Under the developed analytical conditions, the calibration curves for sirolimus in porcine whole blood were linear over a concentration range of 0.5–50 ng/mL. The calibration equation for sirolimus was y = (0.05227 ± 0.00373) x + (0.00766 ± 0.00251), with RThe lower limit of quantification (LLOQ) for the LC-MS/MS assay was established at a concentration of 0.5 ng/mL. In porcine whole blood, the LLOQ showed an accuracy of 95.25% and a precision of 14.14%, whereas, in porcine lung tissue, the LLOQ showed an accuracy of 110.79% and a precision of 1.85% . The sigQuality control (QC) samples were analyzed in five replicates over five different days to determine the intra-day and inter-day accuracy/precision. Four levels of sirolimus in porcine whole blood/lung tissue were investigated, including LLOQ (0.5 ng/mL), low QC , middle QC , and high QC . The extraction recovery was evaluated by comparing the peak responses of sirolimus QC samples and the IS with those of blanks spiked with the analytes after extraction at the same concentrations. The extraction recovery values were similar for all levels of QC within the same type of sample matrix . They raThe matrix effect was investigated in three different porcine lung tissue samples . The peaThe relative matrix effect  was assessed by a direct comparison of the peak areas among samples in the same QC level (set 1). For porcine whole blood samples, the precision of set 1 was in the range 0.77–8.43%, which was comparable with that of set 2. For lung tissue samples, the precision ranged from 1.15% to 6.91%, slightly narrower than the values from set 2. These results indicate that there was no significant effect of the matrix for the analysis of sirolimus and IS in porcine whole blood and lung tissue samples using the developed LC-MS/MS method.At short-term storage conditions , stock solutions of sirolimus (50 ng/mL) and the IS (1 ng/mL) showed the stability of 100.80% and 101.58% compared to fresh-prepared solutions, respectively. After long-term storage at −80 °C for 3 months, the stability was 101.26% for sirolimus and 95.91% for the IS. These data indicate that sirolimus and the IS are stable in the stock solution during both short- and long-term storage.The stability of sirolimus was investigated in porcine whole blood and lung tissue at three different QC  levels over various typical handling and storage conditions . The posCarry-over study was conducted to measure residual analyte from a preceding sample that remains in the analytical instrument. Obtained results for carry-over of sirolimus and IS are shown in max) was 7.79 ng/mL, and the Tmax was 2.26 hr. The systemic exposure up to the last time point  was 156.9 ng/mL·h, and the AUCinf was 164.8 ng/mL·h , ascomycin , formic acid, and ammonium formate were purchased from Sigma-Aldrich . HPLC-grade acetonitrile and water were supplied by Honeywell Burdick & Jackson . All other reagents were of analytical grade and were used without any further purification.TM 4 μm polar-RP 80A column  equipped with a SecurityGuardTM column (4.0 mm × 3.0 mm) was utilized to optimally separate sirolimus and the IS from endogenous substances of porcine whole blood and lung tissue. The mobile phase was a mixture of ammonium formate buffer (5 mM) with 0.1% formic acid (A) and acetonitrile (B). Sample separation was conducted using a flow rate of 0.2 mL/min and an 8.5-min gradient condition . Temperatures of the column and the autosampler were maintained at 30 °C and 4 °C, respectively. The injection volume was 10 μL for whole blood and 5 μL for tissue samples. The SRM transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and IS, respectively. Mass data were acquired using Analyst software version 1.5.2 . Data analysis was processed using SCIEX OS offline software version 1.6 . The working parameters of the LC-MS/MS system are listed in Sirolimus was analyzed using an LC-MS/MS system consisting of an AB SCIEX Triple Quad 5500 mass spectrometer  equipped with a turbo ion spray interface in positive ionization mode, an Agilent LC 1200 Binary pump system , and a CTC analytics autosampler . Ascomycin was used as an IS. A Synergiw/w) cold phosphate-buffered saline (PBS) was added to process the homogenization. Tissues were kept in the ice-bath during the homogenization process, and tissue homogenates were stored at −20 °C until use.Blank lung tissue and pharmacokinetic experimental lung tissue samples were homogenized using the KT 30 homogenizer . Lung tissues were accurately weighed, and four-fold  was appropriately diluted with acetonitrile to obtain sets of working standard solutions (5–500 ng/mL). All the stock and working standard solutions were stored at −80 °C until experimental analysis. Standard samples were prepared in whole blood by spiking 90 μL of blank porcine blood with 10 μL of each working standard solution. Similarly, standard samples in porcine lung tissues were prepared by spiking 15 μL of each working standard solution into 135 μL of blank lung tissue homogenate. The final sirolimus concentrations in blood and lung tissue homogenate were 0.5, 1, 2, 5, 10, 20, and 50 ng/mL. Extraction of the drug and IS was performed using a protein precipitation method as previously described ,39. To eThe LC-MS/MS method for sirolimus analysis was validated according to the bioanalytical method validation guidelines of the US FDA in 2018, as previously described ,40.Blank whole blood and lung tissue homogenates from six or three animals, respectively, were spiked with the IS only or with both sirolimus and IS. Chromatograms of blank blood/lung tissue, blood/lung tissue spiked with the IS only, and blood/lung tissue spiked with both sirolimus and IS were then compared for selectivity of the assay.2 values of calibration curves were used to evaluate the linearity of the assay, using the criteria of ≥ 0.990. Accuracy and precision were evaluated during linearity runs using the deviation from the nominal concentration (RE%) and the CV%, respectively. Criteria of the accuracy and precision of the assay were set within ± 15%, except for the LLOQ, which were set at ± 20%.The linearity of the assay was assessed using the standard samples prepared with sirolimus blood and lung tissue homogenates over the concentration range of 0.5–50 ng/mL. Calibration curves were constructed using the peak area ratios of sirolimus and IS by weighted (1/x) linear regression analysis. RThe LOD was determined by the equation: LOD = 3.3 σ/m, where σ is the standard deviation of the intercept of the regression line, and m is the slope of the calibration curve. LOD was also experimentally determined using a signal-to-noise ratio of ≥ 3. The LLOQ, which is the lowest concentration of sirolimus that can be quantitatively determined with a precision of less than or equal to 20% and accuracy between 80% and 120%, was determined with a signal-to-noise ratio criterion of ≥ 10.QC samples at four different concentrations of 0.5 (LLOQ), 1.5 (LQ), 15 (MQ), and 40 (HQ) ng/mL were used to assess accuracy and precision. For intra-day accuracy and precision, five replicates of QC samples were analyzed within one day. The QC samples were analyzed in five replicates over five different days to examine the inter-day data. The acceptance criteria were set at below ± 15% of RE and within ± 15% of CV for accuracy and precision, respectively, except at the LLOQ, which were set at ± 20%.The extraction recovery of the analyte was assessed to ensure that the sample extraction process is efficient and reproducible. Peak areas of extracted samples at LQ, MQ, and HQ concentrations with those of blanks spiked with sirolimus post-extraction were compared. The matrix effect was evaluated using three sources of blank whole blood or lung tissue homogenates matrix to determine whether the endogenous components of whole blood or lung tissue affect the ionization of sirolimus and IS. To determine the absolute matrix effect, mean peak areas of post-deproteinization samples (set 1) with those of neat solutions of compounds (set 2) in acetonitrile at equivalence concentrations were compared. The variability in peak areas from set 1, expressed as precision , was determined and considered as the relative matrix effect.The stability of stock solutions of sirolimus and IS was assessed by comparing the peak response of freshly prepared acetonitrile solution with that of the solution stored at room temperature for 4 h or at −80 °C for 3 months of sirolimus (50 ng/mL) and IS (1 ng/mL). The stability of QC samples in porcine whole blood  was investigated in terms of short- and long-term stability, post-preparative stability, and freeze–thaw stability. Short-term stability was assessed by keeping QC samples in laboratory condition (room temperature ~25 °C) for 4 h, and long-term stability was evaluated with QC samples stored at −80 °C for 3 months. The post-preparative stability of processed samples stored in an autosampler at 4 °C was evaluated after 24 h. Freeze–thaw stability was tested over three repeated cycles, whereby QC samples were frozen at −80 °C and then thawed at room temperature. Samples were considered stable at the test conditions if the intensity of stored samples differed by less than 15% from that of freshly prepared samples.Carry-over of sirolimus and IS was assessed by analyzing blank samples after the highest sirolimus standard samples . Then, standard samples at LLOQ (0.5 ng/mL) were also analyzed after the blank samples. The experiment was repeated three times for both blood and lung tissue samples. Carry-over in the blank samples should not be greater than 20% of the sirolimus response at the LLOQ and 5% of the response for the IS.In this study, a total of 27 male Defined Health Status pigs were utilized  . Animals were randomly divided into nine groups following the sacrifice time point . As a form of randomization, animals were assigned to the study in order of their implantation. All animals were handled under the National Institutes of Health guideline, and Animal Care and Use Committee policies of the Yonsei University Cardiovascular Product Evaluation Center . TM scaffolds  were implanted in 24 animals, two each. Three animals were used as blank controls. The scaffold is coated with a single layer of poly  (PDLLA), sirolimus (0.9 μg/mm2), and phosphorylcholine polymer. The total amount of sirolimus carried by the two PhoslineTM scaffolds per animal is 326 μg.For the scaffold implantation, anesthesia was induced with a mixture of 0.02–0.04 mg/kg of atropine, 2.0–8.0 mg/kg of azaperone, 0.1–1.0 mg/kg of xylazine, 5.0 mg/kg of alfaxalone, and 1.0–3.0 mg/kg of tramadol (via intramuscular injection), and maintained with a mixture of approximately 2% isoflurane in oxygen . About 200 IU/kg of heparin was administered intravenously before selective catheterization for the scaffold implantation. A total of 48 PhoslineImplantation was performed in two of the coronary arteries  per animal, as the anatomy allowed. The interventionalist decided on the suitability of the vessels, based on angiographic images of the native vessels. The scaffold was introduced into the artery by advancing the scaffolded balloon catheter through the guide catheter and over the guidewire to the deployment site. Four radiopaque markers at each end of the scaffold facilitated precise placement. After implantation, animals received 100 mg of aspirin and 75 mg of clopidogrel daily until termination.n = 24 at 0, 5 min, and 1 h; n = 21 at 2, 4, and 6 h; n = 18 at 1 d; n = 15 at 2 and 3 d; and n = 12 at 7 d. Then, the blood samples were treated with ethylenediaminetetraacetic acid (EDTA). After sacrifice, lung tissues were explanted and washed with PBS. All tissues and whole blood samples were stored at −80 °C until use.Blood samples were obtained at pre-implantation, post-implantation, and at other time points depending on each group until sacrifice . Thus, the numbers of blood samples were as follows: In this study, an LC-MS/MS bioanalytical assay method was successfully developed and validated for the determination of sirolimus levels in both porcine whole blood and lung tissue. The sample preparation required a simple protein precipitation step for sirolimus extraction. This method exhibited adequate selectivity, linearity, sensitivity, accuracy, and precision. The drug and IS were stable during typical handling and processing conditions. The method was successfully applied for pharmacokinetic and bio-distribution studies of sirolimus following the application of stents in porcine coronary arteries. To the best of our knowledge, this is the first report describing the development and validation of an LC–MS/MS assay of sirolimus in both porcine whole blood and lung tissues. This method can be applied in pharmacokinetic and bio-distribution studies to quantitate sirolimus levels in blood, as well as its distribution to various organs."}
{"text": "The incidence of anal squamous cell carcinoma (SCC) has been steadily growing globally in the past decade. Clinical data on anal SCC from China are rare. We conducted this study to describe the clinical and epidemiological characteristics of anal SCC in China and explore prognostic factors of outcomes among patients with anal SCC.We audited demographic characteristics, relevant symptoms, risk factors, treatment modalities and outcomes for patients diagnosed with anal SCC at 11 medical institutions in China between January 2007 and July 2018.n = 93, 64.6%), noticing a lump , and pain . The proportion of patients at the American Joint Committee on Cancer (AJCC) stages I-IV were 10 (6.9%), 22 (15.3%), 61 (42.4%) and 8 (5.6%), respectively, and AJCC stages in 43 (29.9%) patients were unknown. Thirty-six patients (25.0%) underwent abdominoperineal resection initially. Univariable analysis showed that T stage predicted recurrence-free survival (RFS) , and age group , AJCC stage , and N stage  predicted overall survival (OS).A total of 144 patients  were diagnosed with SCC during this period. Median age at initial diagnosis was 52.0 (interquartile range: 46.0–61.8) years. The most common symptoms were bleeding (T stage was identified as prognostic factor of RFS, and age, AJCC stage, and N stage were identified as prognostic factors of OS. Improving symptom awareness and earlier presentation among patients potentially at risk for anal SCC should be encouraged. Familiarity with the standard treatment among health care providers in China should be further improved. Anal cancer is a malignancy accounting for 1–2% of digestive tract tumours and 2–4% of colorectal and anal tumours . In the Before mid-1980s, the standard treatment for SCC was abdominoperineal resection (APR), however because of several disadvantages, such as permanent stoma, this method as first line therapy was abandoned . In 1974One of the key factors that influence the outcomes of anal SCC is the stage at diagnosis , 20. A sIt is not clear whether the findings from studies carried out in Europe or North America are generalizable to China where literature on anal SCC is scarce. We conducted this study to understand the clinical and epidemiological characteristics of anal SCC and prognostic factors of outcomes in patients with anal SCC in China.We audited anal SCC patients recorded in 11 medical institutions in China between January 2007 and July 2018. , Sun Yat-sen University Cancer Center, The Second Affiliated Hospital of Zhejiang University, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Nanfang Hospital, Southern Medical University, Guangdong General Hospital). Patients with anal SCC were identified using the International Classification of Disease (ICD)-10 codes of anal SCC (C 21.0–21.8) . AdenocaFor all identified patients, medical records were reviewed by author PY Li for demographic information , relevant symptoms , risk factors , information of the tumour , treatment received  and outcomes. AJCC stage was determined according to the American Joint Committee on Cancer, 7th edition .p value less than 0.05 was considered to be statistically significant.Data obtained from the medical records were summarized using descriptive statistical analysis. Frequencies and percentages were used to describe categorical variables and median and interquartile range (IQR) were used to describe continuous variables. Chi-square test was used to compare proportion of categorical variables. For analysis, age was divided into two groups: ≤50 years and >50 years. Tumour size was divided into two groups: ≤20 mm and >20 mm. Recurrence-free survival (RFS) was defined as the interval between diagnosis and recurrence . Overall survival (OS) was defined as the interval between diagnosis and death from any cause or last follow-up , 28, 29.This study was approved by the Ethics Committee of the University of New South Wales (IRB ID: HC180393) and the Ethics Committee of the School of Public Health, Sun Yat-sen University (IRB ID: 2018–026). Because the study was retrospective and the data was de-identified, the informed consent requirement was waived.p = 0.637). When grouped by age at initial diagnosis, 41.7% of patients (n = 60) were less than or equal to 50 years, and 58.3% of patients (n = 84) were older than 50 years.A total of 144 patients were identified with anal SCC diagnosed between January 2007 and July 2018 (Table Only one (0.7%) patient  was recorded as HIV positive. Four (2.8%) patients had a history of other cancers . Two (1.4%) patients were diagnosed with sexually transmitted diseases  3 with syphilis and genital warts) while being diagnosed with anal cancer. One (0.7%) patient had a history of genital warts. Among 113 (78.5%) patients with a record of their sexual behaviors, no one reported a history of receptive anal intercourse or homosexual behavior. Seventeen (11.8%) patients were smokers (including current smokers and ex-smokers).n = 93, 64.6%), noticing a lump , pain , altered bowel habit , perianal itch , and tenesmus . Other symptoms  also have been observed. One patient who was diagnosed by physical examination reported no symptom.Median duration of symptoms until initial diagnosis was 90 days (IQR: 30–180 days). The most common symptoms were bleeding  had a tumour less than or equal to 20.0 mm. There were 13 (9.0%) patients diagnosed at T 1 stage, 40 (27.8%), 28 (19.4%), and 36 (25.0%) at stages T 2–4, respectively. The frequencies and proportion of patients in N0–3 stages were 52 (36.1%), 33 (22.9%), 22 (15.3%) and 8 (5.6%), respectively. The frequencies and proportion of patients in AJCC stages I-IV were 10 (6.9%), 22 (15.3%), 61 (42.4%) and 8 (5.6%), respectively, and AJCC stages in 43 (29.9%) patients were unknown.The median tumour size was 30.0 (IQR: 20.0–40.5) mm. About one in four patients (n = 107) of patients, and radiotherapy to 70.1% (n = 101) of patients. Ninety-seven (67.4%) patients underwent CRT. Thirty-six patients (25.0%) underwent APR initially, of which four, five, eight, and zero patients were in AJCC stage I, II, III, and IV, respectively, and AJCC stages of the remaining patients were unknown. Of the 36 patients who underwent surgery initially, 24 patients had performed only APR, and eight, three, and one patient had performed CRT, chemotherapy, and radiotherapy after APR, respectively. Tumour sites treated with APR were anal canal or anal canal and margin. Fifteen (10.4%) patients were treated with local mass resection. Only two patients underwent local mass resection were diagnosed at stage I, and sites of tumour were anal margin.As for diagnosis and treatment modalities, two patients were misdiagnosed with haemorrhoids and underwent haemorrhoidectomy. Chemotherapy was administered to 74.3% , other regiments, such as fluorouracil plus cisplatin (PF), FOLFOX and Capox were also involved. Among the patients diagnosed at M1 stage, the radiation dose varied from 5400 cGy to 6000 cGy, and the frequency of radiotherapy varied 25 to 30 times. Volumetric intensity modulated arc therapy (VMAT) and intensity modulated radiotherapy (IMRT) were the most often adopted technique. Among these patients, five died of cancer, one survived, and one lost to follow-up.The chemotherapy regimens were not uniform and varied across different medical institutions. Among all the 144 patients, TP, PF and fluorouracil (5-FU) plus mitomycin were the most widely used. FOLFOX, Capox, capecitabine monotherapy, 5-FU monotherapy, and cisplatin monotherapy were also used in some hospitals. Concurrent chemotherapy was applied to more than half of the patients, and the others were treated with induction chemotherapy, adjuvant chemotherapy or both. For radiotherapy, apart from some earlier patients who adopted 3-dimensional conformal radiation therapy (3D-CRT), the rest patients all adopted VMAT or IMRT technique. Radiation dose had a wide range, fluctuating between 3780 cGy and 7000 cGy. The median of radiation dose was 5600 cGy (IQR: 5000–6000 cGy). The frequency of radiotherapy varied from 21 to 35. Every single dose, with maximum at 240 cGy and minimum at 180 cGy, was performed five times per week.p = 0.032; Table p = 0.028), AJCC stage , and N stage  predicted OS (Table Within a median follow-up of 44 months (IQR: 25–67 months), 22 patients died of anal SCC and 25 patients developed a recurrence. Estimated 5-year RFS was 79.4%, and 5-year OS was 82.8%. The univariable analysis of RFS showed that T stage was a significant prognostic factor of RFS  had a history of smoking. The smoking rate among males was much higher than that among females. In 2010, Chinese Center for Disease Control and Prevention carried out the Global Adult Tobacco Survey (GATS) in China and reported that smoking rate was 28.1%  among Chinese adults . In the Among 144 SCC patients, only a small proportion were diagnosed at an early stage (10 6.9%] at AJCC stage I and 13 [9.0%] at T1 stage). This was similar to a previous study of anal cancer (129 [71.7%] patients were adenocarcinoma) in China that indicated only 7 of 126 (5.6%) patients were diagnosed at stage I .9% at AJ. The proIn our study we found that about two-thirds of patients received the standard treatment for anal SCC, which was significantly higher than that reported in a previous study carried out in 2011 by Peng et.al where only 9.5% (2/21) received CRT. Peng et al. reported that the reason so few received CRT was that doctors were not familiar with this method as a standard treatment of anal SCC . HoweverClinical practice guidelines suggest that patients who received the standard CRT could achieve a response rate of 80–90%. The remaining 15% of patients whose regional lesion do not respond to CRT can receive APR as salvage treatment . Among aAnal SCC is strongly associated with HPV infection. Recent study regarding cancer burden attributable to HPV infection used 100% as the population-attributable fraction of HPV in anal SCC, which meant that the authors thought nearly all anal SCC were caused by HPV infection . And recIn 2012, HPV 16/18 were responsible for 87.0% anal cancer globally, and proportion rose to 95.9% for HPV 6/11/16/18/31/33/45/52/58 . The HPVFour factors were identified as prognostic factors of outcomes. In our current analysis, we found that advanced T stage at diagnosis was associated with shorter RFS. Previous conducted in Norway reported that advanced T stage significantly increased the risk of recurrence . Ghosn ePrevious studies also mentioned that HPV infection and its surrogate (i.e. p16) were strongly associated with the outcome of anal SCC –54. CompAnal intercourse, a known risk factor for anal SCC, is practiced in a significant proportion of heterosexual couples (6 to 40%)  and nearThere were several limitations that need to be considered when interpreting the data. Our study was a retrospective audit of clinical records. The rarity of anal SCC and the lack of centralised reporting system for anal SCC restricted our ability to find more records which may lead to compromised representativeness of our sample. There is a lack of essential SCC-relevant variables in the current clinical recording system, including detailed sexual behaviors, HPV testing result, HPV-related biomarkers, history of other HPV-related cancers, and HIV status. The tumor registration system in many medical institutions did not systematically separate anal SCC from adenocarcinoma, leading to underreported SCC patients. Meanwhile, the patients in our study were mainly HIV-negative and female. Because of the restricted population, the results of this study may not be generalized. Our study also had several strengths. Our study was the largest one on anal SCC in mainland China. Another strength was the clinical records of patients were collected from 11 medical institutions in various parts of China, which is more representative compared with records obtained from a single institution.Our data illustrated that T stage of anal SCC was a predictive factor of RFS, and age, AJCC stage, and N stage were identified as prognostic factors of OS. Among anal SCC patients, only a small fraction were diagnosed at an early stage. The proportion of patients receiving CRT increased over the past decade. However, the usage of CRT still needs to be improved. Further research about identifying other predictive factors of outcomes e.g. biomarkers, improving the rate of early diagnosis and improving the usage of CRT should be conducted. More efforts are needed to collect necessary information regarding HPV infection, biomarkers of HPV infection, history of HPV-related cancer, and sexual behavioral from SCC patients."}
{"text": "N = 1,500) irrational response to green technological innovation so as to promote their pro-environmental behaviors. Our experiments identify students' mental construal level as an important psychological factor that, when combined with a proper message framing strategy of introducing new green technologies, can remedy their irrational response to new green technologies. Our findings suggest that highlighting the new technology as playing a preventive/promotional role related to climate change can mitigate risk compensation behavior and eventually promote students' pro-environmental behaviors when they are at a high/low mental construal level.Although green technological innovation is designed to combat climate change, recent research suggests that increased attention to technological innovations might decrease climate change risk perception and reduce pro-environmental behaviors due to the feeling of being assured, which is referred to as risk compensation behavior. Although there has been a growing interest in reducing the risk compensation effect related to climate change, the academic literature in this area is very limited. In this study, we propose a psychological intervention to mitigate a sample of university students' ( Most simulations suggest that the change in the global surface temperature between 1,850 and the end of the twenty-first century is likely to exceed 1.5°C. The World Meteorological Organization (WMO) says that if the current warming trend continues, temperatures could rise 3–5°C by the end of this century. Temperature rises of 2°C have long been regarded as the gateway to dangerous warming. The Intergovernmental Panel on Climate Change report of 2014 states that a 70% reduction in anthropogenic GHG emissions between 2010 and 2050 is needed to limit global warming to 2°C above preindustrial levels  based on construal level theory, which is widely used in social psychology, in conjunction with the message framing method to remedy people's irrational responses to new “green” technologies, or “risk compensation” behavior.This paper presents the results of three experiments conducted on 1,500 university students in Chongqing China. The results show that highlighting a new technology as playing a promotional/preventive role related to climate change can significantly mitigate university students' risk compensation behavior so as to promote their pro-environmental behavior when they are at low/high mental construal level , respectively. We examined participants' perceived climate change risk and pro-environmental behavior using a survey before and after interventions. To the best of our knowledge, this is the first study to apply a combination of construal level theory and message framing method on the mitigation of risk compensation behavior among university students related to climate change. We also contribute to the literature that examines the effectiveness of promotional-framed and preventive-framed messages in various contexts. Previous studies have focused on smoking cessation  or a more temporal distance benefit  is highlighted.Lee and Aaker  and ThomTo mitigate risk compensation behavior, we propose that when individuals are in a low construal mindset, learning about a green technological innovation that is framed as playing a promotional role related to climate change can mitigate their risk compensation behavior, due to the belief of abstract thinkers that the preventive action is not an immediate “solution” to climate change, whereas when individuals are in a high construal mindset, learning of a green technological innovation that is framed as playing a preventive role related to climate change can also reduce their risk compensation behavior, due to the belief of abstract thinkers that the preventive action is only a partial and temporary “solution” to climate change. Overall, the incongruent combination of message framing and construal level results in less persuasive messaging and makes participants believe that the conveyed technology is not an efficient remedy for climate change. As a result, this leads to less risk compensation behavior.Experiment one tests for the predicted interaction between mental construal (abstract vs. concrete) and the type of benefit  that is highlighted when advocating the effect of a new “green” technology on the shift of public climate change risk perception.H1a:Advocating “green” technological innovation undermines climate change risk perception due to the feeling of being assured.H1b:The response to “green” technological innovation  varies with individual construal level .H1c: When individuals form an abstract thought , learning of a green technological innovation that is framed as playing a preventive  role related to climate change can mitigate their reduced climate change risk perception (risk compensation behavior) due to the belief of abstract thinkers that preventive action is only a partial and temporary “solution” to climate change and that the new technology is less likely to mitigate climate change risk.H1d:When individuals form a concrete thought , learning of a green technological innovation that is framed as playing a promotional  role related to climate change can mitigate the reduced climate change risk perception (risk compensation behavior) due to the belief of concrete thinkers that the promotional action is not an immediate “solution” to climate change and the technology is not an efficient way to solve the climate change issues.www.wenjuan.com to follow the procedure as follows:The experiment was a 2  × 3 (non vs. mitigation vs. adaptation) mixed design see . In thisStep 1: In the pretest survey, the participants answered a few basic demographic questions and questions from the survey on climate change risk perception and pro-environmental behavior  a group that was asked to complete a task  that manipulated their mental representations into high-level construals; (2) a group that was asked to complete a task  that manipulated their mental representations into low-level construals.Step 3: Then, the participants randomly received one of three versions of the articles , which is the dependent variable. ANOVA was used to test for the significance of differences among the group means in our sample, and regression analysis showed the direction  and magnitude  of these differences.H1athat learning of a “green” technical innovation can undermine climate risk perception and provides suggestions for a risk compensation effect. For those with high-level construals , preventive messaging advocating for technology by highlighting the mitigating benefit of the technology on climate change results in the lowest feeling of assurance  group, and M = 5.01 in the promotional message group) and thus moderates the reduced risk perception  group and M = −3.78 in the promotional message group). This is because the combination of a high construal mindset and a preventive frame is an incongruent intervention. This combination is not effective in persuading participants to believe that the new technology is able to mitigate the climate change risk.M = −1.02 vs. M = 2.62 in the pure technology message (control) group and M = −4.50 in the preventive message group). Overall, the low construal group presented promotional messages advocating green technologies experience a decreased compensation effect and thus exhibit a less reduced risk perception. These findings provide supportive evidence for hypotheses H1b, H1c, and H1d.Likewise, those with low-level construals  were the least assured by promotional messages highlighting the climate-change adaptation (long term) benefit of the technologies. Again, this combination results in an incongruent intervention that is not persuasive enough to make participants believe that the technology is an efficient remedy for climate change. Consequently, this leads to less reduction in risk perception  = 9.85, p = 0.0001], and F is the F statistic. In contrast, the main effects of construal level and message type were not significant  = 0.20, p = 0.6533; and F = 0.33, p = 0.7171]. To further corroborate the interaction effect, we checked the simple main effect of message type by mental construal level and the simple main effect of construal level for the message type. All p-values [see Panel A (a)–(d)], except those for preventative messaging, indicate significance.Next, we examine the regression results in Taken together, we confirm the interaction effect of mental construal level and the way “green” technology is advocated on the shift of public perceived climate risk. In particular, the lack of fit between a mental representation and a message advocating technology as solutions to climate change helps remedy the decrease in climate risk perception. Meanwhile, the results reported in We demonstrated in experiment one that a mismatch between a mental representation and a message framing could effectively attenuate people's lowered climate change concerns caused by risk compensatory behavior. However, it is unclear if greater climate change risk perception would translate into increased individual action to preserve the environment. Consequently, we ran experiment two to examine whether people's heightened concern about climate change would lead to more pro-environmental action intentions.H2a: Changes in climate risk perception can transfer to pro-environmental behavioral intentions.H2b: When individuals form an abstract thought , learning about a green technological innovation that is framed as playing a preventive  role related to climate change can attenuate the reduced pro-environment behavioral intentions caused by the alleviation of lowered climate change risk perception.H2b: When individuals form a concrete thought , learning about a green technological innovation that is framed as playing a promotional  role related to climate change can attenuate the reduced pro-environment behavioral intentions caused by the alleviation of lowered climate change risk perception.www.wenjuan.com. The experimental procedure was the same as that described in Experiment 2, but two differences were presented. First, for the robustness test, we use an alternative way of manipulating participants' mental construal levels. The participants were presented with 10 tasks and asked to indicate either “why” we need to complete the tasks  or “how” we can complete the tasks  Förster et al. -(d)]. The regression results are presented in Panel B. Once again, we observe that learning of “green” technologies reduces people's pro-environmental behavioral intentions  but highlighting mitigating/adapting benefits for people with high/low level mental construals can significantly correct the reduction by 0.764 and 1.097 points, respectively. Taken together, we demonstrate in experiment 2 that individuals' increased climate change concern results in increased pro-environmental action intentions which is consistent with our hypothesis.One inherent challenge associated with surveys eliciting information about pro-environmental behavior is that they are not externally verifiable. Student participants may overstate or fabricate their actual pro-environmental behavior in the survey , learning of a green technological innovation that is framed as playing a preventive  role related to climate change can mitigate the reduced actual pro-environment behavior due to the belief of abstract thinkers that the preventive action is only a partial and temporary “solution” to climate change.H3b: When individuals form a concrete thought , learning of a green technological innovation that is framed as playing a promotion  role related to climate change can mitigate the reduced actual pro-environment behavior due to the belief of concrete thinkers that the promotional action is not an immediate “solution” to climate change.In this experiment, 300 students were recruited from a university in Chongqing, China. The student participants completed the same manipulation of mental construal level as described in Experiment 1 and were then randomly given one of the three versions of the articles. Then, they were asked to play a word decoding effort game. The word game is based on a word decoding effort task, comparable to Dorner , Erkal eEach completed word lowers the amount of money donated to charity for that round. The initial donation for any given round was set at 15 RMB per participant. The charity is a local tree planting organization, and participants are aware that every two RMB given to the organization results in the planting of one seedling. Additionally, participants are aware that each completed word lowers the charity contribution by 2 RMB, which is deducted from the donation of 15 RMB. However, participants may minimize the negative impact of that word on the charity by adding additional letters in the middle right of the paper. The instructions make it obvious to participants that these extra letters are optional. One additional letter reduces the damage to the charity by one-third, two additional letters reduce the damage to the charity by two-thirds, and all three additional letters reduce the damage to the charity to nothing. As a result, participants must choose a trade-off between their private profits and the amount of harm they are prepared to inflict to the charity payout in each round. The current round's cumulative earnings and damage are displayed in the top center of the paper. At the conclusion of the experiment, students were compensated for the amount earned during the activity. The money saved by the participants was then donated to a tree planting charity. The money-incentivized task has the advantage of reflecting participants' real intention to engage in pro-environmental activities.We administered the word game to the participants via paperwork, and one round of a sample game is shown in p = 0.0001. All the p-values for simple main effects, except for those of the effect of message type on people with high construal levels and the effect of construal level when using preventive messages [see Panels A (a)-(d)] on advocating “green” technologies, were significant. The regression results are presented in Panel B. In addition to the demographic characteristics, we also include the productivity indicator we obtained from the practical test to control for the intrinsic word processing ability of the participants. We observe that, on average, presenting preventive/promotional messages about technologies that have mitigating/adapting benefits to people with high/low construals can significantly moderate the donation deduction by 1.084 and 1.921 yuan, respectively. The results are consistent with the hypothesis. The study's design has several advantages, but it also comes with some drawbacks. To begin, we conducted our research on a group of university students, many of whom were living on their own for the first time. This may have influenced their susceptibility to interventions, as people are more inclined to change their habits following a move. This begs the question of whether our intervention's effects can be generalized to more stable household circumstances as well. Nonetheless, this group may be particularly interesting because they represent future environmentalists. Future research should examine the effectiveness of a similar intervention in diverse settings, such as a factory or a household.Another limitation is that participants were aware that they were being monitored throughout the trial in our study. This, of course, could have an impact on their behavior. However, because individuals in different treatment groups received identical information but not interventions, we could account for potential effects associated with the concept of monitoring. Although we randomly assigned participants into different treatment groups to account for potential monitoring effects, it would be interesting to observe if comparable results occur when individuals are unaware they are being monitored.The role of peer effects in decision making has been largely explored in many contexts, such as green product adoption, saving and borrowing decisions  with a targeted messaging method to alter university students' risk compensation behavior. Specifically, we conducted three experiments on mitigating risk compensation behavior in the setting of climate change risk perception and pro-environmental behavior with 1,500 university students from both an online survey platform and a university in Chongqing, China, in 2019. The sample size in our study is substantially greater than comparable studies. For example, Wang et al.  investigThe results demonstrate that highlighting a new technology as playing a preventive/promotional  role related to climate change can mitigate the risk compensation behavior of individuals with high/low mental construals . We provide evidence that this mitigation is driven by the lack of fit between the participants' abstract/concrete thinking and the immediate/long-term solution to climate change. The incongruent combination makes messages less persuasive and allows participants to believe that the technology is not an efficient remedy for climate change. Consequently, this significantly promotes university students' pro-environmental behavior.This paper also has implications for policymakers. The need for measures to address climate change  and efforts to reduce greenhouse gas emissions have been widely acknowledged by scientists and policymakers IPCC, . HoweverThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by School of Finance, Chongqing Technology and Business University. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.This research is supported by The National Social Science Fund of China: Research on Mechanism Innovation and Effect Evaluation of Ecological Poverty Alleviation from the Perspective of Targeted Poverty Alleviation (17BJY131).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": "Scientific Reportshttps://doi.org/10.1038/srep13677, published online 02 September 2015Retraction of: The Editors have retracted this Article.Partial overlap of panels between Figures 2G and 4G, and Figures 2H and 4HDuplication of parts of panels within Figure 4E and 4FDuplication of parts of panels within Figure 2E and 4E, and Figure 4F and 2FThe Editors therefore no longer have confidence in the integrity of the data presented.After publication, it was brought to the Editors' attention that there were irregularities in a number of figures, specifically:Zhaohui Gong disagrees with the retraction of the Article. Lihua Yang, Jie Yang, Jingqiu Li, Xingkai Shen, Yanping Le, Chengwei Zhou, Shaomin Wang, Shun Zhang, and Dazhi Xu did not respond to correspondence in regard to the retraction of this Article."}
{"text": "Adult-onset citrullinaemia type II (CTLN2) is a rare disease in Chinese patients. As a subtype of citrin deficiency (CD), it is an autosomal recessive disease related to the SLC25A13 mutation on chromosome 7q21.3. In this study, we report a case of CTLN2 presenting with paroxysmal altered consciousness and refractory hyperammonaemia. The diagnosis was finally confirmed by gene analysis. The patient recovered after liver transplantation. It can be learned from this case that CD should be considered in patients with refractory hyperammonaemia and paroxysmal mental disorder without a history of liver disease. Metabolic encephalopathies are a group of disorders defined as brain malfunction secondary to systemic metabolic derangements. Hyperammonaemia is one of the aetiologies. As the liver is the main organ of ammonia metabolism, hyperammonaemia is often considered a common complication of chronic liver disease. In fact, any disease involved in the urea cycle can cause hyperammonaemia. Citrin, a liver-type mitochondrial aspartate–glutamate carrier, plays an important role in supplying aspartate to argininosuccinate synthetase in the cytosol to generate argininosuccinate in the urea cycle. Citrin deficiency (CD) can cause urea circulation disorder, which leads to hyperammonaemia. There are two kinds of CD, neonatal intrahepatic cholestasis (NICCD) and adult-onset citrullinaemia type II CTLN2) . In Chin . In Chi2An 18-year-old man with paroxysmal altered consciousness for half a year was admitted to our hospital. The patient complained of episodic delirium, screaming, convulsion, and vomiting every 5–8 days, each time lasting for a few hours before the symptoms spontaneously alleviated. The patients did not provide any precipitating factors before the onset of these symptoms but confessed a preference for foods such as nuts, peanuts, eggs, and meat, as well as an aversion to rice or sweet desserts. The initial local hospital examinations revealed no abnormalities in the brain or abdominal computed tomography but a significant increase in plasma ammonia levels , and a slight elevation of alanine aminotransferase  and aspartate aminotransferase . Tests for EBV, CMV, HAV, HBV, HCV, HIV, thyroid function, ceruloplasmin, markers of autoimmune liver disease, and α-antitrypsin were negative. Data from another hospital 1 month prior also showed a significant increase in plasma ammonia levels (209 μmol/L), and a slight elevation of ALT (77 U/L) and AST (80.15 U/L) levels. Metabolic encephalopathy was diagnosed and treated. As the symptoms recurred with an increased frequency of paroxysmal altered consciousness, the patient was transferred to our hospital.On admission, no abnormalities were found on physical examination. The levels of plasma ammonia (543.9 μmol/L), ALT (88 IU/L), and AST (149 IU/L) were still high. Despite the use of arginine and ornithine aspartate, the patient slipped into a coma without precipitating factors. Both pupils were equally round at approximately 2.5 mm each with a slow light reflex. Active tendon reflexes and positive Babinski signs were elicited and interpreted as the result of cerebral oedema. Mannitol and furosemidum were administered, and the patient recovered consciousness. Brain magnetic resonance imaging (MRI) showed a region of high signal intensity in the bilateral frontal lobes, which indicated the possibility of metabolic encephalopathy or infarction . Urine oSLC25A13 gene analysis showed that the patient was a compound heterozygote for c.851-854del and IVS16ins3kb. His mother had a heterozygous mutation in c.851-854del. Adult-onset citrullinaemia type II (CTLN2) was diagnosed. After conservative treatment including arginine, ornithine aspartate, sodium pyruvate, glutathione, ursodeoxycholic acid, and a low-carbohydrate, high medium-chain triglycerides (MCT) and lactose-free diet, the patient recovered significantly. One month later, the patient received liver transplantation, after which his blood ammonia level dropped to 61 µmol/L. Approximately 1 month after the surgery, brain MRI indicated cerebral atrophy . FortunaConsent for the publication of this case has been obtained from the patient.3+)/NADH ratio through the malate–citrate shuttle and can reduce oxidative stress [CTLN2 is a rare disease in the Chinese population. As a subtype of CD, it is an autosomal recessive disease related to the SLC25A13 mutation on chromosome 7q21.3. Several genotypes of SLC25A13 have been discovered, such as c.851-854del, IVS16ins3kb, IVS6 + 5G > A, and c.1177 + 1G4A . MCT cane stress .Because of the non-specificity of symptoms, CTLN2 is easily misdiagnosed. Elevation of the plasma ammonia concentration is common in CTLN2. Serving as an aspartate–glutamate transporter in the mitochondria, citrin is essential for the urea cycle and NADH shuttle . CD leadIn our case, the patient was very young with a short disease course and rapid progression. Liver cirrhosis and brain atrophy are usually seen in a long disease course. In addition, it is uncommon to find hepatic cirrhosis in a young man with a short duration of CTLN2. Chen reported a case of CTLN2 with a course of 6 months. Brain MRI revealed bilateral symmetric lesions of the cingulate cortex, which disappeared after liver transplantation .4In conclusion, our case indicated that CTLN2 should be considered in patients who present with paroxysmal altered consciousness and refractory hyperammonaemia. Unusual food preferences may provide some clues for the diagnosis. Some conservative treatments may induce a temporary remission of hyperammonaemia and altered consciousness but failed to achieve long-term improvement, and most of the patients finally died of severe brain damage . Therefo"}
{"text": "Background: Acromegaly is a chronic disease caused by an abnormal secretion of growth hormone (GH) by a pituitary adenoma, resulting in an increased circulating concentration of insulin-like growth factor 1 (IGF-1). The main characteristics are a slow progression of signs and symptoms, with multisystemic involvement, leading to acral overgrowth, progressive somatic changes, and a complex range of comorbidities. Most of these comorbidities can be controlled with treatment. The literature reveals that the most evident and early signs are those related to soft tissue thickening and skeletal growth, especially in the head and neck region. Methods: The authors reviewed the available literature on the clinical oro-dental features of acromegaly, selecting articles from PubMed and Google Scholar. The aim of this review was to summarize all the reported clinical oro-dental features of acromegalic patients. Results: The most common facial dimorphisms involved the maxillo-facial district, with hypertrophy of the paranasal sinuses, thickening of the frontal bones, and protruding glabella, which may be associated with joint pain and clicks. Regarding the oro-dental signs, the most frequent are dental diastema (40–43%), mandibular overgrowth (22–24%), mandibular prognathism (20–22%), and macroglossia (54–58%). These signs of acromegaly can be significantly reduced with adequate treatment, which is more effective when initiated early. Conclusions: Increased awareness of acromegaly among dentists and maxillo-facial surgeons, along with the early identification of oro-facial changes, could lead to an earlier diagnosis and treatment, thereby improving patients’ quality of life and prognosis. Acromegaly is a rare chronic disease caused by an excessive secretion of growth hormone (GH), mostly due to an adenoma of the anterior pituitary gland and resulting in an increased circulating concentration of insulin-like growth factor 1 (IGF-1), the main effector of GH activity. It is characterized by a slow progression of signs and symptoms and multisystemic involvement, leading to physical alterations, progressive somatic changes, and a complex range of systemic comorbidities ,7,8,9,10The somatic feature changes are slow and often cause a delay in diagnosis; facial dysmorphism, oro-dental signs, enlargement of extremities, and soft tissue thickening are the most common and earliest manifestations of acromegaly ,14,15,16Focusing on facial changes and oro-dental alterations, these manifestations, in particular, the development of malocclusion due to mandibular prognathism, are associated with reduced self-esteem and decreased quality of life (QoL) ,25, and The authors reviewed the available literature and selected articles using PubMed, Web of Science and Google Scholar. The following keywords were entered separately and in combination: “acromegaly,” “oral manifestations,” “dental manifestations,” “dental manifestation,” “oro-dental acromegaly,” “facial manifestation of acromegaly,” “dentist,” “dentist acromegaly,” and “orthodontics.”All articles published between 1950 and 2021 were considered for inclusion. We included only papers published in English. We excluded case reports and papers for which only the abstract was available. The literature was analyzed specifically for the collection and rationalization of all the reported clinical oro-dental features shown by the acromegalic patients. The aim of this review was to collect all the clinical oro-dental features observed and reported in acromegalic patients.Acromegaly prevalence ranges from 20 to 130 people per million inhabitants ,13,14,15The most frequent signs and symptoms of acromegaly are: menses abnormalities, physical changes , headache, paresthesia, impaired glucose metabolism, erectile dysfunction, arthropathy, hypertension, fatigue, daytime sleepiness, and increased perspiration . SymptomThe suspicion of acromegaly in a subject is based on the clinical manifestations and it can be confirmed with testing. The standard blood tests include IGF-1 levels and GH nadir after an oral glucose tolerance test. Imaging of the pituitary gland and magnetic resonance or computerized tomography (if MR is not applicable) are also used to confirm diagnosis. According to the guidelines, screening for excess of GH and IGF-1 is important in patients with clinical features of acromegaly associated with multimorbidity  who are unresponsive to treatment. Similarly, if comorbidities that cause GH excess have an earlier than usual onset or if these comorbidities are associated with a pituitary mass , patients should also be tested for acromegaly.Treatment of GH pituitary adenoma includes different therapeutic approaches, such as neurosurgery (the first-line treatment), medical treatments , and radiotherapy in select patients. Therapeutic goals in patients with acromegaly are the biochemical control of GH and IGF-1 secretion, the control of systemic comorbidities, the reduction of mortality, and the restoration of QoL.Among acromegaly symptoms, oral manifestations and maxillo-facial features are reported by most patients to have been present up to 10 years before diagnosis, preceded only by increased size of hands and feet. These manifestations may also be present in the later phases of the disease depending on duration of active disease, entity of diagnostic delay, and GH and IGF-1 secretion levels. Mandibular growth, reported in 22–24% of patients, can lead to prognathism in 20–22% of these patients and the development of a class III dental and skeletal pattern , panel AMacroglossia is seen in 54–58% of acromegaly patients ,6,19,22;Acromegaly patients can also develop a dental malocclusion , panel CRegarding dental elements, acromegaly patients may report diastemata , panel ESome studies also describe an increased mineralization of the alveolar bone at the level of the roots of the molar elements, which outlines the features of hypercementosis ,24,28,29The effects of the disease on the alteration of gingival tissues and on the development of periodontal and periradicular pathologies are still being debated, and the link with the disease remains questionable . Some auAt the palatine and mandibular level, a study  identifiClinically, not only dental components are modified by GH and IGF-1 excess but also soft tissue may be involved in facial changes due to acromegaly. In fact, it has been found that acromegalic patients commonly have enlarged submandibular glands (regardless of the activity of the disease) ,32,33, tFinally, hypertrophy of the paranasal sinuses, especially of the frontal ones, may also occur. This aspect, together with laryngeal hypertrophy, can cause a deepening and sound resonance of the patient’s voice ,6,9.These oro-facial alterations in acromegaly patients may be associated with pain in the mouth, especially in the maxillo-facial area  and joinBiochemical treatment for the limitation of GH hypersecretion can only partially improve facial appearance, especially regarding soft tissues, but in most cases, oro-facial changes are not completely reversible and persist despite pharmacological treatment ,25. TablSome studies were also conducted using teleradiography examination to capture the cephalometric characteristics of acromegalic patients. The results showed that these patients generally report an increase in overall facial height and in the length of the cranial bases, an increase in the gonial angle, and an increase in the size of the branch  and of tAccording to the literature, orthopantomography does not play a significant role in identifying the pathognomonic signs of the disease. It has been reported that only the panoramic and intraoral radiographs of these patients can reliably show radiopaque lesions at the level of the roots of the molar elements, defined as “hypercementosis” ,29,43,47Nevertheless, clinicians should pay attention to these aspects when reading the results of radiographic examinations. Dentists and orthodontists  are adviAbout 80% of acromegaly patients present with oro-dento-facial signs, such as dental diastema, mandibular overgrowth, mandibular prognathism, and macroglossia. Dentists, orthodontists, and dental hygienists are in an ideal position to detect these signs early in acromegaly patients, which can lead to earlier diagnosis and improved prognosis. Moreover, with early diagnosis and subsequent appropriate endocrinological treatment earlier in the disease course, these signs can be significantly reduced. This would significantly impact patients’ QoL and self-esteem. It should be noted that even if the signs are not reduced by endocrinological treatment, they can be addressed with specific oral and maxillo-facial treatments.For these reasons, oro-facial alterations, as distinctive and early signs of acromegaly, should be considered a primary target to be identified and evaluated properly. It is imperative to increase awareness among specialists, including dentists, orthodontists, and dental hygienists, with the specific goal of recognition of the pathology and earlier diagnosis of the disease. This would result in earlier initiation of treatment, which has been shown to improve patient outcomes in a myriad ways."}
{"text": "Stem cell therapy is one of the novel and prospective fields. The ability of stem cells to differentiate into different lineages makes them attractive candidates for several therapies. It is essential to understand the cell fate, distribution, and function of transplanted cells in the local microenvironment before their applications. Therefore, it is necessary to develop an accurate and reliable labeling method of stem cells for imaging techniques to track their translocation after transplantation. The graphitic quantum dots (GQDs) are selected among various stem cell labeling and tracking strategies which have high photoluminescence ability, photostability, relatively low cytotoxicity, tunable surface functional groups, and delivering capacity. Since GQDs interact easily with the cell and interfere with cell behavior through surface functional groups, an appropriate surface modification needs to be considered to get close to the ideal labeling nanoprobes. In this study, polyethylene glycol (PEG) is used to improve biocompatibility while simultaneously maintaining the photoluminescent potentials of GQDs. The biochemically inert PEG successfully covered the surface of GQDs. The PEG-GQDs composites show adequate bioimaging capabilities when internalized into neural stem/progenitor cells (NSPCs). Furthermore, the bio-inertness of the PEG-GQDs is confirmed. Herein, we introduce the PEG-GQDs as a valuable tool for stem cell labeling and tracking for biomedical therapies in the field of neural regeneration. Stem cell based therapy is currently a promising and advanced research field that attracts attention as a critical player for the treatments of intractable diseases, such as cancer, neurological disorders, cardiac diseases, diabetes, and liver diseases ,2. ThereQuantum dot (QD) is a promising solution for more capable bioassay and bioimaging technology. QDs emerged as a result of the discovery that the size of the nanomaterials determines the properties of QDs ,9. In te2 or OH functionalized ones. However, all three different surface chemistry influenced the biological pathway, such as phosphorylation [Recently, the cytotoxicity related to the surface functional group and the size which influences the biological effects and the uptake of GQDs were addressed. Weihu Shang et al. showed that GQDs smaller than 10 nm demonstrated direct and easy penetration into stem cells without affecting their viability, proliferation, or differentiation capacity . Furtherrylation . Thereforylation , drug derylation ,26, and rylation . PEG funrylation ,29,30,31rylation ,33,34.For these reasons, the PEG-GQDs were fabricated and investigated as a platform of the theranostic agents for the vulnerable and fragile nerve tissues. The GQDs were fabricated by the graphite intercalation compounds mediated exfoliation method and the PEGylation proceeded on the GQDs’ surface through the solvothermal method. The size, surface charge, surface functional groups, and photoluminescence property of GQDs before and after PEGylation were investigated. Then, the experiments that dealt with the concentration dependant cytotoxicity established the available dose of material when the fetal rat NSPCs were employed. Moreoever, it was confirmed that the PEG-GQDs are well internalized into the cell. Finally, the presence of the PEG-GQDs’ influences on cell differentiation was closely investigated. Through this study, a method to utilize the low-cytotoxic, bio-inert, and bioimaging nanoparticle is proposed.4H4O6·4H2O, Sigma Aldrich, Burlington, MA, USA) precursor was introduced in the graphite by mixing and the exfoliation proceeded at 250 °C for 24 h. The size distribution of the GQDs was controlled by sieving with a dialysis membrane . The particles were dried for several days under vacuum conditions. The surface functional groups of GQD were reacted with polyethylene glycol (PEG-bis(amine), MW: 2000, Sigma Aldrich, Burlington, MA, USA) via an amide bond [The GQDs were fabricated by the graphite intercalation compounds (GICs) method described previously ,36,37. Bide bond . The aqu−1) analyses were conducted.X-ray diffractometer , dynamic light scattering and zeta potential , X-ray photoelectron spectroscopy , transmission electron microscopy , Fourier transform infrared spectroscopy . The amount of 100 µL PEG-GQDs was added to the 96-well plate and fluorescence images were taken by a fluorescence microscope .5 rNSPCs was seeded into a 6 cm low-attachment culture dish and incubated in a 37 °C, 5% CO2, and 100% humidity incubator. Every 2 days, half of the media was replaced with a fresh one. Cells were passaged once a week when the neurospheres were larger than 200 µm in diameter. For consistency, cells between passage 2 and 5 were used in this study.Neural stem/progenitor cells (NSPCs) were prepared using a previously reported method ,40. The The complete growth media contains DMEM: F12 , L-GlutaMAX , 2% B27 minus vitamin A , 1% Penicillin/ Streptomycin , 20 ng/mL epidermal growth factor , and 20 ng/mL basic fibroblast growth factor .The differentiation media contains Neurobasal plus medium , 2% B27 Plus Supplement , 0.5 mM L-GlutaMAX, and 1% Penicillin/Streptomycin.For identification of the differentiation potential of rNSPCs, cells were firstly seeded in the 96-well plate and incubated for 24 h under the growth media. Then, the growth media was changed into differentiation media to induce the differentiation of rNSPCs and cells were cultured for another 7 days. The media was changed every 2–3 days.4 rNSPCs in 100 µL growth media was seeded in a precoated 96-well plate and permitted to grow for 24 h. Subsequently, different concentrations of PEG-GQDs  were added to the cells. Twenty-four hours later, after discarding the supernatant, cells were carefully washed once with warm PBS and treated with CCK-8 reagent  diluted with prewarmed media and incubated for another 2 h. Then the absorbances were measured with a microplate reader at 450 nm. For detecting the viable cells, the LIVE/DEAD™ Viability/Cytotoxicity Kit  was used. The cells were incubated with Calcein AM (2 µM) and Ethidium homodimer-1 (4 µM) and were diluted with prewarmed DMEM: F12 media for 30 min at 37 °C. Before capturing, cells were washed once with prewarmed DMEM: F12 media. Images were captured with a fluorescence microscope  at 40×.For the culture of NSPCs, the plates were coated with 1% Matrigel , a good basement membrane biomaterial. In detail, the original Matrigel was diluted to 1% with ice-cold DMEM/F12 media. The amount of 100 µL of 1% Matrigel solution was added to the 96-well plate and the plate was then placed into an incubator at 37 °C for 1 h. Before seeding cells, the plate was gently washed once with DMEM/F12 media. The amount of 1 × 104 cells in 500 µL growth media) in a 24-well plate and incubated for 24 h. PEG-GQDs were added to the cells at a concentration of 320 µg/mL. After 24 h of incubation, the supernatant was removed and cells were washed twice with PBS to remove residual PEG-GQDs. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA)  for 30 min, following three times of PBS washing. For better observation of cell morphology, Alexa Flour 488 Phalloidin  solution was added to cells and incubated for another 30 min at room temperature. After washing it three times in PBS, the fluorescence images were captured using a Zeiss LSM 700 confocal microscope .To check the visualization ability and intracellular location of PEG-GQDs, confocal laser-scanning microscopy  was used. The rNSPCs were seeding on the 1% Matrigel pre-coated 15 mm cover-glasses , and then resuspended in cell staining buffer . The uptake of PEG-GQDs into rNSPCs was analyzed by fluorescence-activated cell sorting (FACS) analysis , based on the blue fluorescence signals of PEG-GQDs.The rNSPCs were seeded in a 24-well plate at 1.25 × 10For identifying the stemness and differentiation potential of primary culture rNSPCs, immunocytochemical experiments were performed and the data are shown in n = 15). The total cell numbers were quantified by Hoechst staining.For quantitative analysis, Tuj1 and GFAP-positive cells were manually quantified in a visual field at 400× (p < 0.05) between groups.All results are presented as means ± standard deviations (SD). For all assays, one-way ANOVA and Tukey’s comparison tests were performed to determine possible significant differences , sp3 (~284.8 eV), and C–O or C–OH (~286.6 eV); and functional groups amide (N–C=O) (~288.7 eV), carboxyl (COOH), or ester (~290 eV), respectively. The binding energy of GQDs presented a predominant peak near 284 eV. This is close to the sp2 binding energy with an asymmetric shape, which is preferably derived from graphite or graphene samples. After the PEG coating, the portion of sp3 was increased. Moreover, the peak of the PEG and amide group was revealed at around 286.6 and 289 eV. These XPS results confirm the presence of PEG on the GQDs after successful encapsulation.The XPS spectra of carbon 1s were investigated to obtain more information about the surface functional groups. As shown in −1; C=C, C=O stretching, ester, aliphatic COOH, and conjugated C=O ~1600 cm−1; C-H stretching ~3000 cm−1; and O–H stretching ~3500 cm−1, respectively. After surface modification, the distinctively changed locations were highlighted. The C–O stretching epoxide peak at 1024 cm−1 appeared prominently in PEG-GQDs. The ratio between C-H stretching band (~2880 cm−1) and the C–O stretching band (1130 cm−1) was also changed after the PEGylation. On the surface of PEG-GQDs, the ratio of the C–H/C–O was lowered and the carboxyl groups at 1660 cm−1 completely disappeared. These results are evidence of the successful modification of the PEG layer on the GQDs.This result was also crosschecked by the FT-IR spectra analysis and is shown in In summary, it was confirmed that the PEG modification successfully proceeded on the GQDs surface. It is expected that they could increase the utilization of nanoparticles as a bio labeling probe without an accumulation of unwanted locations caused by nonspecific binding on the cell and proteins and simultaneously reduce the biological interaction than the pristine GQDs.The samples were prepared at various concentrations in the DW to investigate the visualization ability of the PEG-GQDs. The images of PEG-GQDs were captured with a fluorescence microscope with three different wavelengths. As shown in In neural regeneration, neural stem/progenitor cells (NSPCs) are one of the major cell types for stem cell based therapies because the NSPCs’ self-renewing and multipotent characteristics can generate the critical phenotype cells of the nervous system such as neurons, astrocytes, and oligodendrocytes ,45,46,47After seeding rNSPC for 24 h, various concentrations of PEG-GQDs  were added and then incubated with cells for an additional 24 h. As shown in Fluorescence-positive cells were sorted by performing FACS analysis to quantify the rate of intracellular uptake ratio of PEG-GQD in rNSPC with respect to incubation time. Percentages of blue fluorescence-positive cells were then calculated based on the FACS analysis. As shown in The differentiation potential of rNSPCs with internalized nanomaterials is an essential characteristic for stem cell based therapy that should not be changed by the containing material for further bio applications. To investigate whether PEG-GQDs impair the differentiation potential of rNSPCs, we induced a differentiation process of rNSPCs. After 7 days of differentiation, neuron-specific class III beta-tubulin (Tuj1) and glial fibrillary acidic protein (GFAP) were examined using immunocytochemistry. As shown in The successful PEGylation did not interfere with the photoluminescence properties of GQDs while passivating the surface from other reactive substances. The assays related to the biological effects on rNSPCs showed that the PEG-GQDs exhibited excellent biocompatibilities, bioimaging property without weakening the rNSPCs’ activity, and differentiation ability under concentrations up to 320 µg/mL. Moreover, the PEGylation of GQDs may extend the in vivo circulation time and improve the penetration efficiency to nerve tissue, leading to enhanced bio-imaging, delivering, and therapeutic effects to the nerve tissue injury sites.It was confirmed that the interaction between PEG-GQDs and rNSPCs may provide some useful information for future explorations of a biocompatible and visible on-target probe for the different therapies for neurological disorders. The uniform-sized, water-soluble, and light-emitting GQDs are successfully combined with the PEG that is used to enhance the colloidal stability with a hydrophilic surface modification. Our approach takes advantage of the photoluminescence property of GQDs without distinct losses and takes advantage of the non-reactive hydrophilic surface of the PEGs. The synthesized material showed no specific cytotoxicity up to a concentration of 320 µg/mL and exhibited an ability to be imaged when the material was internalized inside the cells. The embryonic rNSPCs that internalized the substances were investigated on whether they sustain their differentiation ability. The result showed that the differentiation potentials of rNSPCs were not adversely affected by PEG-GQDs.In conclusion, PEG-GQDs showed proper bio labeling ability and biocompatibility without attenuation of rNSPCs differentiation, suggesting that PEG-GQDs are useful material tools at the visible nano-delivery system for the regeneration therapies against neurological disorders."}
{"text": "There is an increasing recognition of the importance of sex in susceptibility, clinical presentation, and outcomes for heart failure. This review focusses on heart failure with reduced ejection fraction (HFrEF), unravelling differences in biology, clinical and demographic features and evidence for diagnostic and therapeutic strategies. This is intended to inform clinicians and researchers regarding state-of-the-art evidence relevant to women, as well as areas of unmet need.Females are well recognised to be under-represented in clinical trials, but there have been some improvements in recent years. Data from the last 5 years reaffirms that women presenting with HFrEF women are older and have more comorbidities like hypertension, diabetes and obesity compared with men and are less likely to have ischaemic heart disease. Non-ischaemic aetiologies are more likely to be the cause of HFrEF in women, and women are more often symptomatic. Whilst mortality is less than in their male counterparts, HFrEF is associated with a bigger impact on quality of life in females. The implications of this for improved prevention, treatment and outcomes are discussed.This review reveals distinct sex differences in HFrEF pathophysiology, types of presentation, morbidity and mortality. In light of this, in order for future research and clinical medicine to be able to manage HFrEF adequately, there must be more representation of women in clinical trials as well as collaboration for the development of sex-specific management guidelines. Future research might also elucidate the biochemical foundation of the sex discrepancy in HFrEF. Heart failure (HF) continues to increase in prevalence in our community in men and women, estimated to affect 64.34 million people globally . The preThere has been a growing need, and widespread call, for increased representation of women in clinical trials, for studies to be powered for sex disaggregated analyses, and for guidelines to consider and reflect sex differences. Here, we review the most recent studies and literature and summarise the sex differences in demographics, mechanism, clinical presentation, biomarkers, outcomes, clinical pathways, management and representation in the literature for HFrEF.In addition to referring to key early studies in the field, we systematically searched databases on Embase using the search strategy (HF with reduced ejection fraction OR HFrEF) and , limited to the English language, humans and the years 2016 to 2021. After removing duplicates, there were 488 results. After abstracts were screened, there were 42 studies and after reading the full texts, 33 were relevant to this review, with key themes summarized in  –7. They New data from the UK has found that women are more obese at presentation, and this obesity portends worse outcomes in women compared to men . This maThere are key sex differences in the underlying mechanisms contributing to HFrEF, as well as differences in the susceptibility to each and the relative contribution to the burden of HFrEF.Whilst a driving precipitant of HFrEF in males is macrovascular coronary artery disease, myocardial ischaemia and infarction , HFrEF iThere are some HF aetiologies that are unique to women, such as peripartum cardiomyopathy, with risk factors including advanced maternal age, and pre-eclampsia . AdditioIn patients with underlying myocardial dysfunction and cardiomyopathy, inflammation is increasingly recognised as playing a key role in a sex-specific manner. Sex discrepancies have recently been demonstrated in antibody-mediated immune response on cardiac remodelling in HFrEF, affecting progression of HF. We know that IgG can be found in the myocardium in patients with end-stage HFrEF associated with ischaemic heart disease (IHD). One study indicated that, in the early stages of remodelling, IgG1 and IgG3 levels differ between men and women . FurtherTakotsubo cardiomyopathy is also more common in postmenopausal women. Whilst classically understood to be an acute and reversible condition, there is increasing appreciation of longer-term myocardial abnormalities and many patients continue to suffer symptoms which may be attributed to the recovery process , 17.More evidence is emerging on the impact of sex hormones on sex discrepancy in HFrEF. There is increasing research on the protective effects of oestrogen on HF. Additionally, an oestrogen depletes postmenopausal state may contribute to women having higher left ventricular systolic and diastolic stiffness when compared to men as oestrogen is involved in blood pressure and arterial tone regulation . FurtherIn terms of presenting features of HFrEF, most recent data concurred with older data , 21 showEchocardiography analysis highlights that women have higher ejection fractions with smaller left atria, higher longitudinal strain and higher circumferential strain , 22. AsiAs coronary microvascular and macrovascular dysfunction have similar risk factors, unique biological risk factor profiling is a promising method of early diagnosis . CurrentBiomarker identification in HF could identify potential targets for HF prevention in higher-risk individuals . One bioImportantly, data from 12 years’ worth of follow-up, after adjustment of risk factors, showed that a more androgenic hormone profile among post-menopausal women was associated with increased risk of HF . Whilst International guidelines such as those of the European Society of Cardiology  or the ANumerous recent studies have demonstrated that women are less likely to receive evidence-based medical therapy for treatment of HFrEF compared to their male colleagues , 6, 34. Differences in tolerability and efficacy between drug therapies provide evidence that women require different guidelines for medical therapy of HFrEF , 38. ForAdvances in electric and mechanical devices have continued with substantial benefits for patient symptoms, hospitalisation and outcomes, with strong evidence in both sexes  Table 11. The raThere is strong evidence, reflected in the guidelines, for cardiac rehabilitation (including education), and lifestyle modifications including salt reduction, weight loss, and exercise (strong evidence) and a whole grain plant-based diet (less evidence) , in imprp = 0.001), and this survival difference was not observed in men [The literature reports on multiple outcomes in HF, exploring mortality, quality of life, rates of hospitalisation and readmission, susceptibility to arrhythmia, and recovery of LV function. The previous body of evidence found no difference in-hospital mortality between sexes, which also correlated with several recent studies , 44. A fd in men .Previous literature had described that females with HF report lower quality of life, and this was reiterated in new data . DespiteRegarding rates of hospitalisations, most data, including more recent studies, showed that women have a lower risk of hospitalisation . Only onp = 0.04) [Although many studies have explored predictive factors for HF with recovered ejection fraction, few have sought to understand whether there is a gender discrepancy. More recent data has identified a link between female sex and left ventricular ejection fraction recovery –54. One  = 0.04) . Since p = 0.04) .An important consideration in the interpretation of the literature in regard to heart failure outcomes is the often-poor stratification of analyses and reports by acute versus ambulant or chronic heart failure, with less data available regarding sex differences in the acute heart failure setting. Data that is available suggests that, whilst co-morbidities at the time of presentation differ between the sexes, acute in-hospital outcomes are similar. For example, in the ALARM-HF multinational survey of over 4000 patient hospitalizations, men were more likely to have acute coronary syndrome as the precipitating event and underlying coronary artery disease, and women were more likely to have concomitant AF, obesity, anaemia and depression. In hospital, mortality, however, was similar in both sexes (11%) . This coFour studies closely analysed and quantified female representation in trials. Female participation in landmark HF trials ranged from 0 to 40% with an average of 20% . In an aUnder representation of females in clinical trials and lack of sex-disaggregated data means studies are often underpowered to apply results to clinical practice. For more than 25 years, there has been an acknowledgement of the sex discrepancy in HFrEF , and desThe sex differences in HF with reduced ejection fraction are undeniable. The pathophysiology, types of presentation, morbidity and mortality differ between the sexes. However, without more representation of women in clinical trials, the development of sex-specific management guidelines appears improbable."}
{"text": "Heart disease is the leading cause of death in both men and women in developed countries. Heart failure (HF) contributes to significant morbidity and mortality and continues to remain on the rise. While advances in pharmacological therapies have improved its prognosis, there remain a number of unanswered questions regarding the impact of these therapies in women. Current HF guidelines recommend up-titration of neurohormonal blockade, to the same target doses in both men and women but several factors may impair achieving this goal in women: more adverse drug reactions, reduced adherence and even lack of evidence on the optimal drug dose. Systematic under-representation of women in cardiovascular drug trials hinders the identification of sex differences in the efficacy and safety of cardiovascular medications. Women are also under-represented in device therapy trials and are 30% less likely to receive a device in clinical practice. Despite presenting with fewer ventricular arrythmias and having an increased risk of implant complications, women show better response to resynchronization therapy, with lower mortality and HF hospitalizations. Fewer women receive advanced HF therapies. They have a better post-heart transplant survival compared to men, but an increased immunological risk needs to be acknowledged. Technological advances in mechanical circulatory support, with smaller and more hemocompatible devices, will likely increase their implantation in women. This review outlines current evidence regarding sex-related differences in prescription, adherence, adverse events, and prognostic impact of the main management strategies for HF. Men and women have the same risk of developing heart failure (HF) throughout life. However, it is well-known that women develop the disease later in life. In addition, women have a higher prevalence of HF with preserved ejection fraction (HFpEF), the prevalence of which increases with age. This may partly explain the under-representation of women in pharmacologic and device therapy trials designed to treat HF with reduced EF (HFrEF) .Sex based differences in pharmacokinetics and pharmacodynamics of pharmacological agents may explain the variable effects in men and women. However, given the smaller number of women included in clinical trials of HFrEF, where they represent less than one-third of the study population, we do not have accurate information. Unfortunately, the results of large clinical trials are often not analyzed separately by sex and we only have subgroup analyses so they cannot be fully extrapolated to women . The samThere are important sex-dependent differences in pharmacokinetics (PK) and pharmacodynamics (PD) that need to be acknowledged to understand how specific cardiovascular drugs can affect women and men differently. The differences can affect absorption, metabolism, distribution, and elimination.For orally administered drugs, two main factors need to be acknowledged: compared to men, women (1) produce less gastric fluid, which can lead to a decrease in the absorption of weak acids and an increase in the absorption of weak bases and (2) have longer intestinal transit time , 4. The Total body water is greater in men, while women have a higher proportion of adipose tissue. Therefore, distribution volume for hydrophilic or lipophilic drugs varies according to sex.Plasmatic proteins involved in drug transport can be modulated by estrogens, resulting in a sex-dependent distribution , 6.Lower hepatic flow in women, sex-dependent activity of metabolic enzymes, increased proportion of adipose tissue and lower basal metabolic rate can explain differences in drug metabolism , 7, 8.In general, glomerular filtration, tubular secretion, and tubular reabsorption are higher in men , howeverLiver enzyme activity decreases in presence of elevated female hormone levels which may decrease drug elimination. Therefore, metabolism can change throughout the menstrual cycle, during pregnancy, with oral contraceptives intake or after menopause .An increased risk of death in women was reported in the DIG trial. Although it may have been related to higher digoxin levels in women, it could not be proven since digoxin levels were available in less than one third of the study patients .Women have higher plasma levels of beta-blocker (BB) due to decreased renal clearance (Cl) and smaller distribution volume (Vd) . Despitemax) and area under the curve (AUC) were found in women, the differences disappeared when adjusted for weigh (Sex-based differences have not been identified on the antihypertensive effects of angiotensin-converting enzyme inhibitors (ACEI), angiotensin-receptor blockers (ARB) and aliskiren . Althougor weigh .The potential effects of age and sex on the PK of Sacubitril/Valsartan were assessed in a study that enrolled 36 subjects, 50% male and 50% female: No sex-dependent differences were found in PK .max and AUC of torsemide are 30–40% higher in women due to reduced elimination . But safety and efficacy studies suggest the importance of dose adjustment based on body weight. In a DOAC meta-analysis including 66,389 patients (37.8% women), DOACs were associated with a significantly lower risk of major bleeding in women compared to men  and a higher risk of stroke and systemic embolism compared with men   established guidelines for the inclusion of women and minorities in clinical research. They recommend that clinical trials should enroll equal numbers of men and women in order to understand sex differences. Shortly thereafter, Congress approved these recommendations, and they became law. The Food and Drug Administration (FDA) published another regulation requiring detailed information by sex in clinical trials investigating new drugs, and therapies .Clinical trials however, unfortunately, remain underpowered to identify statistically significant treatment effects in both sexes.A recent study assessed the enrollment of women in 36 cardiovascular trials evaluating different drugs approved by FDA from 2005 to 2015. Adequacy between the percentage of women included in the trials and the prevalence of the female sex in the disease studied, was evaluated using the participation to prevalence ratio (PPR). A relationship between 0.8 and 1.2 was considered to reflect a good representation of women population. It should be noted that in the 3 HF trials included in this study women inclusion ranged from 22 to 40%. The overall PPR was 0.5, reflecting an inclusion of women in the trials well below their prevalence of the disease. More recent trials show the same pattern, ranging from 21 to 29% inclusion of women .In heart failure (HF) clinical trials, women represent approximately a quarter of patients with HFrEF and over half of those with HFpEF. However, epidemiologic data demonstrate a much higher proportion of women suffering the disease in the real world , 23 Fig.The differences between real world proportion of women with heart failure and their representation in clinical trials may depend on a variety of factors: more comorbidities, older age in women, fulfilling exclusion criteria more frequently, lower proportion of HFrEF, less investigator counseling or less personal availability, and willingness to enroll.The first clinical trials with ACEI date back to the late 1980s. A sub-analysis of the SOLVD trial  revealedWhen large trials of ARB in HFrEF populations  explored sex-specific treatment effect they found no differences in mortality or HF hospitalization between women and men . ELITE Ip < 0.0001). Conversely, there was no survival difference between ACEI or ARB in men   and 9,475 men , showed that women on ARBs had a better survival than those on ACE inhibitors, with a 31% relative risk reduction in all-cause mortality  .Hormone effects on angiotensin II receptor expression or differences in adverse events may explain the potential superiority of ARB in women.The studies assessing the role of mineralocorticoid receptor antagonist (MRA) in HFrEF (spironolactone in RALES and eplerenone in EPHESUS), showed no sex differences in prognosis , 31. In p = 0.051)  .The PARADIGM-HF trial, showed superiority of sacubitril/valsartan compared to enalapril at reducing mortality and HF hospitalization in patients with HFrEF . In subgp = 0.017)  compared to men 1.03  , primariIn conclusion, PARAGONF_HF subanalysis suggest that sacubitril-valsartan may lead to greater reduction in HF hospitalizations in women with HFpEF.post-hoc pooled analysis confirmed similar and significant benefits of BB  on combined end-point of all-cause mortality and all-cause hospitalizations in both women and men  in sinus rhythm with heart rate >70 bpm, showed a reduction in the composite primary outcome of CV death or hospital admission for worsening HF. Subgroup analyses did not show any sex-differences in efficacy or safety of ivabradine .In the last 6 years, several large cardiovascular outcome trials evaluated the effect of iSGLT2 in patients with type 2 diabetes and established cardiovascular disease or those with high cardiovascular risk, they have consistently shown to reduce the risk of hospitalization for heart failure –49.p interaction ≥ 0.17).A meta-analysis of SGLT2i including patients with type 2 diabetes enrolled in the EMPA-REG OUTCOME, CANVAS Program, DECLARE TIMI-58, and CREDENCE trials, showed  no sex dRecently, a meta-analysis condensing two single large-scale trials  in patients with HFrEF with or without diabetes assessing the effects of SGLT2i on cardiovascular outcomes have been published. SGLT2i reduced hospitalizations for HF and death, with an improvement in renal outcomes, regardless of sex and other conditions such as age, diabetes status, or baseline heart failure medications .The effects of diuretics on mortality and morbidity in chronic heart failure have not been studied in large clinical trials. There are no reported sex-related differences with diuretic therapy. Observational studies have shown a relationship between diuretics dose and mortality risk, which was maintained after adjusting for sex .p = 0.011) and no excess mortality in women at serum concentrations between 0.5 and 0.9 ng/ml, whereas serum concentrations ≥1.2 ng/ml was harmful .In the DIG trial, digoxin was associated with a significantly higher risk of death among women , with no increased risk in men . SubsequOverall, whereas higher digoxin levels tend to increase mortality in women, low concentrations seem to be safe and associated with improved symptoms.The A-HEFT trial enrolled more than 5,000 black women  with moderate to severe heart failure (NYHA class III-IV)  to test Treatment with hydralazine and isosorbide showed a significant reduction in mortality, first heart failure hospitalization, and change in quality of life at 6 months, with no differences between men and women.Women are known to have an increased adverse reaction (AR) to cardiovascular drugs compared to men (1.5–1.7-fold)  and haveWomen experience greater electrolyte imbalance with diuretic use, which in turn increases the arrhythmic risk. For instance, women have an increased risk of drug-induced torsades de pointes (2.3-fold) related to a longer corrected QT interval induced by the effects of estradiol on potassium and calcium channel modulation .A post hoc analyses of the DIG study  showed aWomen present higher plasma levels of beta-blocker due to a lower distribution volume  and a slower clearance. Dosage needs to be adjusted according to these differences to prevent AR.An increased risk of angioedema and cough (2-fold) has been described in women . MoreoveNo significant sex-differences in risk of kidney impairment, hypotension, or hyperkalemia have been described with the use of Losartan .There seems to be a higher withdrawal rate in men due to the appearance of gynecomastia (seen in 5.3% of the men) .Implantable Cardioverter Defibrillators (ICD) have shown to reduce sudden death risk in heart failure patients with reduced ejection fraction, especially of ischemic etiology. Therefore, they have a class 1A indication according to current guidelines for primary prevention in patients with left ventricular ejection fraction (LVEF) <35% despite optimal medical therapy .p < 0.001) were offered ICD implantation. Of note, the same proportion of men and women underwent the implant once it was advised . In another observational study 32.2 per 1,000 men and 8.6 per 1,000 women received ICD therapy. After controlling for demographic variables and comorbidity, men were 2.44 (95% CI 2.30–2.59) times more likely to receive an ICD compared to women.These recommendations are based on classical studies, such as SCD-HeFT , MADIT IThe reduced rate of ICD implantations in women may be related in part to the controversies regarding efficacy and higher risk of complications in women compared to men.p ≤ 0.001) compared to men and no significant benefit on mortality. In a European study . The reasons for the differences in the complication rate are unclear but could be related to delayed presentation or greater severity of illness. Smaller vessel size and a thinner walled right ventricle may explain a higher rate of pneumothorax or perforation. Increased bleeding risk have also been reported in women.ICD related complications have been reported more frequently in women. In the Ontario ICD Database  women weOverall, studies show sex differences in arrhythmic risk and ICD-related complications. Nevertheless, there is a risk of sudden death in women with HF and reduced LVEF that could be prevented by ICD implantation. Careful and individualized assessment is required to identify patients that would benefit the most from this therapy.Cardiac Resynchronization Therapy (CRT) has shown to improve functional capacity and survival amongst patients with LVEF <35%, left bundle block >130–150 ms and NYHA functional class II-IV and therefore has a class 1A indication in current HF guidelines . Severaln = 752, 28% female) and COMPANION  trials demonstrated similar reduction in mortality and time to hospitalization in both sexes after CRT implant. In the MASCOT  women showed better left ventricular remodeling and lower mortality and HF hospitalizations after adjustment for cardiovascular risk factors. Remarkably, women showed wider QRS and smaller left ventricle size at enrollment. In another study that only included patients with non-ischemic cardiomyopathy  CRT response among women was greater  than in men, despite similar baseline QRS duration (p = 0.025) and showed a trend toward lower heart failure hospitalization. In a metanalyses , QRS duration was the only independent predictor of CRT benefit. Further analysis showed the benefit was even more significant at lower height. There was a higher proportion of women amongst the wider QRS and shorter patients.The CARE-HF   mainly related to pneumothorax, infection or bleeding. The main risk factor for complications seemed to be size and body mass index both in women and men.As we discussed for ICD, complication rate seems to be higher in women after CRT implant. In the MADIT-CRT trial, women were twice as likely as men to experience a major procedure-related adverse event  female sex was associated with an increased risk of first neurological event , with no difference in other complications. In a later paper focusing on stroke rates during support with continuous-flow LVAD, female sex was also a predictor of stroke . The same was reported in an analysis of more than 900 HeartMate II outpatients  is the therapy of choice to improve survival in patients with end-stage HF. Mean survival after HT nowadays is 12.5 years for the adult population and 12–21 years for the pediatric population . Rejectip < 0.001)  .Women are younger than men at the time of listing (mean 48 year for women vs. 56 years for men), have less ischemic heart disease and more idiopathic dilated cardiomyopathy, and fewer cardiovascular risk factors such as smoking, diabetes mellitus, hypertension, or tobacco use . On the In the post HT period, women are at a lower risk of coronary allograft vasculopathy and malignancy. These differences could explain longer survival in female HT recipients.Pre-HT sensitization and post-HT rejection risk are higher in women, related predominantly to the presence of circulating preformed HLA antibodies due to sensitization from previous pregnancies . Donor-rIn summary, fewer women receive HT, despite their better long-term survival. Sex specificities need to be considered in the pre-HT evaluation , at the time of transplant (sex and size donor-recipient matching) and in the long-term post-HT follow-up  .Adherence to long-term therapies for chronic diseases in developed countries averages only about 50–75% . Inadequn = 7,599) and they found that 11% were poor adherers . Poor adherers were more likely to be women , have a higher heart rate, and a greater number of concomitant illnesses  between January 2000 and December 2008 who had a follow-up >1 year after HF drug treatment initiation. In this study women were more likely than men to be adherent to their treatment  (We can conclude that non-adherence to disease-modifying drugs is associated with an increased mortality and HF readmissions, but adherence seems to be similar between sexes .Although women represent 50% of the world population and despite similar overall prevalence of heart failure among men and women  women arWomen are generally more symptomatic than men when they present with HFrEF , which cWomen are also less likely to receive lifesaving therapies such as LVAD  and HT. Reproductive health counseling, teratogenic effect of HF medications and pregnancy management for women with HF are some important topics that affect women uniquely and that need to be a focus for future research and discussion, especially for those in need of advanced HF therapies and devices.Despite high prevalence of HF in women, there is lack of data on the use of drugs and HF therapies, with limited enrolment in randomized control trials and limited access to lifesaving strategies. Future trials should focus on greater enrollment of women in heart failure therapeutics and devote resources to understand the pathophysiology of the sex differences and disparities in access to advanced therapies.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."}
{"text": "The complement system plays an important role in the development of left ventricular hypertrophy. Complement C1q is an initial component of the classical complement pathway and is related to many inflammatory diseases. We aimed to determine whether there was an association between serum complement C1q and left ventricular hypertrophy induced by coarctation of the aorta (CoA).n = 15) and a complex CoA group (n = 13). Meanwhile, we selected simple large VSD (n = 14) patients and normal children (n = 28) as the control group. The serum complement C1q level was compared using immunity transmission turbidity among different groups.Based on whether CoA was combined with a large ventricular septal defect (VSD) or patent ductus arteriosus (PDA), the patients were divided into a simple CoA group (P < 0.05). There was no significant difference in the preoperative content of C1q between the complex CoA group and the large VSD group (P > 0.05). There was a negative correlation between the preoperative complement C1q content and the interventricular septal thickness and left ventricular posterior wall thickness . The percentage of postoperative decrease in C1q in children with simple CoA or complex CoA was positively correlated with the time of cardiopulmonary bypass and aortic cross clamp, respectively . There was no significant difference in the content of preoperative triglycerides (TG), total cholesterol (TCHO), high-density lipoprotein cholesterol (HDL-C) or low-density lipoprotein cholesterol (LDL-C) among the different groups (P > 0.05). In the simple CoA group and complex CoA group, the preoperative complement C1q, TG, TCHO, HDL-C and LDL-C levels were significantly higher than those after the operation (P < 0.05). There was no significant correlation between preoperative complement C1q and TG, TCHO, HDL-C or LDL-C (P > 0.05).The preoperative content of C1q in the simple CoA group was significantly lower than that in the complex CoA group and normal group (96.97 ± 20.66 vs. 130.73 ± 35.78, 96.97 ± 20.66 vs. 156.21 ± 29.14, Complement C1q has an inhibitory effect on the formation of left ventricular hypertrophy, which may not be mediated by regulating lipid metabolism. During cardiac surgery, complement C1q may have a protective effect against myocardial injury. Congenital coarctation of the aorta (CoA) is a common congenital heart disease. Its incidence is approximately 1/2500 live birth infants, and it accounts for 6 ~ 8% . Accordiβ-Catenin pathway [The complement system is an essential part of innate and adaptive immunity. It comprises of a group of proteins with enzyme activity in the blood, body fluids and on the surface of the cell membrane. It plays a biological function in regulating phagocytosis, clearing aging apoptotic cells, participating in the immune response and mediating the inflammatory response . C1q is  pathway .ThereforTherefore, determining the level of complement C1q in patients with CoA will be of great significance to further clarify the pathogenesis of left ventricular hypertrophy induced by CoA. In this study, we analyzed the content of complement C1q and the changes in complement C1q before and after surgery in patients with CoA and clarified the regulatory effect of complement C1q on CoA-induced left ventricular hypertrophy and the correlation between operation-related factors and complement C1q.Patients with CoA hospitalized in the pediatric heart center of Beijing Anzhen Hospital between January 1, 2017 and December 31, 2019 were included in this study. Inclusion criteria: (1) Meets the diagnostic criteria of CoA; (2) Clinical diagnosis of simple CoA or complex CoA  (defect diameter ≥ 10 mm) or CoA with a thick patent ductus arteriosus (PDA) , and a left to right shunt at ventricular level or large artery level); (3) Complement C1q examination was performed before and 24 h after the operation, and the clinical data were complete; (4) There were no other systemic diseases. Exclusion criteria: (1) patients with CoA who did not undergo surgery; (2) patients with an early death; (3) CoA with a small ventricular septal defect (defect diameter < 5 mm) or a small patent ductus arteriosus (pulmonary end of the ductus arteriosus < 2 mm), or CoA with other cardiovascular malformations; (4) CoA with a large ventricular septal defect or a thick patent ductus arteriosus and a right to left shunt at the ventricular or large artery level; (5) combined with other systemic diseases; (6) incomplete clinical data before and after the operation. Meanwhile, we selected children with simple large VSD with complete clinical data  and normal children as controls. The institutional review board for clinical research at Beijing Anzhen Hospital approved the use of patient medical records for this retrospective review.Based on whether CoA patients had a large VSD or PDA, the patients were divided into a simple CoA group and a complex CoA group . The control group was divided into a simple large VSD group and normal group.All patients were measured by transthoracic echocardiography with an ultrasonic instrument (PHILIP IE33) one day before the operation. The position and length of the aortic constriction were measured by two-dimensional echocardiography through the long axis section of the suprasternal aortic arch, and the blood flow velocity at the constriction was measured by color Doppler flow imaging. Left ventricular end diastolic diameter (LVDd), interventricular septal thickness (IVST) and left ventricular posterior wall thickness (LVPWT) were measured by M-mode echocardiography through the parasternal left ventricular long axis section. The diameter of the VSD and the left to right shunt velocity at the ventricular level were measured through multiple sections by two-dimensional echocardiography and color Doppler flow imaging. The diameter, length and left to right shunt velocity of the patent ductus arteriosus were measured by two-dimensional echocardiography and color Doppler flow imaging through the short axis section of the parasternal artery.IVST, LVPWT and LVDd were converted to Z scores (number of standard deviations from the expected mean) using the formula from Sluymans and Colan .Venous blood was taken from patients with an empty stomach in the case group one day before the operation and 24 h after the operation in the morning. Venous blood was taken from the children in the control group with an empty stomach in the morning. The serum was separated from the blood samples. The content of complement C1q was determined by a complement C1q determination kit (immunoturbidimetry) .The venous blood of patients with an empty stomach was taken before the operation, and serum samples were used to determine the contents of TG, TCHO, HDL-C and LDL-C.The intraoperative cardiopulmonary bypass time, aortic cross clamp time and mechanical ventilation time were collected.χ2 test was performed to analyze the sex distribution. A t-test was used to compare the differences between the two groups of normally distributed measurement data, and one-way ANOVA was used to compare the differences among three or more groups. The Mann–Whitney U test was used to compare the nonnormally distributed measurement data between the two groups. For the correlation between complement C1q and IVSTZ, LVPWTZ, LVDdZ, TG, TCHO, HDL-C, LDL-C, cardiopulmonary bypass time, aortic cross clamp time and mechanical ventilation time, if the data conformed to a normal distribution, pearson correlation analysis was used; if the data did not conform to a normal distribution, rank correlation analysis was used. P < 0.05 was considered statistically significant.SPSS 20.0 was used for statistical analysis. Measurement data are presented as the mean ± standard deviation  in the simple CoA group and 13 cases (46.4%) in the complex CoA group (11 cases (39.3%) were CoA combined with large VSD, and 2 cases (7.1%) were CoA combined with thick PDA). There were 42 cases in the control group, including 14 cases (33.3%) in the simple large VSD group and 28 cases (66.7%) in the normal group.There was no significant difference in sex, age, weight or height among the simple CoA group, complex CoA group, simple large VSD group and normal group (P > 0.05) Table .Table 1TP < 0.05). There was no significant difference in LVDdZ between the complex CoA group and the normal group (P > 0.05). There was no significant difference in IVSTZ and LVPWTZ between patients in the complex CoA group and the large VSD group (P > 0.05), but the LVDdZ in the complex CoA group was significantly lower than that in the large VSD group (P < 0.05)  Table .Table 2TP < 0.05)    (Table P < 0.05) (Table P > 0.05) (Table P > 0.05)   Table . In simp5) Table . There w5) Table . In the 5) Table .However,5) Table .Table 4Tr = − 0.035, r = − 0.288, P < 0.05)   Fig. .Fig. 2Tht = 0.515, P > 0.05). There was no significant difference in cardiopulmonary bypass time, aortic cross clamp time or mechanical ventilation time between the simple CoA group and the complex CoA group (P > 0.05)  (Table P < 0.05)   is a kind of localized aortic stenosis near the arterial catheter that was first described by Morgagni in 1760 . CoA canThe mechanisms of cardiac hypertrophy include cellular metabolism, proliferation, noncoding RNAs, immune responses, translational regulation, and epigenetic modifications . Immune β-catenin activation [The complement system is an important part of innate and adaptive immunity. Under physiological conditions, the activation and regulation of the complement system are in a balanced state. When the balance is broken, the complement system will attack the body’s own cells and tissues, which will lead to a variety of inflammatory reactions and autoimmune diseases . C1q is tivation . The C1qtivation . In 2019tivation . TherefoBased on our study, we found that the content of complement C1q in the plasma of patients with simple CoA was significantly lower, and their IVSTZ and LVPWTZ were significantly higher. By further analyzing the relationship between complement C1q and left ventricular hypertrophy, we found that the content of complement C1q before the operation was negatively correlated with IVSTZ and LVPWTZ. These data indicate that complement C1q has a certain inhibitory effect on the formation of left ventricular hypertrophy. Meanwhile, we also found that the IVSTZ and LVPWTZ were not significantly different between the complex CoA group and the large VSD group, which may be related to the large left to right shunts at the ventricular level, but the LVDdZ in the complex CoA group was significantly lower than that in the large VSD group. We think that this may be related to the reduction in the left to right shunt flow through the ventricular septum, which was induced by the obstruction of the left ventricular outflow tract caused by CoA.During cardiac surgery with cardiopulmonary bypass and aortic cross clamp, the heart is isolated from the circulation. This inevitably induces myocardial ischemia. In addition to this ischemic insult, an additional hit will occur upon reperfusion, which may worsen the extent of tissue damage and organ dysfunction . In 2020Mechanical ventilation can also promote the release of inflammatory mediators and activate the inflammatory response. Silvia Marchesi et al. reported that the blood IL6 and TNF-α ratio increased significantly in patients on mechanical ventilation . DhanireBy analyzing complement C1q in children with CoA before and 24 h after the operation, we found that the decreased percentage of C1q in children with simple CoA or complex CoA was positively correlated with the time of cardiopulmonary bypass and aortic cross clamp. In the simple CoA group, the percentage of postoperative decrease in C1q was positively correlated with the time of mechanical ventilation. However, in the complex CoA group, there was no significant correlation between the percentage of postoperative decrease in C1q and the time of mechanical ventilation. These results show that with the extension of cardiopulmonary bypass time and aortic cross clamp time, complement C1q is gradually consumed and reduced. Complement C1q may have a certain protective effect on myocardial ischemia reperfusion during cardiopulmonary bypass and aortic cross clamp. In terms of mechanical ventilation time, with the extension of mechanical ventilation time, complement C1q gradually decreases, which may be due to mechanical ventilation causing inflammatory reactions, and complement C1q protects other organs and tissues by inhibiting inflammatory reactions. The continuous activation and consumption of complement C1q in this process eventually leads to a gradual decrease in complement C1q.Abnormal lipid metabolism of cardiomyocytes can cause left ventricular hypertrophy. Celentano A found that hypercholesterolemia in normotensive nondiabetic adults is independently associated with a mildly concentric left ventricular geometry . IncreasIn 2004, Wong GM et al. identified C1q complement/TNF-related protein (CTRP) . To dateIn conclusion, in this study, by analyzing the content of complement C1q in the plasma of children with simple and complex CoA and the relationship between the content of complement C1q and LVPW, IVS and LVDd, we found that complement C1q has an inhibitory effect on CoA-induced left ventricular hypertrophy, which is negatively correlated with left ventricular myocardial hypertrophy. By analyzing the content of complement C1q in children with CoA before and 24 h after the operation, it was found that the decreased percentage of complement C1q after the operation was positively correlated with the time of cardiopulmonary bypass, aortic cross clamp and mechanical ventilation. We also found that there was no significant correlation between complement C1q and related indicators of lipid metabolism. The above results have certain clinical significance for clarifying the pathogenesis of left ventricular hypertrophy caused by congenital CoA and the role of complement C1q in myocardial injury during cardiac surgery.There were some limitations in our study. First, this was a retrospective study, there was only the value of complement C1q at 24 h after operation, and the values at other times after operation were incomplete. Therefore, we only analyzed the complement C1q level at 24 h after the operation. The time of echocardiogram reexamination after operation was inconsistent, so the correlation between postoperative complement C1q and LVDd, IVST and LVPWT could not be analyzed. Second, the number of patients included in this study is small. In the future, we will conduct a large clinical prospective study to further investigate the correlation between complement C1q levels and left ventricular hypertrophy induced by coarctation of the aorta.C1q is an important promoter of the classical complement pathway. In our study, we found that serum complement C1q levels in simple CoA patients showed lower level. In CoA patients, serum complement C1q had a negative association with interventricular septal thickness and left ventricular posterior wall thickness. The decreasing of serum complement C1q was an unfavorable factor for acute myocardial injury during cardiac surgery. Complement C1q may be related to the process of left ventricular hypertrophy induced by CoA and have a protective effect against myocardial injury during cardiac surgery."}
{"text": "Patients with Alzheimer’s disease show a sex-dependent decline of cognitive and behavioral performance. It is controversially discussed whether general anesthesia itself can aggravate or even cause this neurocognitive decline. Therefore, we investigated the effect of general anesthesia on neurocognitive and behavioral function and amyloidopathy in a mouse model of early-stage Alzheimer’s disease with respect to sex.After governmental approval 10 months old Tg2576 mice and wild type  either underwent general anesthesia with 1.0 minimal alveolar concentration of isoflurane for 2 h or were not exposed to isoflurane (controls). Following cognitive and behavioral testing using the modified hole board test (mHBT), brains were investigated regarding amyloidopathy, inflammation, and apoptosis. Data were analyzed using repeated measure analysis of variance (ANOVA) and univariate analysis of variance (UNIANOVA).p < 0.001), less anxiety (p = 0.022 and p = 0.024), increased locomotor activity (p = 0.025), and impaired fine motor skills (p < 0.001). Amyloid precursor protein (p < 0.001), soluble amyloid-beta (p < 0.001) and insoluble amyloid deposits (p < 0.001) were increased in Tg2576 animals. Neither sex nor exposure to isoflurane had an effect on cognitive or behavioral testing or expression of amyloid-related biomarkers.Tg2576 mice showed a decline in memory function (We found that 10 months old Tg2576 showed typical signs of early-stage Alzheimer’s disease and corresponding histopathological alterations. Relevant sex-specific differences or an effect of isoflurane anesthesia could not be detected at this early stage of the disease. Alzheimer’s disease (AD) is the most common neurodegenerative disorder worldwide. Due to medical progress and a rising life expectancy, the prevalence is increasing with an estimated total number of people living with AD dementia of 13.8 million in the U.S. in 2050 . While tAccording to epidemiological studies, women have a higher lifetime risk for developing AD . NeurodeWhile an interaction between anesthetics and amyloid-beta has been demonstrated , the relMice and humans are analogous regarding higher cognitive functions such as cognition, behavior and fine motor skills . The Tg2This study aimed to investigate the effects of general anesthesia on cognitive function and behavioral parameters in a model of early-stage AD with respect to sex. We therefore decided to expose 10 months old Tg2576 to general anesthesia with isoflurane and afterward compare them to their non-transgenic littermates  using the modified hole board test (mHBT). In order to assess amyloidopathy, neuroinflammation, and apoptosis we examined the brains for corresponding biomarkers.This study was carried out in strict accordance with the recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). The following experimental procedures on animals were approved by the Governmental Animal Care Committee . All surgical procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering. Animal welfare was assessed daily.°C, 60% humidity and free access to water and standard mouse chow) 2576Kha mouse model of AD, also referred to as Tg2576. With the approval of Taconic , male B6;SJL-Tg(APPSWE)2576Kha mice were crossed with female C57B6/SJL mice  in a separate breeding facility. The genotype was confirmed by PCR, using DNA from tail tissues . Mice homozygous for the rd1-mutation were excluded from the analysis as these mice suffer from blindness. At least 14 days prior to anesthesia and cognitive and behavioral testing, mice were transferred to a test facility for acclimatization. Mice were housed under standard laboratory conditions  .n = 9–12 mice per group) regarding isoflurane anesthesia or sham procedure using a computer-generated randomization list. The experimental groups were designed as depicted in For this study, 85 mice with 10 months of age were used , 44 Tg2576; 41 male, 44 female). Mice were randomly assigned to one of eight groups (°C ± 0.5°C). General anesthesia was maintained for 2 h with 1.6 Vol% isoflurane (MAC 1.0) and a fraction of inspired oxygen of 50% (FiO2 0.5) administered via a nose chamber. Mice breathed spontaneously with an applied positive end-expiratory pressure of 5 mbar.For induction of general anesthesia mice were placed in an acrylic glass chamber that had been pre-flushed with 4.5 Vol% isoflurane  and 50% oxygen. After loss of postural reflexes, mice were placed on a warming pad , gas concentrations, and rectal temperature were measured .After 2 h mice were again placed in the acrylic glass chamber with 50% oxygen now without isoflurane until full recovery from anesthesia. Afterward the animals were weighed and placed in single cages. During general anesthesia respiratory rate, heart rate .We evaluated two different parameters regarding behavior, cognition and motor skills, respectively. Parameters focusing on behavior are the latency with which the mice first visited the holes on the area of interest . Each brain was separated into hemicortices. One hemicortex was sliced into 21 sagittal slides of 50 μm and 21 sagittal slides of 10 μm . The other hemicortex was separated into prefrontal motor cortex, sensory cortex, and hippocampus. A total of 56 brains  were further analyzed.On day 9 after general anesthesia mice were anesthetized deeply using isoflurane and brains were harvested by decapitation using a guillotine . The samples were stored at −80n = 56 brains), we homogenized brain slices of prefrontal motor cortex in 10 volumes of cold guanidine–HCl buffer  and mixed them for 3–4 h at room temperature. The homogenates were diluted in 10 volumes of cold casein buffer , centrifuged , and the supernatant stored at −80°C  was used for the following ELISA. Samples and the standard solution were diluted with standard diluent buffer (Sample concentration: 0.075 μg/μl) and 100 μl of each were added to the appropriate wells and the plate was incubated in the refrigerator at 8°C on a 3D-shaker  overnight. The next day the wells were washed four times with washing solution, 100 μl of HRP-conjugated antibody solution were dispensed into the wells and the plate was incubated at room temperature on a different 3D-shaker  for 30 min. Wells were washed another four times with wash buffer and 100 μl Chromogen (Tetramethybenzidin) solution was added.To determine the total amount of Aβ per brain  and the standard curve was generated using the Magellan® software . We performed two determinations for each brain region and averaged the results for the final analysis.After then adding 100 μl of stop solution, the absorbance at 450 nm optical density was determined using the Sunrisen = 21 per brain) were investigated. The slices including sensory cortex and hippocampus were fixed on microscope slides  in −20°C acetone for 20 min. The staining protocol has been described previously  were suspended separately in grinding tubes  and 300 μl extraction-solution were added . The samples were then homogenized using pestles  for 2–3 min and afterward centrifuged for 30 min (13.000 rounds per minute at 4°C). After centrifugation the supernatant was stored at −80°C. The protein-concentration (by Bradford Assay) was standardized with Laemmli buffer . The samples were transferred onto the gel  in equal amounts (20 μl) and equal protein-concentrations (1 μg/μl) per lane for separation by gel electrophoresis and blotted onto a membrane . The membrane was blocked for 1 h and incubated afterward with the first antibody  overnight at 8°C on a 3D-shaker. After washing with TBS/T  the membrane was incubated with the second antibody  for 1 h on a 3D-shaker.Sensory cortex and hippocampus of seven animals per group . For analysis and normalization ImageLab® was used in addition to the Stain-Free® Technology to assess the total protein amount . A standard lane was included in every blot.Following another washing step, the membrane was incubated in the dark with Clarity™ Western ECL Substrate  for 1 min. The labeled proteins were detected with camera imaging . The material was then observed under a light microscope  with a 10×, 40×, and 100× objective. The full cycle in mice can be divided into 4 stages: Proestrus, estrus, metestrus, and diestrus. The determination of the respective cycle phase is based on the proportion among epithelial cells, cornified cells and leukocytes.Following decapitation and preparation of the brains, the reproductive cycle of each female mouse was assessed by vaginal cytology . TherefoParameters obtained in the mHBT were analyzed using repeated measure analysis of variance (ANOVA) to represent the learning process over eight consecutive days as well as univariate analysis of variance (UNIANOVA) to assess the mice’ performance on day 8 of neurocognitive and behavioral testing.Using repeated measure ANOVA, the dependent variables were tested by the between-group factors sex , genotype (Tg2576 and wild type), intervention (isoflurane anesthesia and sham), the within-group factor time (day 1–8), and their interaction terms. The values of testing on eight consecutive days are included in the model and effects of time were analyzed in a linear fashion (time) due to the strictly monotonic character of learning curves in these tests.In calculations using UNIANOVA sex , genotype (Tg2576 and wild type) and intervention (isoflurane anesthesia and sham) served as independent variables while parameters obtained in the mHBT on day 8 served as dependent variables.Post-hoc tests were performed if the interaction terms sex × intervention or genotype × intervention had proven to be significant.For determination of the effect size, we calculated mean differences with 95% confidence interval. post hoc comparisons, nine animals per group were found to be sufficient. In order to account for drop-outs we decided to aim for 12 animals per group in accordance with the Governmental Animal Care Committee.Regarding sample-size calculations, variables of the mHBT are considered relevant if two groups differ two times the given standard deviation. We planned a hierarchical model addressing three between-subject factors  resulting in eight randomized groups. Based on a type I error of 0.05, a type II error of 0.20 and with 64 possible Since distribution of the amount of amyloid-beta, the concentration of APP, Tumor necrosis factor (TNF) alpha and Caspase 3 in the western blot analysis, and as the percentage of brain area covered with amyloid deposits were positively skewed, the statistical analyses were performed following logarithmic transformation.Results of ELISA (amyloid-beta), western blot  and methoxy staining (amyloid deposits) (dependent variable) were compared using UNIANOVA with the additional factors sex, genotype and intervention, and their interaction terms (independent variable).p < 0.05. Calculations were done with SPSS Statistics® .The significance level was set at p < 0.001). Tg2576 visited a hole earlier during the trials than WT throughout the 8 days of testing  and on day 8 . Tg2576 spent more time on board than WT over the course of the mHBT  and on day 8  on the gray board of the mHBT and the time spent on this board  evaluated exploratory motivation and anxiety. Mice underwent 4 trials per day on 8 consecutive days. Each trial ended as soon as the mice found and ate the third bait or after a maximum of 300 s. LFHV decreased from day 1 to day 8 , irrespective of sex or intervention. On day 8 of the mHBT, Tg2576 visited more already emptied holes compared to WT . Tg2576 did less WCT compared to WT over the course of day 1 to day 8  as well as on day 8  and wrong choices total  were evaluated. If an animal revisited a previously emptied baited hole again, it counted as RC. If an animal did not visit a baited hole  or visited a non-baited hole (= wrong choice (WC), [number per trial]) it was later summed up as wrong choices total . Tg2576 did more RC than WT  and on day 8  during one trial and time for food intake . The floor of the four-sided mHBT apparatus is divided into 9 squares by two parallel lines between opposite walls and the gray board with the cylinders is placed in the middle . Wheneven = 44, WT = 21, Tg2576 = 23). Fourteen mice  were in estrus, 4 mice  in metestrus, 25 mice  in pro-estrus and 1 WT mouse (2.3%) in diestrus.The reproductive cycle of each female mouse was assessed . There were no significant differences regarding sex or intervention (The expression of APP was significantly higher in both the sensory cortex (SC) and hippocampus (HC) of Tg2576  than WT, where no amyloid deposits were present, irrespective of sex or intervention (The area [%] of isocortex (IC) and hippocampus (HC) covered with amyloid deposits was higher in Tg2576  of Tg2576 contained significantly more soluble amyloid-beta 1–42 compared to WT (mean difference = 1.23 × 106 units) in the HC was higher in male mice (Tg2576 and WT) compared to female mice (Tg2576 and WT) irrespective of genotype or intervention  in the HC was higher in Tg2576 compared to WT irrespective of sex or intervention , less anxiety (reduced LFHV and increased TOB), increased locomotor activity (increased LC), and impaired fine motor skills (increased TFI). The animals had pathognomonic signs of Alzheimer’s disease in the brains like increased APP, soluble amyloid-beta, and insoluble amyloid deposits. At this early stage of the disease, we did not detect any relevant sex-specific differences nor an effect of general anesthesia.During the course of behavioral testing all mice showed signs of habituation to the test situation : increasAPP was expressed significantly more in the HC and SC in Tg2576 regardless of sex or intervention . This reCaspase 3 as a marker for apoptosis was found significantly more in the HC of male mice, regardless of genotype or intervention . Female TNF alpha as a sign of neuroinflammation was expressed higher in the HC in Tg2576 . This fiThere is not only controversy whether or not general anesthesia might influence neurodegenerative diseases but also which anesthetics and what duration of exposure might be harmful or even beneficial . Han et The lack of sex-differences and the missing effect of isoflurane might be explained by our study conception. In a previous study from our department, female Tg2576 showed a pronounced cognitive decline at the age of 12 months when compared to male Tg2576 at 12 months. A female sex-dependent correlation of cognitive impairment to the amount of soluble amyloid-beta and amyloid deposits has also been demonstrated . We therBesides the missing effect of our intervention our study has other limitations. We chose to use an AD mouse model restricted to amyloidopathy, although in humans living with AD both amyloidopathy and tauopathy are found . Recent Tg2576 mice show typical signs of early-stage Alzheimer’s disease at 10 months of age: decline in working and declarative memory, less anxiety, increased locomotor activity, and impaired fine motor skills. We also found histopathological alterations in the brains typical for Alzheimer’s disease. Sex or general anesthesia do not seem to have an effect on cognition, behavior, or motor skills at this early stage of the disease.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Regierung von Oberbayern, Maximilianstr. 39, 80538 Munich, Germany, Chair: Dr. B. Wirrer, Registration number: 55.2-1-54-2532-67-2016, July 28th, 2016.LB: investigation, formal analysis, validation, writing – original draft and review, and editing. SB: investigation, writing – review, and editing. MB: methodology, resources, formal analysis, supervision, writing – review, and editing. CP: investigation and formal analysis. BU: formal analysis and validation. BJ: conceptualization, methodology, formal analysis, validation, supervision, writing – review, and editing. SS: conceptualization, methodology, investigation, formal analysis, validation, data curation, writing – original draft and review, and editing. All authors contributed to the article and approved the submitted version."}
{"text": "To study the clinical effect and influencing factors of kyphoplasty in the treatment of osteoporotic thoracolumbar compression fractures (OTCF) complicated with type 2 diabetes mellitus (T2DM).A total of 472 patients with OTCF complicated with diabetes who were enrolled in our hospital from January to December 2019 were selected as the study subjects, and all patients were treated with percutaneous kyphoplasty (PKP). The effects of gender, age, smoking, drinking, body mass index (BMI), bone mass density (T score), fasting blood glucose level, fasting C-peptide, glycosylated hemoglobin, course of T2DM, vertebral segment and surgical instrument on postoperative improvement were analyzed. The quality of life was evaluated by visual analog score (VAS) and Oswestry disability index (ODI) before PKP and 7 days, and 6 months after PKP, and the patient satisfaction was assessed by the modified Macnab criteria at 6 months postoperatively.P = 0.025), fasting blood glucose ≥8 mmol/L , glycosylated hemoglobin ≥6.5 mmol/L , duration ≥8 years  and Kyphon instrument  were independent influencing factors of OTCF complicated with DM.The overall excellent and good rate of evaluation result was satisfactory. In multivariate regression, independent risk factors for poor patient satisfaction included: age ≥70 years (odds ratio (OR) = 2.298, 95% confidence interval [CI] 1.290–4.245, Kyphoplasty for patients with osteoporotic thoracolumbar compression fractures complicated with diabetes can achieve a satisfactory clinical effect, the curative effect is affected by many factors, attention to these factors can improve the clinical effect. Diabetes mellitus (DM) and osteoporosis are major public health problems in the aging population affecting people all over the world and are of serious health and financial concern . DM can This was a retrospective cohort study, which included 472 patients with OTCF complicated with diabetes at our hospital from January to December 2019. We reviewed each patient's clinical records and built the database.Inclusion criteria: (1) OTCF was confirmed by low-intensity signal changes on T1-weighted image, high-intensity changes on T2-weighted image preoperatively; (2) bone mass density (BMD) T score≤−2.5 SD; (3) OTCF patients who underwent PKP surgery; (4) patients with T2DM; (5) follow-up time >6 months; (6) complete clinical and imaging data.Exclusion criteria: (1) acute vertebral compression fracture caused by severe trauma such as car accidents and falling injury; (2) primary or metastatic tumors; (3) multiple myeloma or other systemic diseases; (4) infection; (5) previous spinal surgery; (6) allergic to surgical supplies. This study was approved by the Ethics Committee of Xi'an Honghui Hospital, and all patients obtained preoperative informed consent.All patients were placed in prone position on a spinal reduction stent prior to surgery to maximize reduction of the compressed vertebra. Routine skin disinfection was carried out in the surgical area, sterile towel sheets were laid down and skin care membranes were pasted. The compressed vertebral body was fluoroscopically located, local anesthesia was performed with 1% lidocaine, and incisions about 5 mm long were made on either side of the injured vertebral body. Under fluoroscopy, the pedicle surface of the injured vertebral body was slotted using the apex, and the apex was driven into the pedicle. Pull out the inner core of the apex and insert the guide wire into the vertebral body under fluoroscopy. At this point, pull out the sleeve of the apex vertebra and insert it into the channel along the direction of the guide needle under fluoroscopy. Pull out the guide pin and insert the drill bit into the channel to drill. After that, the drill bit was taken and put into the dilator, and the contrast agent was injected into the balloon. Under fluoroscopy, whether the contrast agent leaked into the blood vessel was observed. After seeing the balloon, it was opened to make the fracture part reset. Remove the contrast agent from the balloon and draw out the dilator. The bone cement sleeve was inserted into the channel, and an appropriate amount of bone cement was injected into the vertebra from both sides of the pedicle under fluoroscopy, and satisfactory filling effect was achieved. The sleeve and channel were pulled out after the cement hardened. The incision was sutured and covered with compresses. All patients were prescribed calcium tablets , calcitriol (0.25–0.5 μg/day) and alendronate (70 mg/week), postoperatively.The effects of gender, age, smoking, drinking, BMI, BMD T score, fasting blood glucose level, fasting C-peptide, glycosylated hemoglobin, course of T2DM, vertebral segment and surgical instrumentation postoperative improvement were analyzed. The Visual Analog Scale (VAS) and the Oswestry Disability Index (ODI) were routinely administered to all patients. Meanwhile, the quality of life was evaluated by VAS scores for low back pain and ODI indices for the functional assessment preoperatively, 7 days and 6 months postoperatively, and the modified Macnab criteria were used to assess the patient satisfaction . Accordit-test, Mann–Whitney U-test, chi-square test, and Fisher exact test were used to evaluate differences between these groups. Multivariate binary Logistic regression analysis were performed under single factor. P-value < 0.05 was considered statistically significant.EpiData3.5 software  was used to establish the database. All analyses were conducted using SPSS version 26  and GraphPad Prism version 9.3.0 . Specifically, Student 2 and −3.0 ± 0.3 SD, respectively. The mean fasting blood glucose, fasting C-peptide and glycosylated hemoglobin were 8.0 ± 2.4 mmol/L, 0.67 ± 0.24 nmol/L and 6.5 ± 1.3 mmol/L. The mean course of disease was 8 ± 1.5 years. In terms of instrumentation, KMC system was used in 334 patients and Kyphon was used in 138 patients. There were 108 cases with T11 fracture, 120 cases with T12 fracture, 138 cases with L1 fracture, 106 cases with L2 fracture.In this study, a total of 472 patients meeting the criteria were included with a follow-up period of 6 months. The general characteristics of the patients are summarized in P < 0.001). The ODI score was 72.5 ± 17.7 preoperatively, which declined to 30.2 ± 16.8 at the final follow-up with a mean decrease of 42.3 ± 9.8 (P < 0.05). According to the modified Macnab criteria for patient satisfaction, the clinical good-to-excellent rate was 93.6% for all parentis: excellent- 228 (48.3%), good- 214 (45.3%), fair- 20 (4.2%), and poor- 10 (2.1%). Among the follow-up cases, there were 26 cases of adjacent vertebral body fracture and 4 cases of distal vertebral body fracture.All patients were followed up for 6 months. Preoperative and postoperative VAS scores of low back pain and ODI evaluations are shown in P > 0.05). Age, fasting blood glucose, fasting C-peptide, glycosylated hemoglobin, duration and instrument were correlated with patient satisfaction after PKP (P < 0.05). As shown in P = 0.025], fasting blood glucose , glycosylated hemoglobin , duration  and instrument .The results of univariate analysis were shown in In this study, it was found in the 6-month follow-up after surgery that the overall excellent and good rate of patient satisfaction was 93.6%, indicating that the use of kyphoplasty can achieve relatively satisfactory clinical results, but there were 15 patients with poor results, and it was found that the blood glucose control of these 15 patients was not ideal, all of them were severe osteoporosis. Furthermore, during the follow-up of this study, 13 cases of compression fractures occurred in the adjacent vertebrae of surgically reshaped vertebrae and 2 cases of distal vertebral fractures. Therefore, effective control of blood glucose is an effective prerequisite for preventing fractures in diabetic patients. In addition, the results of this study showed that age ≥70 years, fasting blood glucose ≥8 mmol/L, glycosylated hemoglobin ≥6.5 mmol/L, duration ≥8 years and Kyphon instrument were independent influencing factors of OTCF complicated with T2DM.Age is an important factor affecting the degree of osteoporosis, and bone loss increases with age . PreviouFasting blood glucose is one of the most frequently used factors reflecting blood glucose level in patients with T2DM. Previous studies have shown that long-term poor blood glucose control can lead to an increase in advanced glycation end products, while the continuous accumulation of advanced glycation end products can inhibit osteoblast differentiation, thus promote osteoblast apoptosis and osteoclast formation, resulting in a decrease in bone mass and aggravation of osteoporosis , 15. In At present, most studies have confirmed that glycosylated hemoglobin ≥6.5% (≥48 mmol/mol) can irremediable long-term risk for diabetic complications and mortality . Lim et The present study demonstrated that the duration of diabetes ≥8 years was an independent risk factor for poor patient satisfaction after PKP treatment in OTCF patients with diabetes. An increasing number of studies have shown that the longer duration of diabetes affects the health-related quality of life , 23. TakIn our study, patients who used surgical instruments using KMC had a higher recovery rate after surgery than those who used Kyphon. Combined with the result of logistic regression analysis in this study, Kyphon instrument was independent influencing factor of OTCF complicated with T2DM. After analyzing for underlying reasons, we found some possibilities as follows: (1) When KMC was used in surgery to expand the coronal surface centered on the channel, it was not easy to produce pressure on the lateral wall of the vertebral body, so the possibility of bone cement exudating and pressing blood vessels and nerves could be minimized to the greatest extent. (2) KMC mainly expands in the height direction of the vertebral body, which can effectively restore the height of the compressed vertebral body. It is point-shaped when opened, so that the bone cement can pour into the vertebral body from all directions, while Kyphon evenly spreads the balloon, which is not easy to control in the direction and the permeability of the bone cement is not satisfactory , 26. TheThe limitations of the present study mainly include the following items: (1) the clinical data were collected in our hospital, so it lacked comparison with different centers. (2) The patient was followed up for a short time, so the results of long-term follow-up are needed to support our conclusions. (3) The clinical efficacy of kyphoplasty is affected by many factors, the indicators used in this study are not comprehensive, so the results are inevitably biased.To sum up, kyphoplasty for treatment of OTCF with diabetes can play a more satisfactory clinical effect, its curative effect is affected by age, duration, fasting blood glucose levels, glycosylated hemoglobin, duration and type of instrument."}
{"text": "This cross-sectional study uses health administrative data to examine trends in diabetes incidence among children during the COVID-19 pandemic in Ontario, Canada. There is no clear mechanism by which COVID-19 infection might cause new-onset diabetes.2 Kamrath et al3 recently reported an increase in type 1 diabetes incidence among children in Germany during the pandemic. They did not observe an association of COVID-19 and increased type 1 diabetes incidence in the months after infection or an increase in the frequency of autoantibody-negative type 1 diabetes, which prompts the question of whether COVID-19 infection is associated with incident type 1 diabetes.3 Given the challenges of ascertaining a COVID-19 infection history for children with new-onset diabetes, additional population-based studies investigating changes in diabetes incidence among children during the pandemic are needed. Canada has one of the highest incident rates of type 1 diabetes worldwide. Therefore, this study examined whether diabetes incidence increased during the COVID-19 pandemic among children and youths (aged <18 years) in Ontario, Canada.A recent study reported an association between COVID-19 infection and new-onset diabetes among people younger than 18 years in the US.STROBE reporting guideline.For this population-based, repeated cross-sectional study, data use was authorized under Section 45 of Ontario’s Personal Health Information Protection Act and therefore did not require research ethics board review or informed consent. The study followed the We used health administrative data (January 2017 to September 2021) linked using unique encoded identifiers, held and analyzed at ICES  in Ontario, Canada. We included all children and youth (aged 1-17 years) eligible for universal health care insurance  on January 1 of each year (2017 to -2021).4 We used generalized estimating equations for Poisson regression to model 3-year pre–COVID-19 rates adjusting for age group, sex, pre–COVID-19 month, and secular trend. We then used these models to estimate expected post–COVID-19 monthly rates (95% CIs) using 2-sided hypothesis tests .Ontario has a population of approximately 14.8 million people; 3 million are younger than 18 years. Between November 2020 and April 2021, an estimated 3.3% of children in Ontario had SARS-CoV-2 infection.There were 2 700 178 children in the 2021 cohort; the mean (SD) age was 9.2 (4.9) years, and 48.7% were girls. Overall, there was no difference in observed vs expected relative rates (RRs) of new diabetes presentations  . However3 who reported a 1.15-fold increase in type 1 diabetes incidence among children in Germany during the pandemic.3 Limitations of our study include its smaller population and therefore lower power; thus, we cannot rule out a 1.3-fold increase in RRs. An advantage of our study is that we report monthly variations in post–COVID-19 diabetes incidence showing a decline then an increase in rates, suggesting possible delays in diabetes diagnosis for children early in the pandemic with a catch-up effect. Although we are unable to differentiate type 1 and 2 diabetes, 95% of children with diabetes in Ontario have type 1.6 The lack of both an observable increase in overall diabetes incidence among children during the 18-month pandemic restrictions and a plausible biological mechanism calls into question an association between COVID-19 and new-onset diabetes. Given the variability in monthly RRs, additional population-based, longer-term data are needed to examine the direct and indirect effects of COVID-19 and diabetes risk among children.In this cross-sectional study, we observed a slightly higher but nonsignificant increase in diabetes incidence among children during the COVID-19 pandemic. Our overall rate ratio is similar to that of Kamrath et al,"}
{"text": "HighlightsWe question certain aspects of the recent Centers for Disease Control and Prevention (CDC) Morbidity and Mortality Weekly Report (January 2022) showing a significant increase in the incidence of diabetes in children after COVID‐19 infection.We are concerned at the source of data being limited to commercial health insurances and lack of factoring race, ethnicity, social determinants of health, body mass index, type of diabetes, and viral variants in the interpretation of these data. It is well known that multiple viruses  are associated with triggering development of type 1 diabetes in genetically susceptible individuals. In January 2022, the CDC, in their Morbidity and Mortality Weekly Report, published a study suggesting that youth under 18 years old had a higher rate of developing diabetes more than 30 days after COVID‐19 infection.Significantly higher rates of diabetic ketoacidosis (DKA) were noted in patients with a previous COVID‐19 diagnosis who went on to develop diabetes, 40.2%  and 48.5% (IQVIA), compared to DKA reported in patients with new onset diabetes who did not have a recent COVID‐19 diagnosis .The report's strengths are that it utilizes large sample sizes  and captures data in the USA since the advent of the pandemic for at least 12 months. Having age‐ and sex‐matched control groups adds to the strengths of the study. It also corroborates with prior US data showing an increased risk for diabetes in the adult population with COVID‐19 and is consistent with studies from Europe that similarly report an increased incidence of pediatric diabetes associated with COVID‐19 infection. Further, the incidence of diabetes in the non‐COVID cohort in the IQVIA database was 32.6/100000 children, consistent with data from the SEARCH study, which reported incidence of diabetes in youth at 36.1/100000 children.The authors appropriately list several limitations that are important to consider. Significant details regarding glycosylated hemoglobin levels, presence of autoantibodies, and characterization of diabetes into type 1 or type 2 were not presented. Patients were only followed for limited periods of time and longer‐term follow up would be required to assess whether diabetes was transient or persistent. It is well established that the risk for both types of diabetes varies significantly between different racial and ethnic groups and that COVID‐19 has disproportionately affected disadvantaged groups.Additionally, the data do not represent uninsured children (5%) or those on Medicaid (29%), who encompass over one‐third of the pediatric population in the USA, a substantial portion that may be disproportionately affected by COVID‐19 and type 2 diabetes. Thus, the true incidence of diabetes may be much higher, and risk of developing type 1 vs type 2 diabetes is likely quite different and considerably more complex than what we can glean from the data presented.The timing of presentation for diagnosis of diabetes should also be considered. Because children with prediabetes and diabetes are often asymptomatic, diabetes may have been incidentally diagnosed when presenting to the clinician with COVID‐19 symptoms. Thus, the acute infection may have brought children to medical attention sooner, allowing for the diagnosis of diabetes to be made at presentation.Also, this report is only applicable to the COVID‐19 viral variants present from March 2020 to June 2021 and does not represent the omicron surge that has been sweeping our country and has affected larger numbers of children than any prior wave of the pandemic.Lastly, while the conclusions in the article highlight vaccination as a strategy to prevent new onset diabetes after COVID‐19 infection, this study did not evaluate how vaccination status affected diagnoses of new onset diabetes in the pediatric population. While vaccination is important to help prevention of well‐documented sequelae, to our knowledge, there are no data to support that vaccination will prevent diabetes, and this takeaway message should be interpreted with caution.In conclusion, this observational study presents preliminary data of an association between new onset diabetes and recent COVID‐19 infection; it does not establish causation. Further prospective studies are required to confirm these findings.None.The authors have no conflicts of interest to disclose."}
{"text": "Bile cast nephropathy (BCN) or cholemic nephropathy (CN) is an acute renal dysfunction, including acute kidney injury (AKI) in the setting of liver injury. It is a common phenomenon in patients with liver disease and is associated with significant morbidity and mortality. CN is characterized by hemodynamic changes in the liver, kidney, systemic circulation, intratubular cast formation, and tubular epithelial cell injury. CN has been overlooked as a differential diagnosis in chronic liver disease patients due to more importance to hepatic injury. However, frequent and considerable reporting of case reports recently has further investigated this topic in the last two decades. This review determines the evidence behind the potential role of bile acids and bilirubin in acute renal dysfunction in liver injury, summarizing the implied pathophysiology risk factors, and incorporating the therapeutic mechanisms and outcomes. The term bile cast nephropathy (BCN), also known as cholemic nephropathy, icteric nephrosis, or cholemic nephrosis, is described as acute renal dysfunction, including acute kidney injury (AKI) in the setting of liver injury . QuinckeThe AKI in cholestatic liver dysfunction is usually linked with other unfavorable factors such as hypovolemia, endotoxemia, and exposure to nephrotoxins . On the BCN is underdiagnosed and is witnessed as a neglected injury found in the autopsy of patients with hyperbilirubinemia and renal dysfunction. The pathophysiology of AKI in the setting of hyperbilirubinemia is multifactorial and involves a wide range of mechanisms. This review aims to determine the evidence behind the potential role of bile acids and bilirubin in acute renal dysfunction in liver injury. It also summarizes the implied pathophysiology risk factors and incorporates the therapeutic mechanisms and outcomes.Bilirubin and cholemic nephropathyExcess bilirubin has detrimental effects on kidney tubule function and intracellular mitochondrial function. Bilirubin aids in oxidative stress to kidney tubular epithelium leading to damage to the tubules and associated renal structures ,4,8. A sMoreover, hyperbilirubinemia can cause uncoupling oxidative phosphorylation in the mitochondria ,8. OxidaIn contrast to bilirubin’s harmful effects, it also exhibits debatable renoprotective effects . It is aBile acids and cholemic nephropathyMost bile acids are usually reabsorbed in the ileum and transported via portal blood circulation back to the liver. It is estimated that the liver does not take up about 10-50% of reabsorbed bile acids, making them escape in the peripheral circulation leaving behind 100 μmol bile acids per day prone to glomerular filtration . OrganicWith the above theory in context, it is worth noting how these mechanisms are affected in the event of cholestatic diseases such as obstructive jaundice, primary biliary cirrhosis, and primary sclerosing cholangitis. To counteract the rising bile salts in these pathologies, hepatocytes heighten basolateral hepatocellular export and enhance renal filtration and tubular secretion in the proximal tubules via organic anion transporters and multidrug resistance-associated proteins ,6. ThereHemodynamic changes due to hyperbilirubinemiaHRS is a life-threatening complication that compromises renal function, especially in patients with advanced liver disease -10. The Moreover, in patients with liver cirrhosis, endotoxins are released due to the translocation of bacteria and pathogen-associated molecular patterns (PAMPs) from the gut and prompt systemic inflammation ,4. SpeciApproach and diagnosisCurrently, a kidney biopsy is the gold standard diagnostic test for CN. In autopsy evaluations of CN patients, the kidney’s cortex and medulla appear yellow due to bilirubin. After fixation with formalin, color changes to green due to bilirubin oxidation and conversion to biliverdin ,27. On hBiomarkersAKI is associated with high morbidity and mortality and can occur in patients with severe liver disease. Proximal tubule cells are sensitive to hypoxic injury, leading to a release of proteins into the urine. Several promising urinary biomarkers may be used to evaluate AKI. The most studied is neutrophil gelatinase-associated lipocalin (NGAL), a 25 kDa iron-transporting protein excreted in nephrotoxic or ischemic kidney injury. Urinary(u) NGAL levels in CN were shown to be suitable to monitor tubular epithelial damage and therapeutic effects under experimental conditions ,45. The Treatment strategies and outcomesThe definitive diagnosis and subsequent treatment of CN are challenging, especially considering that a few case reports are diagnosed postmortem through autopsy, and adequate treatment was not provided. Treatment of CN is primarily based on treating the underlying cause of hyperbilirubinemia to prevent kidney injury . In the Extracorporeal therapies are possible treatment options in patients with CN and are divided into biologic and nonbiologic. Biologic therapies use living liver cells, while nonbiologic therapies use artificial membranes and adsorbents . These therapies can reduce inflammatory cytokines and bilirubin levels. Plasmapheresis is when plasma is separated from the blood, filtered, and returned to the patient. This therapy aids in the removal of excess toxins and bilirubin and replenishes albumin, coagulation factors, and hepatic regenerative stimulating substances to improve the symptoms of the patient .On the other hand, hemodialysis is a complicated process involving blood filtration and regulating fluid balance. The case report by Sens et al.  utilizesFuture implicationsThere are no systematic guidelines for diagnosing, treating, and managing a patient presenting with CN. CN is a diagnostic dilemma, and more reasonable diagnostic alternatives are crucial in approaching suspecting patients with CN. The transjugular approach for kidney biopsy may be an effective option considering the associated coagulopathic risks with traditional kidney biopsy in liver cirrhosis patients . HoweverCN is an uncommon diagnosis but a common finding in patients with liver disease. Suspicion and meaningful consideration should be given to BCN in non-respondents to HRS treatment. The kidney biopsy is an accurate diagnostic, and the transjugular approach can be a better alternative to traditional biopsy to expedite the diagnosis by simultaneously obtaining liver and kidney biopsies, also lessening the bleeding risk in high-risk patients. This review is crucial in suggesting the various mechanisms, diagnostic techniques, and treatment approaches to BCN."}
{"text": "Adcy1 reduced morphine-induced hypersensitivity compared to control mice. In contrast, acute morphine antinociception, along with thermal paw withdrawal latencies, motor performance, exploration in an open field test, and burrowing behaviors were not affected by intrathecal Adcy1 knockdown. Knockdown of Adcy1 by intrathecal injection also decreased inflammatory mechanical hyperalgesia and increased burrowing and nesting activity after intraplantar administration of Complete Freund’s Adjuvant (CFA) one-week post-injection.Opioid tolerance, opioid-induced hyperalgesia during repeated opioid administration, and chronic pain are associated with upregulation of adenylyl cyclase activity. The objective of this study was to test the hypothesis that a reduction in adenylyl cyclase 1 (AC1) activity or expression would attenuate morphine tolerance and hypersensitivity, and inflammatory pain using murine models. To investigate opioid tolerance and opioid-induced hyperalgesia, mice were subjected to twice daily treatments of saline or morphine using either a static  or an escalating tolerance paradigm . Systemic treatment with an AC1 inhibitor, ST03437 , reduced morphine-induced hyperalgesia in mice. Lumbar intrathecal administration of a viral vector incorporating a short-hairpin RNA targeting  On a cein vitro  and in via (DRG) .To date, nine membrane-bound AC isoforms (AC1-9) and one soluble isoform (AC 10) have been confirmed in mammalian nervous systems . AC1 is Adcy1 expression in mice. Mechanical withdrawal thresholds were measured in morphine tolerant and morphine withdrawn mice. Similarly, several evoked and spontaneous behavioral measures were used to determine if Adcy1 knockdown would also decrease hypersensitivity or improve mobility in a mouse model of inflammatory pain.Although all of the underlying mechanisms behind tolerance and opioid-induced hyperalgesia are not currently known, increased AC expression and activity have been suggested to be one of the major causative agents. To date, it is unknown if selectively inhibiting AC1 activity or reducing AC1 expression after chronic MOR stimulation alters the development of opioid tolerance and opioid-induced hypersensitivity. The purpose of this study was to better understand how the activity of AC1 in the spinal cord and dorsal root ganglia contributes to hypersensitivity seen during morphine tolerance, opioid-induced hypersensitivity, and chronic inflammatory pain. To accomplish this, systemic pharmacological inhibition of AC1 or intrathecal delivery of a short hairpin RNA (shRNA) through a viral vector was used to decrease ad libitum. Mice were kept in conventional microisolator cages with no more than five animals per cage. The cages contained irradiated corn cob bedding enriched with aspen and cotton nesting materials. After drug administration and CFA injections, mice were monitored for overall wellbeing and any adverse reactions. Mice were acclimated to individual testing apparatuses before behavioral testing. All experiments were conducted during the 14 h day cycle except for nesting behaviors. Mice were euthanized by isoflurane anesthesia (5%) followed by decapitation at the end of the study. A Table of experimental studies in provided in All experimental procedures involving animals were approved and performed under the University of Minnesota Institutional Animal Care and Use Committee guidelines. Adult male and female C57Bl6 mice were obtained via Charles River  having an average weight of ∼25 g (23–31 g). Mice were housed in a facility ranging from 20 to 26°C on a 14 h light/10 h dark cycle with water and rodent chow (Purina 5015) In behavioral studies, 5–10 male and female mice were randomly assigned to either a control or ST034307 treatment group. Viral vector studies consisted of two treatment groups, each containing 10 randomly assigned male and female mice. Use of male and female animals is consistent with the National Institute of Health’s Sex as a Biological Variable policy. For all experiments, male mice were tested before female mice and equipment cleaned in between testing of sexes.Morphine  was administered through a 100 µl subcutaneous injection in saline. For morphine tolerance experiments, baseline mechanical paw withdrawal testing was performed before and after administration of 15 mg/kg of morphine for 5 days . BaselinST034307  was dissolved in 10% β-cyclodextrin with 5% DMSO in saline and administered in a 100 µl intraperitoneal injection. ST034307 or vehicle was administered 15 min after morphine in tolerance experiments.In separate experiments, Complete Freund’s Adjuvant  was administered through an intraplantar injection  into the left hind paw.® 2450, IITC Life Science, Woodland Hills, CA, United States). The plantar surface of the hind paws was gently pressed with the probe until a nocifensive response  was elicited. Baseline measurements were collected five times from both the right and left hind paw and averaged, with an interstimulus interval of at least 1 minute.Mice were acclimated to the testing environment on at least two separate occasions for 30–60 min before formal testing. The testing environment consisted of a mesh floor, allowing access to animal hind paws, and individual clear acrylic chambers. Mechanical paw withdrawal thresholds, in grams, were determined by electronic von Frey testing equipment . Viruses were delivered by direct lumbar puncture (10 uL) in awake mice and behavioral assessments were performed 3–8 weeks post-injection . CFA was injected into a cohort of mice 7-weeks post inoculation. CFA treated mice underwent a series of behavioral tests 3–4 weeks after virus injections but before CFA administration  and after CFA administration .Four weeks post-virus injections morphine efficacy was determined using an escalating dose-response curve (5–20 mg/kg) waiting 30 min after each injection . MorphinAgility assessment was conducted using Rotamex-5 automated rotarod system . Mice were placed onto a stationary knurled PCV rod suspended in the air. The initial rotation speed of 4 rpm was gradually increased by 1 rpm in 30-s intervals until animals fell off the rod or reached a speed of 14 rpm (300 s). Two tests were administered per animal and averaged.Latencies to a radiant light beam focused on the plantar surface of each mouse hind paw were obtained using a modified Hargreaves Method  . The avehttp://ethowatcher.paginas.ufsc.br/) . The distance traveled (cm), time spent immobile (sec), average velocity (cm/s), and the change in orientation angle (degrees) were computed by using data output from the Ethowatcher computational tool software  .Adcy1 and AAV9-scramble mice were subjected to burrowing testing. Mice were acclimated to empty burrowing tubes for ∼2 h on at least two separate occasions before formal testing. The burrows were made from a 6 cm diameter plastic pipe and 5 cm machine screws were used to elevate the open end by 3 cm , AAV9- by 3 cm . During ad libitum overnight. A single 2” Nestlets™  square was weighed and added to each cage. The next morning (∼14 h) untorn pieces of each nesting square were weighed and the resulting nests were photographed and scored on a 5-point scale as described previously  and RNeasy Mini Kit  according to the manufacturer’s protocol with 30 min DNase 1 digestion. Complementary DNA synthesis was performed with 50 ng total mRNA using Omniscript RT Kit  and random nonamers  according to the manufacturer’s protocol.6 µmoles) (6.022 × 1023copies/mole). Internal controls included negative RT-PCR samples and comparative expression versus housekeeping genes, 18S and Gapdh. Amplification efficiencies were >1.8 and the targeted ΔCt between two dilutions was around -3.3. Fold expression of each gene of interest was determined by: (mean gene concentration/mean 18s concentration)/. See Quantitative PCR was performed using SYBR Green I dye with a LightCycler 480 machine . The cDNA copy number was typically quantified against a ≥5 point, 10-fold serial dilution of a gene-specific cDNA standard (∼5e1 to 5e6 copies/µL) cDNA standards were created using block PCR for one gene  and purified using Qiaquick (Qiagen). Standards were quantified using a UV-Vis spectrophotometer and DNA copies/µL calculated using the equation: (DNA µg/µl) (µmoles/DNA m. w. µg)  ELISA Kit  kit according to manufacturer instructions. Briefly, spinal cords were homogenized in RIPA lysis buffer  with added protease  and phosphatase  inhibitors. Spinal cord homogenates were centrifuged and further diluted in Sample Diluent provided by ABclonal. After incubation in Biotin Conjugate Antibody, Strepavidin-HRP, and TMB substrate, each well was read in microplate reader  at 450 nm with a wavelength correction at 570 nm. Samples were compared against an 8-point standard calibration provided by ABclonal.Histological sections were taken from spinal cord, DRG, and sciatic nerves to verify the delivery of the AAV9 virus within the lumbar intrathecal space 8-weeks post inoculation. Verification of virus inoculation was visible by the presence of green fluorescent protein (GFP). Sections  were mounted onto electrostatically charged slides and images were collected using a Nikon TiS Microscope and associated software.4, 26.2 mM NaCO3, 1.67 mM NaH2PO4, 1.5 mM CaCl2, 9.64 mM Na+ gluconate, 5.5 mM d-glucose, and 7.6 mM sucrose, pH 7.4 . Electrical stimulation was performed at a frequency of 0.3 Hz with electric pulses of 100-µs duration at 100–10,000 µA delivered by a pulse stimulator . Evoked CAPs were recorded with electrodes placed ∼5 mm from the stimulating electrodes. Dapsys software was used for data capture and analysis . The lowest stimulus producing a detectable response in the nerve was determined as the threshold stimulus and the peak amplitude determined when the response no longer increased in amplitude. The conduction velocity was calculated by dividing the latency period, the time from stimulus application to neuronal initial response, by the stimulus-to-recording electrode distance. The sciatic nerves were collected in mice previously used in the morphine escalating dose-response curve studies.Compound action potentials (CAPs) were measured from both left and right desheathed sciatic nerves from AAV9-GFP-U6-m-Adcy1-shRNA and AAV9-GFP-U6-scramble-shRNA8 weeks after intrathecal injection. Sciatic nerves were dissected from the hind limbs of mice and recordings were performed the same day. Each nerve was mounted in a chamber filled with superficial interstitial fluid composed of 107.7 mM NaCl, 3.5 mM KCl, 0.69 mM MgSOt-test, one-way, two-way, or repeated-measures ANOVA followed by either Dunnett’s or Bonferroni’s post hoc analysis was used to determine significance for mechanical thresholds, thermal latencies, gene expression, burrowing, open field testing, rotarod assessments, and CAP recordings. A Mann–Whitney U test was used for nesting behaviors. All statistical analyses were carried out using GraphPad Prism version 9 . Data presented as mean ± SEM unless otherwise indicated with p < 0.05 considered statistically significant.Data were collected by personnel blinded to the animal’s condition and treatment. The appropriate Adcy isoforms in nervous system tissues of morphine tolerant mice. Of the isoforms examined, an increased expression of Adcy1 is seen in DRG and spinal cord  mRNA were also elevated in dorsal root ganglia, while Rapgef3 (protein: Epac1) was also elevated in the spinal cord.Chronic agonist exposure of the MOR decreases inhibitory intracellular responses and increases adenylyl cyclase/cyclic-AMP activity . We attenal cord . This suTo further understand the physiological role of AC1 during tolerance and withdrawal with chronic morphine administration, pharmacological and gene knock-down strategies were implemented with behavioral assays. Previous research demonstrated ST034307 acts as an AC1 inhibitor and as an analgesic in a mouse chronic inflammatory pain model . In bothin vivo, mice were subjected to twice daily morphine injections  in combination with either an injection of vehicle or ST034307  and DRG  compared to the AAV9-scramble mice. Changes to the expression levels of Adcy5, Adcy8, and Oprm1 were also analyzed, but no significant differences were seen for any of these genes in either tissue  indicating AAV9-Adcy1 mice spent less time stationary compared to AAV9-scramble injected mice. This data indicates the AAV9-Adcy1 shRNA does not cause any major mobility changes in mice. Lastly, the downregulation of Adcy1 did not have any impact on thresholds, amplitude, or conduction velocity of C-fiber CAPs  Sin vitro . In chroin vitro , spinal in vitro , and priin vitro . Opioid in vitro . In our in vitro . This fuAdcy1. Static dosing of morphine  and escalating doses of morphine over 4 days  both resulted in enhanced baseline mechanical sensitivity. After intrathecal administration of AAV9-Adcy1, mice had higher mechanical paw withdrawal thresholds before (pre) and after (post) morphine administration compared to control mice. Our data agree with previous studies, as a global loss of either/both AC1 or AC8, appear to have a role in attenuating morphine tolerance and withdrawal , could be due the deferred hypertrophy of adenylyl cyclases after tissue injury, or the delayed role of adenylyl cyclases in enhanced transcription of pro-inflammatory molecules, taking several days to manifest , or an increase in spontaneous pain, or lowering the nociceptive threshold, the clinical effect is the same . Further"}
{"text": "Brassica rapa var. rapa L.) (syn. B. campestris L. ssp. rapifera Sinsk) is an important crop species belonging to the Brassicaceae family. The 185 accessions belonging to this crop were collected from several areas of Toodshak region in Isfahan province, Iran, and their tubers were cultivated under homogeneous conditions in loamy clay soil. The morphological traits of different organs of those accessions were evaluated. Significant variations were detected among the accessions studied based on the traits recorded. Tuber shape showed high variation and included globose, oblong, ovate, obovate, and fusiform. Also, tuber skin color was highly variable, including white, bicolor white–violet, light violet, and dark violet. Tuber weight ranged from 1.56 to 35.70 g, while total soluble solids (TSS) of tuber flesh ranged from 7.00 to 11.80%. Principal component analysis (PCA) showed that 18 components were extracted by explaining 74.88% of total variance. The dendrogram obtained based on all the characters measured clustered the accessions into two major clusters. Sixteen accessions were placed into the first cluster, while the remaining accessions were placed into the second cluster which was divided into six subclusters. High level of morphological variabilities was observed among the accessions, which is applicable and useful for B. rapa var. rapa breeding programs. Based on the commercial and quality traits, 17 accessions could be selected for direct cultivation. Also, the promising accessions identified here can be utilized directly in breeding programs for genetic enhancement of this crop.Turnip ( Brassica rapa var. rapa L.) could be selected for direct cultivation. Also, the promising accessions identified here can be utilized directly in breeding programs for genetic enhancement of this crop. Tuber skin color was highly variable, including white, bicolor white–violet, light violet, and dark violet. Tuber weight ranged from 1.56 to 35.70 g. Tuber flesh TSS ranged from 7.00 to 11.80%. Principal component analysis (PCA) showed that 18 components were extracted by explaining 74.88% of total variance. Based on the commercial and quality traits, 17 accessions of turnip ( B. campestris L. ssp. rapifera Sinsk) is an important crop species belonging to the Brassicaceae family. It is an annual or biennial plant with wide variation in size, shape, and color. Its classification is based on morphological characteristics, leading to a division of the cultivated forms into three main subspecies: turnip, oilseed, and leafy types. Thus, it plays a vital role in agriculture, and contributes to both national economies and human health. The B. rapa var. rapa is a vital cruciferous vegetable species, with unique physiology and morphology traits. Its plant parts, including both roots and leaves, are important for human consumption and also for animal feed  was performed to evaluate the variation among accessions based on the traits measured using SAS software , followed by leaf lamina blistering (262.27%) and vivipary (228.13%). The lowest CV belonged to flower length (10.18%), total soluble solids (TSS) of tuber flesh (10.44%), petal width (11.22%), flower width (12.82%), and petal length (13.44%)  constitute only stem tissue, and carrot (Daucus carota L.) constitutes root tissue , oblong (34), ovate (33), obovate (40), and fusiform (40). Also, tuber skin color was highly variable, including white (50 accessions), bicolor white–violet (40), light violet (49), and dark violet (46). Tuber surface was smooth 52), bit wrinkled (79), and very wrinkled (54). Central tuber maximum transverse diameter position was predominant (112 accessions). Tuber flesh was not bitter in most of the accessions (167). Tuber flesh texture was firm in the majority of accessions (150)  and green (77), while the color of floral leaves was strongly variable, including light green (4 accessions), green (87), dark green (87), and green–silver (7). Leaf apex shape was rounded in the majority of accessions 102). Leaf blade shape outline (lamina) was highly variable, ranging from deltoid to oblong. Flat leaf lamina was predominant (119 accessions). Leaf division was lyrate in the majority of accessions (126). Leaf margin showed strong variability, including crenate (37 accessions), broadly crenate (13), sinuate (57), broadly sinuate (55), serrate (8), and broadly serrate (15)  regions or genes that regulate flowering and vernalization . The range of flower‐related characters was as follows: flower length: 10.75–18.62 mm, flower width: 7.80–15.21 mm, petal length: 4.95–13.14 mm, and petal width: 3.29–5.83 mm. Flowering is an important step in plant growth and defines the agriculture setting of the crop. Vernalization is the promotion of flowering after exposure to cold, where plants do not necessarily initiate flowering but acquire the competence to do so. In Silique surface outline was smooth (105 accessions), undulating (55), and constricted between seeds (65). Silique shape was cylindrical (41 accessions), spathulate (5), oblong (19), lanceolate (108), and deltoid (12). Dry silique color was highly variable, including light golden yellow (49), golden yellow (25), cream–light brown (10), light brown (86), and brown (15). Silique pedicel node color was highly variable, ranging from homochromatic to black. The range of silique number in main stem range was 2–39, while total silique number per plant was 5–820. The range of other silique‐related characters was as follows: silique length: 24.45–59.17 mm, silique width: 2.43–6.05 mm, silique thickness: 1.67–5.15 mm, silique beak length: 3.47–31.25 mm, silique pedicel length: 5.61–38.66 mm, and silique weight: 0.02–0.11 g , early June (47), and mid‐June (39) Table . The ranr = 0.73), tuber middle diameter (r = 0.63), tuber flesh texture (r = 0.51), and leaf length (r = 0.53).Pearson correlation analysis showed significant correlation between some characters (data not shown). Tuber weight showed positive correlations with tuber length , tuber middle diameter (0.91), tuber maximum transverse diameter (0.88), and tuber collar diameter (neck) (0.70) with positive correlations were found to be influential in PC1. The PC2 was correlated with four leaf‐related traits, including leaf length (0.70), petiole width (0.83), lamina length (lamina blade length) (0.68), and lamina width (lamina blade width) (0.76) with positive correlations. The PC3 was correlated with plant height until first flower (0.54), flowering branch number (0.79), leaf number until first flower (0.82), and total leaf number (0.84). The remaining characters loaded significantly in the rest components (PC4–PC18) and explained less variability.The scatter plot created based on the PC1 and PC2 Figure  showed tThe dendrogram obtained based on all the characters measured clustered the accessions into two major clusters Figure . SixteenB. rapa var. rapa breeding programs. Matthaus et al. (Brassica napus L.) cultivars from Turkey. Agro‐morphological‐based variation is important to screen best accessions in field experiment. The diverse agro‐morphological‐based accessions are useful for further biochemical and molecular evaluation. Genetic diversity study is used for efficient utilization and for development of improved cultivar/varieties (Jan et al., Here, a high level of morphological variabilities was observed among the accessions, which is applicable and useful for s et al.  reported4B. rapa var. rapa. At the present time, breeding programs, particularly with an obligate outcrossing crop such as turnip, present a challenge. Based on the commercial and quality traits, 17 accessions, including no. 40, 72, 17, 10, 7, 19, 112, 98, 83, 2, 4, 1, 5, 9, 37, 6, and 8, could be selected for direct cultivation. Also, the promising accessions identified here can be utilized directly in breeding programs for genetic enhancement of this crop.Estimation of different qualitative and quantitative traits offers a distinct means to improve varied accessions. Significant variations were recorded for various morphological traits among the accessions of The authors declare no conflict of interest.None.None."}
{"text": "Structured education of healthcare professionals around infection prevention practices and protocols is currently lacking. We launched a pilot program of an infection prevention interprofessional education (IPE) elective for students in their clinical years of medical, pharmacy, nursing, and physician assistant school.Both quantitative and qualitative assessments prior to initiation and at completion of the 2-week multidisciplinary interactive and practical IPE elective were performed. We assessed students’ confidence with specific infection prevention skills knowledge, and attitudes towards IPE, and obtained qualitative feedback. Wilcoxon signed rank test was used to compare assessment results before and after IPE elective.Twelve students completed the IPE elective. There was a significant increase in Lickert scale rankings of students’ comfort level with various infection prevention concepts and skills after IPE (p< 0.001). Similarly, a significant improvement in students’ assessment of their IPE attitudes was noted after completion of the IPE elective (p=0.001).Feedback comments included: “I really enjoyed learning the ‘why’ behind everything [because] it's made me comfortable passing the information to others and speaking up if needed--even still as a student”; “learned information I will use every day”; “people from different backgrounds”; “knowledgeable facilitators, excited to teach & facilitate learning.”Infection prevention education was impactful via the incorporation of an IPE elective into clinical rotations, and use of knowledgeable and adept teachers from multiple disciplines. Leaders emphasized the “why” behind protocols, and allowed students to practice the hands-on skills of infection prevention.Julie Ann Justo, PharmD, MS, FIDSA, BCPS, Gilead Sciences: Advisor/Consultant|Shionogi: Advisor/Consultant|Vaxart: Stocks/Bonds"}
{"text": "The study aims to evaluate the neurodevelopmental outcomes of neonates with myelomeningocele (MMC) operated in the postnatal period.This is a prospective follow-up study in a tertiary neonatal intensive care unit. Neurodevelopmental outcomes of term neonates operated for MMC and healthy term newborns were compared with the Bayley Scales of Infant and Toddler Development-Third Edition (BSID III) at 12–18 months.A total of 57 cases were included in the study . Demographic data between the groups were similar. Cognitive, linguistic, and motor composite scores of the patient group were lower than those of the control group (p < 0.001). In the patient group, those who underwent ventriculoperitoneal shunt had lower cognitive, language and motor scores than those without shunt (p < 0.05). The cognitive, linguistic, and motor composite scores in the patient group who underwent surgery before 72 h were better than those who underwent surgery after 72 h.In our study, it was found that the neurodevelopmental prognosis of MMC cases requiring ventriculoperitoneal shunt in the postnatal period was significantly worse than those without shunt. It is the first study in which the neurodevelopment of patients with MMC who were operated in the postnatal period was evaluated with BSID III evaluated and delays in all areas were shown in cases with MMC compared to normal cases. Better neurodevelopmental outcomes in patients operated in the first 72 h suggest that early surgery will improve neurodevelopmental outcomes in patients with MMC. Neural tube defects (NTD) are the most common congenital malformations after congenital heart defects and occur due to the failure of the neural tube to close spontaneously, which should be closed by the third to fourth week in intrauterine life. Myelomeningocele (MMC) is the most common NTD, characterized by protrusion of the meninges and spinal cord through open vertebral arches leading to paralysis .With nearly 60 cases per 100,000 births in the United States, the prevalence of MMC varies among countries. MMCs may be part of a genetic syndrome or may occur as an isolated defect. With both environmental and genetic factors affecting the emergence of the defect, the causes remain uncertain. It has been reported that many factors may lead to the development of MMC including maternal exposure to hyperthermia during pregnancy, use of alcohol, valproic acid, carbamazepine or isotretinoin, maternal malnutrition or obesity, low folic acid levels, maternal diabetes, and genetic markers (mutations in folic acid-responsive or folic-acid-dependent enzyme pathways) ,3,4.A surgical correction should be performed as soon as possible following the diagnosis of neural tube defects. However, since other system disorders such as mental and motor retardation, bowel and bladder dysfunction, orthopedic problems can accompany NTDs in varying degrees, the treatment applied may bring limited success. Patients suffering from NTD often require long-term care, leading to a significant financial burden on healthcare costs .Today, NTD patients who lost their lives in childhood in the past can reach adulthood to a large extent. A close follow-up with a multidisciplinary approach is required from birth to preserve the neurological functions of these patients as much as possible and to overcome orthopedic and urinary complications as they reach adulthood. It is of great importance to follow the neurodevelopment of patients and to support them with appropriate methods when needed in terms of their neurological prognosis. However, unfortunately, the literature contains very few studies evaluating the neurodevelopmental outcomes of cases with MMC and investigating the factors that may positively affect these results.The current study aims to determine the demographic data of term newborns who were operated in the postnatal period with the diagnosis of MMC in the neonatal intensive care unit of our hospital and to evaluate the neurodevelopmental results with BSID III.This is a prospective follow-up study. The study protocol was approved by the local committee .The cases included in the study were divided into two groups:Patient group (group 1): The patient group includes term newborn babies who were operated in the postnatal period with the diagnosis of MMC in the neonatal intensive care unit of our hospital between January 2018 and December 2019.Control group (group 2): The control group includes healthy term newborn babies who applied to the pediatric outpatient clinics for different reasons.Criteria for exclusion from the study:Premature cases (week of gestation <37 weeks), cases with multiple organ anomalies, cases diagnosed with metabolic disease or hypoxic ischemic encephalopathy, and with low birth weight according to gestational week or with intracranial bleeding were excluded from the study.Demographic information and clinical characteristics of the patients in the patient and control groups were compiled retrospectively from their hospital records.Neurodevelopmental evaluations including cognitive, language, motor, social-sensory and adaptation behaviors of both the patients in the patient group and the control group were performed by a certified child development specialist with the BSID III at 12–18 months.In this study, all data were analyzed with the SPSS 20.0 program. Numerical data were expressed as arithmetic mean ± standard deviation (min-max) or median, and categorical data were expressed as numbers and percentages, depending on whether they showed parametric properties or not. Student-t, Mann-Whitney U and chi-square tests were used to compare two independent groups. A p value of <0.05 was considered statistically significant.A total of 57 cases, 27 in the patient group and 30 in the control group, were included in the study. Demographic information of both groups is shown in When the mothers of the cases in Group 1 were evaluated in terms of neural tube defect etiology, it was seen that there were no mothers who were exposed to the drug or another teratogen, except for two mothers who used valproic acid due to epilepsy and were learned that they did not take folic acid supplementation during their pregnancies. Mothers of the cases in group 2 did not have radiation, hyperthermia, and drug exposure during pregnancy.It was seen that 81% of the cases in group 1 had a prenatal diagnosis and three of them had NTD individuals in their families. The other cases in this group had no prenatal diagnosis.Distribution of cases according to MMC localization and accompanying anomalies is shown in Epilepsy developed in 3 (11.1%) of the patients in grup 1 during the follow-up and antiepileptic treatment was started.It was seen that 55% of the cases in group 1 were operated within the postnatal 72 h and the patients were operated on the mean postnatal 4th ± 3.54 day (1–13 days).It was determined that ventriculoperitoneal (V/P) shunt was placed in thirteen of the cases in group 1, shunt revision was required in four cases, and shunt infection developed in three of the cases that underwent shunt revision. Ventriculitis was not observed in any patient. While the cases in group 1 were followed up in the neonatal intensive care unit, clinical sepsis developed in nine, proven sepsis in four, and meningitis in one.Bayley Scales of Infant and Toddler Development-Third Edition test application age was 16.07 ± 5.53 months in group 1 and 17.4 ± 4.43 months in the control group. The two groups were found to be similar in terms of test administration time (p = 0.326).In the study, the cases in group 1 were divided into two subgroups as cases with V/P shunts (group 1a) and cases without (group 1b) to determine the factors that negatively affect their neurodevelopment. While the number of cases was 13 in group 1a, it was 14 in group 1b.The distribution of Bayley cognitive scale score, Bayley language scale score, Bayley motor scale score and Bayley composite score of the cases in groups 1a and 1b are shown in The Bayley cognitive composite score (p = 0.004) and Bayley total language composite score were found to be lower in the subjects in group 1a compared to the subjects in group 1b (p = 0.001). Bayley motor composite score was found to be lower in the cases in group 1a compared to the cases in group 1b (p < 0.001).In addition, the cases in group 1 were divided into two subgroups as those who had surgery before the postnatal 72 h and those who did not. When both groups were evaluated with BSID III, the cognitive, language and motor composite scores of the patients who had surgery before the postnatal 72 h were better than those who had surgery after 72 h, but there was no statistically significant difference between them (p > 0.05).Myelomeningocele is a common congenital malformation that occurs in the first month of embryological life and often causes permanent deformities throughout life. Despite the improvement of antenatal diagnosis possibilities and the increasing effectiveness of folic acid as a preventive factor, MMC continues to be an important health problem in our country and in the world ,7.There is not yet a consensus in the literature about how the delivery method of myelomeningocele cases should be. In some studies in the literature, it is accepted that delivery by normal spontaneous vaginal route causes spinal cord injury and poses a risk factor for a poor neurological condition, while other studies report that the mode of delivery does not affect the neurological prognosis . In the It has been reported that the management of the delivery room of babies with neural tube defects is important for the neurological prognosis of the cases. Especially, cases with MMC who do not have an intact skin tissue should be laid on their side immediately after birth, care should be taken not to tear the sac, and the defect should be covered with gauze moistened with saline . All theIt was thought that correction surgeries to be performed in the prenatal period in myelomeningocele cases would positively affect the neurodevelopmental prognosis. However, in the literature, prematurity is seen more frequently in MMC cases who were operated on prenatally, but neurodevelopmental damage is reported to be similar when compared with cases operated postnatally . All of In the literature, it has been reported that cases with MMC have a high risk of cognitive dysfunction, which becomes especially evident with increasing age. Studies show that verbal and performance IQ scores and arithmetic and reading skills of children with MMC are lower than those of children without MMC –13. HoweAlthough it has been reported in the past that the presence of hydrocephalus and V/P shunts in patients with MMC cause significantly lower scores in neurodevelopmental and cognitive tests, today, it is accepted that the presence or absence of shunt does not affect neurodevelopmental tests unless there are additional problems such as central nervous system infection or intracranial bleeding –17. In tDue to the high risk of meningitis in myelomeningocele cases, prophylactic antibiotics are recommended in patients until the defect is closed with surgery. When antibiotic prophylaxis was compared in MMC cases, it was reported that the rate of ventriculitis development was higher in those who were not given antibiotics . In the It has been reported that early and aggressive surgical intervention is associated with low morbidity and mortality in cases with myelomeningocele. In a study by Öncel et al., including 30 MMC cases, early surgical intervention (≤5 days), short hospital stay, and antibiotic therapy were associated with a low complication rate . In anotTo the best of our knowledge, there is no study in the literature comparing the effects of early or late surgery on the neurodevelopment of MMC cases in the postnatal period with the BSID III. In the current study, although the cognitive, language and motor composite scores of the patients who underwent surgery before 72 h were better than those who underwent surgery after 72 h, no statistically significant difference was found between them. Due to the small number of cases in our study, we recommend to support our outcomes by studies with a higher number of cases investigating the effect of early/late surgical intervention on neurodevelopmental outcomes.In this study, it was found that the neurodevelopmental prognosis of MMC cases requiring ventriculoperitoneal shunt in the postnatal period was significantly worse than those without shunt. It is the first study in which the neurodevelopment of patients with MMC who were operated in the postnatal period was evaluated with BSID III and delays in all areas were shown in cases with MMC compared to normal cases. Better neurodevelopmental outcomes in patients operated in the first 72 h suggest that early surgery will positively change neurodevelopmental outcomes in patients with MMC.Detection of problems at an early age with early and regular follow-up in MMC cases increases the quality of life in these babies. Close follow-up of these patients with developmental tests and after surgery, evaluation and treatment of the problems positively affect the neurodevelopmental prognosis of these patients."}
{"text": "Little is known about the demographics of people who use cannabis, including how use trends within population subgroups have evolved over time. It is therefore challenging to know if the demographics of participants enrolled in cannabis clinical trials are representative of those who use cannabis. To fill this knowledge gap, data from the National Survey on Drug Use and Health (NSDUH) on “past-month” cannabis use across various population subgroups in the United States was examined from 2002 to 2021. The most notable increases in “past-month” cannabis use prevalence occurred in those aged 65 and older  and 50–64-year-olds (472.4%). In 2021, people reporting “past-month” cannabis use were 56.6% male and 43.4% female. Distribution across self-reported race and ethnicity was 64.1% White, 14.3% Black, 14.1% Hispanic, and 3.1% more than one race. And many ages were represented as 24.4% were 26–34, 24.1% were 35–49, 22.4% were 18–25, and 17.6% were 50–64 years old. To understand if these population subgroups are represented in cannabis clinical trials, participant demographics were extracted from peer-reviewed clinical trials reporting on pharmacokinetic and/or pharmacodynamic models of cannabis or cannabinoids. Literature was grouped by publication year (2000–2014 and 2015–2022) and participant prior exposure to cannabis. Results identified that cannabis clinical trial participants are skewed toward overrepresentation by White males in their 20s and 30s. This represents structural discrimination in the research landscape that perpetuates social and health inequities. Cannabis and cannabinoid pharmacology and epidemiology are the focus of several federal Requests for Proposals and Notices of Special Interest. Despite renewed pharmacological research interest, little is known about the demographics of people who currently or have recently used cannabis, including how use trends have evolved over time. In 2018, an extensive review on national trends in adult cannabis use was published5, considering data from 2002 through 2014 in participants aged 18–25 and those aged 26 and older7. It was noted that increases in cannabis use occurred across sex, region, educational level, and employment status8. However, the detailed demographics of people who use cannabis was not critically assessed nor reported. In 2019, Cerdá et. al. found that recreational cannabis legalization was associated with increases in frequent cannabis use among adults9. However, impacts of demographic variables beyond age were not assessed. Also in 2019, Hasin et. al. published a narrative review on cannabis use trends by sociodemographic subgroups, summarizing data from several national surveys and a literature review10. They concluded that cannabis use had increased across all sociodemographic subgroups including age sex, race, ethnicity, educational level, and location10. While this work was comprehensive and complete, survey results through 2015 were considered, and an update is required. In 2022, Waddell et. al. reported on the age, sex, and race-varying rates of cannabis use in veterans and non-veterans11. Although the demographic characteristics of these populations were assessed, this work focused on interactions of demographic details and veteran status as risk factors for alcohol and cannabis use11.The shifting legal status of cannabis in the United States (US) and internationally is resulting in increased research interest into cannabis and cannabinoid pharmacokinetics and pharmacodynamics12. Cannabis effects may be moderated by a wide range of factors such as sex, age, race, and ethnicity. For example, preclinical work from rodent ∆9-tetrahydrocannabinol (∆9-THC) dosing studies demonstrates that metabolism and bioaccumulation of ∆9-THC and psychoactive metabolites are significantly impacted by rodent sex. Human studies have also identified sex differences in subjective cannabis effects16, although additional exploration is needed. Similarly, aging is associated with metabolic changes, morbidities, and an overall decline in functioning17, likely impacting cannabis pharmacology. However, only a few studies have evaluated the pharmacology of cannabis in older adults21. The authors were unable to find literature describing demographics of people who currently or have recently used cannabis. However, two recent reviews and meta-analysis of published works on cannabis use disorder and behavioral health found that approximately 70% of study participants were male, 72% were non-Hispanic White, and the median participant age (SD) was 29.9 (9)24. However, balanced clinical trial participant pools must be demographically representative of those who use cannabis to gather generalizable results translatable to public policy. We hypothesize that cannabis clinical trial participants do not represent the sex, race, ethnicity, and age characteristics of people who use cannabis. One may argue that most fundamental pharmacokinetics and pharmacodynamics assessments of cannabis or cannabinoids do not aim to inform statutory or policy language. However, the lack of knowledge surrounding cannabis pharmacokinetics and impairment forces policy makers, enforcement officials, and other stakeholders to apply any available works to their immediate public health and safety needs. That is, results from any cannabis pharmacokinetics or pharmacodynamics studies in humans are likely to be read by and applied to those tasked with crafting evidence-based policies, recommendations, or assessments. This begs the question, what are the demographics of cannabis clinical trial participants, and do they reflect those of people who use cannabis? To begin answering this question, we will consider two data sources: (1) participant demographics extracted from a systematic review of cannabis pharmacokinetics and/or pharmacodynamics studies and (2) results from the United States National Survey on Drug Use and Health (NSDUH) from years 2002–2021. To the best of our knowledge, this is the first study comparing the demographics of cannabis clinical trial participants to those of people who use cannabis.It is well known that cannabis pharmacokinetics and pharmacodynamics vary intra- and inter-personally25. It is a key source of national and state-level data on the prevalence of substance use and health in the US.The National Survey on Drug Use and Health (NSDUH) is a nationally representative and cross-sectional survey of individuals aged 12 years or older living in households or non-institutional group housing  or with no permanent housing . The NSDUH uses a multistage area probability sample for each US state and the District of Columbia and an audio computer-assisted interviewing method to support confidential and private responsesSelf-reported “past-month” cannabis use was examined by demographic characteristics within the 2002–2021 NSDUH data. All analyses used the Substance Abuse & Mental Health Data Archive (SAMHDA) Public-use Data Analysis System (PDAS) to query the NSDUH data. Self-reported “past-month” cannabis use  was considered as a function of respondent sex . Similarly, other variables were considered such as reported race and ethnicity . This included the following options: non-Hispanic White, non-Hispanic Black/African American, non-Hispanic Native American or Alaskan Native, non-Hispanic Native Hawaiian or Other Pacific Islander, non-Hispanic Asian, non-Hispanic more than one race, or Hispanic. A combined sex by race variable was also used  for those who identify as non-Hispanic White, non-Hispanic Black, or Hispanic. Similarly, age was considered  in the ranges of 12–17, 18–25, 26–34, 35–49, 50–64, and 65 + years old. Prior to 2005, the 6-Level age category was not available. Therefore, CATAG5  was used for years 2002 through 2004 for the age ranges of 12–17, 18–25, 26–34, and 35–49 years old. A combination of sex and age category was also used  which identified the distribution of males and females within 12–17 and 18–25 age groups.“Past-month” cannabis use was reported in eSupplement Table 26.Our study was exempt from IRB approval per the University of Wisconsin-Madison’s policy on publicly available, de-identified data sets. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines for cross-sectional studies 27. Regression lines were fitted to prevalence estimates using linear regression fits in which the dependent variable was the annual prevalence estimates. Linear regression models include slope (β), y-intercept (α), and their standard errors. Results from linear regression models are included as eSupplement Table 2) values. Residual standard error was found as the standard deviation of residuals using 18 degrees of freedom. A right-tailed F-test was used to identify model significance through the F-statistic (F) and p-value (p). Due to methodological changes in the 2005 NSDUH, the 6-level age category was not available for the 50–64 and 65 and older age ranges in years 2002 through 2004.Weighted crosstab analysis was used to identify and extract self-reported “past-month” cannabis use by age, sex, and race and ethnicity from 2002 through 2021. Results were displayed as weighted count, prevalence, and distribution Both authors independently extracted the following data from each article: (1) participant type , (2) previous cannabis exposure , (3) total number of participants, (4) administration route, (5) time of last plasma sample, (6) pharmacokinetics model (yes/no), (7) pharmacodynamics model (yes/no), (8) pharmacodynamics indicator reported, (9) participant sex, (10) participant age, and (11) participant race and ethnicity. Not all articles reported participant sex, age, race, and ethnicity.30. This striking proportion of the population using cannabis recently and/or regularly is the culmination of a steady increase in societal acceptance of cannabis and cannabis use. Linear regression analysis identified trend directionality, and detailed results are provided in eSupplement Table In 2021, NSDUH estimates 12.9% of Americans  used cannabis in the “past-month”Increasing cannabis use is also observed, although unevenly, across sex and racial and ethnic groups, Fig. The demographics of people who have recently used cannabis was identified from 2021 NSDUH data. In 2021, a total of 69,850 US residents completed the NSDUH. Weighted demographics of respondents, as shown in eSupplement Table Overall, 12.9% of the 2021 respondents reported “past-month” cannabis use. Considering all people engaging in “past-month” cannabis use, demographic distributions were 43.4% female, 64.1% White, 14.3% Black or African American, 1.3% Native American or Alaskan Native, 2.5% Asian, 3.2% two or more races, and 14.1% Hispanic  and females . Only 20 publications70 reported at least some participant race information, see eSupplement Table 70 (11.9%), including 139 (16.4%) total participants and 14 (10.1%) who identified as Hispanic.Results of the literature search and selection process are detailed in Fig. 63 including 155 participants. Those works included participants in the following age ranges: 14–15 , 16–17 , 18–19 , 20–29 , 30–39 , 40–49 , 50–59 , and 60–69 . In addition, 37 publications70 reported age ranges including 907 participants. The minimum age considered was 15 and maximum was 79 years old. Considering the minimum age, the mean was 23.9, the median was 21, and the range was 15–52 years old. Considering the maximum age, the mean was 44.3, the median was 43, and the range was 25–79 years old.Detailed participant age information was available in 6 publications42 were published in 2000–2014 that met the inclusion criteria of this work. These studies included 187 participants of which most  were male. When race was reported, 100.0% of participants were White. Conversely, 29 manuscripts70 were published in 2015–2022 and met the inclusion criteria of this work. These studies included 780 participants of which 429 (65.0%) were male. Race was reported for 338 participants, including While , Black , Asian  Asian, two or more races , Native American , or \"other\"  participants. In this cohort (2015–2022), ethnicity was reported for 139 participants, with 14 (10.1%) identifying as Hispanic. Minimum and maximum age ranges were similar, when reported, for these works.To assess for shifts in cannabis clinical trial participant demographics over time, the above described data was partitioned into works published in 2000–2014 and those published in 2015–2022. Summaries of participant demographic characteristics for each literature analysis subgroup are included in eSupplement Table 67 including 715 participants of which 430 (72.3%) were male. Race was reported for 240 of these participants, including White , Black , Asian , two or more races , Native American , or other . Ethnicity was reported for 78 participants and 10 (12.8%) were Hispanic.When applying cannabis clinical trial results to people who use cannabis, readers must consider if the clinical trial participants were patients or healthy and if they have prior experience with cannabis. To that end, we also considered cannabis clinical trial participant demographic characteristics (published in 2000-2022) when all “patients” and “no prior cannabis exposure” participants were removed, see eSupplement Table 67 and 545 participants that should be most similar to the demographic characteristics of NSDUH “past-month” cannabis use survey results. Within this group, 297 (69.9%) participants were male. Race was reported for 187 participants including those who identified as White , Black , Asian , two or more races , Native American , or \"other\" . The distribution of racial groups (when available) included in cannabis clinical trials reporting pharmacokinetics and/or pharmacodynamics models are shown in Fig. 60 reported the participant’s ethnicity, including 78 total participants and 10 (12.8%) who identified as Hispanic. Participant ages were primarily reported as minimum to maximum age ranges. The range of minimum ages was 15–29 years old, with an average minimum age of 21.2 years old. The range of maximum ages was 25–52 years old, with an average maximum age of 35.7 years old.Within the above described “prior cannabis exposure” cohort, 10 manuscripts were published between 2000 and 2014. Removing those, a final subgroup was created including “prior cannabis exposure” participants that were published between 2015 and 2022, see eSupplement Table Asian 7, .7%, two Asian 7, .7%, two 71. Therefore, any pharmacokinetics or pharmacodynamics models generated from these cannabis clinical trials may not be generalizable to the increasingly diverse population of people who use cannabis.This is the first study to compare the NSDUH demographics of people reporting “past-month” cannabis use to cannabis clinical trial participants. The main finding of this study is that demographics of those reporting “past-month” cannabis use are quite diverse, with significant prevalence increases between 2002 and 2021 in older Americans, women, and historically underrepresented groups. However, cannabis clinical trial participants continue to be majority White males in their 20s and 30s. Literature published recently (2015–2022) have included more women and racial minorities compared to literature from 2000–2014. However, this chasm between the demographics of people reporting recent cannabis use and clinical trial participants is concerning. It is well known that demographic differences are an important factor in interpersonal variability in the pharmacokinetics and pharmacodynamics of a substance74. In cannabis clinical trials, age ranges were reported for most participants (94.0%), but detailed age information down to the decade was only reported on some (15.6%) participants. Furthermore, removal of patient participant works reduced the overall age range of participants included in studies. Pharmacokinetics and pharmacodynamics clinical trials are inherently limited by small sample size and stringent inclusion and exclusion criteria. Despite these limitations, the rapid expansion of “past-month” cannabis use by adults aged 50 and older warrants additional clinical investigation.Over the past 18 years, the prevalence of “past-month” cannabis use increased across all age categories except 12–17-year-olds. While decreasing prevalence of “past-month” cannabis use in the pediatric population is encouraging, significant increases in other age groups warrants increased awareness and scientific inquiry. Increases in “past-month” cannabis use prevalence was not evenly distributed across all age groups considered. For example, “past-month” cannabis use prevalence increased 50.9% in 18–25-year-olds from 2002 to 2021 whereas the increase was 224.9% in 26–34 and 148.0% in 35–49-year-olds. Increases were even larger for 50–64-year-olds at 472.4% and use by those aged 65 and older increased an astonishing 2,066.1%. Significant increases in “past-month” cannabis use by older Americans has not received a commiserate increase in research interest and clinical investigationTo understand how cannabis clinical trial participant demographic characteristics have changed over time, data extracted from the literature was divided into several subgroups. These subgroups were: works published in 2000–2014, works published in 2015–2022, works including healthy participants with prior cannabis experience, and works including healthy participants with prior cannabis experience which were published from 2015 to 2022. With respect to publication date, promising improvements in participant demographic characteristics are apparent. Works published in the years 2015–2022 included more women and historically underrepresented racial and ethnic groups than those published in 2000–2014. Including participant eligibility criteria  alongside publication years of 2015–2022 boosted the racial and ethnic diversity of participants, but decreased female representation. Furthermore, removal of patients (studies assessing cannabis for therapeutic applications) reduced the age range of included participants.Cannabis clinical trial participant demographic information was significantly lacking in the works considered here. Participant sex was commonly (87.9%) reported, but race (39.1%) and ethnicity (14.0%) details were deficient. When participant demographic data is missing, the reader is unable to critically assess the translatability of reported results to “real world” cannabis use. Even when available, race and ethnicity data is oversimplified and incomplete. For example, NSDUH crudely aggregates all those identifying as Hispanic together, limiting our ability to consider how ethnicity overlaps with different racial groups in those who report “past-month” cannabis use. However, cannabis clinical trial participant demographics largely failed to capture any ethnicity information. This represents structural discrimination in the research landscape that perpetuates social and health inequities. Overall, there needs to be greater transparency and reporting of participant demographics.This study had several limitations. Cannabis clinical trial participant data was derived from peer-reviewed literature, which introduces publication bias. That is, this work fails to capture any studies that were completed but not published, for whatever reason. Additionally, cannabis clinical trials often include intentionally stringent participant eligibility and ineligibility criteria. For example, females of reproductive potential may be ineligible due to teratogenicity risk. Additionally, the small sample sizes used in pharmacokinetics and pharmacodynamics studies may result in narrow eligible age ranges, preventing consideration of older adults. This is exemplified by studies on therapeutic potential of cannabis and cannabinoids  for certain indications which tended to include older adults.https://pdas.samhsa.gov/). Therefore, demographic details on people reporting cannabis use, including “past-month”, “past-year”, or “lifetime” cannabis use, is available. This data and online data exploration tool was chosen to promote its use when designing future cannabis clinical trials. As shown in this work, the demographics of people reporting “past-month” cannabis use has changed over time. These changes across age, sex, and race and ethnicity necessitate annual reconsideration of the demographics of people who use cannabis. However, cannabis clinical trials seeking to generate pharmacokinetics and/or pharmacodynamics models of cannabis or cannabinoids do not include participants whose demographics are representative of those who use cannabis. This disconnect is problematic for those seeking translatable data to inform public policy on cannabis use and cannabis products. Well-crafted clinical trials should consider the demographics described in this work and updated annually through the Substance Abuse and Mental Health Data Archive. Funding agencies should also consider this data when evaluating funding proposals and crafting Requests for Proposals and Notices of Special Interest announcements.Significant investments have facilitated the wide availability of NSDUH results and datasets to the public through the Substance Abuse and Mental Health Data Archive (Supplementary Tables."}
{"text": "Non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ALD), both of them accounting for fatty liver disease (FLD), are among the most common chronic liver diseases globally, contributing to substantial public health burden. Both NAFLD and ALD share a similar picture of clinical presentation yet may have differences in prognosis and treatment, which renders early and accurate diagnosis difficult but necessary. While NAFLD is the fastest increasing chronic liver disease, the prevalence of ALD has seemingly remained stable in recent years. Lately, the term steatotic liver disease (SLD) has been introduced, replacing FLD to reduce stigma. SLD represents an overarching term to primarily comprise metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), as well as alcohol-related liver disease (ALD), and MetALD, defined as a continuum across which the contribution of MASLD and ALD varies. The present review discusses current knowledge on common denominators of NAFLD/MASLD and ALD in order to highlight clinical and research needs to improve our understanding of SLD. Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and shows a continuously increasing global prevalence of 38% in recent years, driven by the obesity pandemic ,2. In coNAFLD is closely linked to obesity and the metabolic syndrome and represents the most rapidly increasing cause for chronic liver failure, hepatocellular carcinoma (HCC), and the need for liver transplantation (LT) . While tThe nomenclature of NAFLD has been a matter of debate in recent years. It is perceived that the term NAFLD may have contributed to the trivialization of the disease, prolonging patient journeys until adequate treatment was offered . AdditioThus, joint efforts of international scientific societies and patient associations have been made to rename and redefine NAFLD to destigmatize patients, improve diagnostic accuracy, and raise awareness . SupportSince progression of hepatic steatosis to cirrhosis and HCC is an important feature of both NAFLD and ALD, it is relevant to identify potentially harmful alcohol consumption and metabolic risk factors for FLD early in order to offer adequate treatment.This review summarizes current knowledge on common denominators and specific differences between NAFLD/MASLD and ALD in clinical presentation, pathogenesis, diagnosis, and treatment in order to uncover hitherto unresearched fields and catalyze our understanding of unmet medical need.Throughout this review article, the previous nomenclature will be used where appropriate to allow for an accurate reflection of the published literature.NAFLD is the most rapidly growing indication for liver transplantation in the US and has become the most common indication for liver transplantation in the US elderly population ,14.According to a recent systematic review, global NAFLD prevalence in the general population increased by ≈50% from 25% in the time period of 1990–2006 to 38% in 2016–2019. Its prevalence has been estimated at 31% in North America and Australasia, 44% in Latin America, 25% in Western Europe, 37% in the Middle East/North Africa, 34% in South Asia, 33% in Southeast Asia, and 28% in Asia-Pacific countries .The prevalence of NAFLD is rising in parallel with the global obesity epidemic. Among metabolic risk groups, prevalence of NAFLD is even higher, with >70% in patients with type 2 diabetes mellitus (T2DM) and 90% in patients with severe and morbid obesity undergoing bariatric surgery ,17. Currently, about 13% of children and adolescents are affected by NAFLD . The shaALD accounts for 5.1% of all disease and injury globally, 5.3% of deaths are attributed to alcohol consumption exceeding those caused by diabetes, and the main cause of death among men aged 25–45 years is alcohol consumption . In contCurrently available data on NAFLD and ALD epidemiology need to be interpreted with caution given that underlying databases largely did not account for systematic testing for alcohol consumption or systematic screening for alcohol use disorder (AUD) in NAFLD patients. Thus, the continuous increase in NAFLD prevalence might be biased by undetected harmful alcohol consumption .Within NAFLD/MASLD, two stages are discerned: (i) non-alcoholic fatty liver (NAFL) or metabolic dysfunction-associated steatotic liver (MAFL) which shows low liver-related morbidity, and (ii) non-alcoholic steatohepatitis (NASH) or metabolic dysfunction-associated steatohepatitis (MASH) which has a higher risk for progressive hepatic fibrosis and shows substantial liver-related mortality . ImportaAs in other etiologies of chronic liver disease, fibrosis stage emerged as a major determinant of prognosis. Angulo et al. identified the superior prognostic value of fibrosis stage as compared to presence or absence of NASH on liver histology . This fiOn the other hand, prognosis is also limited by extrahepatic factors, such as cardiovascular risk and non-hepatic malignancies. A diagnosis of NAFLD should, therefore, prompt screening for cardiovascular disease, e.g., by exercise testing or cerebrovascular sonography. It should be noted that the presence of NAFLD may indicate the cardiovascular manifestations of the underlying metabolic syndrome. Recently, in a retrospective study on 460 patients with stroke, we could show an association of mortality with increased FIB-4 values on admission .Similar to NAFLD/MASLD, ALD encompasses a spectrum of simple hepatic steatosis, steatohepatitis, fibrosis, cirrhosis, and HCC. Additionally, patients may suffer from severe alcoholic steatohepatitis (ASH) that may present as acute-on-chronic liver failure associated with high liver-related short-term mortality . NotablyBesides the well-known impact of abstinence during follow-up, prognosis of ALD, similarly to NAFLD, is mainly determined by fibrosis stage. In a large multinational cohort of 450 patients with ALD of varying severity, SALVE fibrosis stage (SFS) at baseline was found to be an excellent predictor of outcome after 10 years  31]..31].However, alcohol consumption not only increases liver-related morbidity and mortality but affects numerous extrahepatic organs. Similar to NAFLD, diabetes, cardiovascular disease, and obesity are highly prevalent in patients with ALD. According to a recent meta-analysis by Theodoreson et al., major causes of extrahepatic morbidity and mortality comprise cardiovascular disease, non-hepatic malignancies, as well as infection associated with increased risk of death, with a relative risk of 2.4, 2.2, and 8.2, respectively . NAFLD/MASLD and ALD share a common picture of clinical presentation, have large overlaps in associated comorbidities, such as cardiovascular disease, obesity, and cancer, and also share fibrosis as determinant of prognosis. However, longitudinal long-term studies on the natural history of NAFLD/MASLD and ALD are needed to better understand the dynamic of both diseases as well as to facilitate public health initiatives to improve prevention and therapy. Recently, using a Delphi process, a multidisciplinary panel defined a global research priority agenda to enhance public health responses to SLD . This paNAFLD pathogenesis is multifactorial and seems to be mainly driven by lipotoxicity, insulin resistance, and inflammatory pathways. It remains unclear why only 20% of NAFLD patients progress to NASH .Overload of free fatty acids leads to increased fat accumulation from de-novo lipogenesis as well as impaired lipolysis, resulting in lipotoxicity. The latter leads to hepatocellular injury, which provokes inflammation and activation of hepatic stellate cells to myofibroblasts, resulting in enhanced fibrogenesis.Multiple hits stressing the liver, including gut-derived pathogen-associated molecular patterns (PAMPs) and or damage-associated molecular patterns (DAMPs) enhance the transition of NAFL to NASH .In addition, genetic factors, such as patatin-like phospholipase domain-containing protein 3 (PNPLA3) variants, but also altered gut microbiota (dysbiosis), may contribute to the progression to NASH. Interestingly, a recently proposed hypothesis suggests that saccharomyces and candida species contained in the gut mycobiome are an important source of endogenous EtOH generation from ingested fructose (“autobrewery syndrome”) . AdditioBased on current knowledge of pathogenetic factors, potential therapeutic targets have been identified, such as a reduction in fat accumulation, improvements in insulin signaling, suppression of inflammatory response, a reduction in oxidative stress, and the inhibition of fibrogenic signaling  37]..37].Development of ALD and associated mortality is largely dependent on the amount of alcohol consumed. As shown by a meta-analysis by Rehm et al., >2 standard drinks per day for women and >3 standard drinks per day for men is associated with a significantly increased risk for liver cirrhosis morbidity . In a moAlcohol consumption leads to cell injury via direct and indirect toxic effects of EtOH and its even more toxic metabolite acetaldehyde. EtOH is metabolized to acetaldehyde predominantly via ADH (oxidative pathway), which is mainly expressed in the cytosol of hepatocytes  . Via the2O2 and O2−), DNA damage or lipid peroxidation of cell membranes, and increased endotoxin generation and inflammation. During chronic heavy drinking, the continuous reduction of NAD+ to NADH in the reactions catalyzed by ADH and acetaldehyde dehydrogenase (ALDH) during EtOH and acetaldehyde metabolism causes a significant metabolic shift towards fatty acid synthesis as well as increased production of lactate causing metabolic acidosis (REF). Structural mitochondrial alterations caused by acetaldehyde lead to decreased ATP generation and the production of reactive oxygen species causing oxidative stress  , which can be induced by increasing amounts of alcohol as EtOH protects CYP2E1 from being degraded by the ubiquitin proteasome system . This leEtOH and acetaldehyde also show direct toxic effects onto the gastric and intestinal mucosa which may lead to maldigestion and malabsorption of nutrients, vitamins, and trace elements. In the small and large intestine, EtOH is metabolized by the residing microbiota leading to an additional increase in acetaldehyde, cellular damage, and dysbiosis, including changes in the mycobiome and virome (mainly bacteriophages) . Despite the toxic effects of alcohol, only up to 20% of patients with continued excessive alcohol consumption develop alcohol-related liver cirrhosis, pointing to additional protective and risk factors in ALD pathogenesis. As well as the amount of alcohol consumed, the time period of continued alcohol consumption and the drinking pattern are important contributors: daily alcohol consumption, drinking while being fasted and binge drinking have been identified as determinants of increased risk ,9. Although conflicting data exist, the type of alcohol may also be associated with the risk of ALD development. While beer and red wine have been found to be associated with less harm to the liver in animal models and human studies, others did not confirm these findings ,48,49,50Additionally, genetic ancestry and sex modify the risk and severity of ALD and may predispose individuals to AUD ,52. PatiSimilar to NAFLD, several single nucleotide polymorphisms (SNP) have also been identified as modifiers of the risk and severity of ALD ,56. BaseFurthermore, cultural influences, environmental factors, and diet are factors contributing to the development and severity of ALD ,54,60,61As well as epidemiology and clinical presentation, NAFLD/MASLD and ALD have several pathogenetic determinants in common. However, pathogenesis of NAFLD and ALD is still incompletely understood. Elucidating the interaction of EtOH and food intake/weight, as well as the role of microbiota, including the intestinal barrier, might help identify biomarkers for early diagnosis and new therapeutic targets. Due to the high prevalence of NAFLD risk stratification by simple noninvasive tests, it is of utmost importance to provide optimal linkage to care for those patients with high risk of liver-related events. Previous studies have identified advanced fibrosis (F ≥ 3) as the major prognostic factor in NAFLD ,27,62. TWhile there is currently no approved biomarker for the presence of NASH, several noninvasive tests are available for the estimation of fibrosis stage in NAFLD. Simple tests based on routine clinical and laboratory parameters are readily available and are, therefore, useful for broad risk stratification in primary care. NAFLD fibrosis score (NFS), using a combination of the parameters of age, fasting glucose, body mass index (BMI), platelet count, albumin, and AST/ALT ratio , as wellIn addition, several proprietary liver fibrosis panels have been developed. The enhanced liver fibrosis (ELF™) test is based on three extracellular matrix proteins including hyaluronic acid (HA), procollagen-3 N-terminal peptide (P3NP), and tissue inhibitor of metalloproteinase-1 (TIMP-1), and has been studied in adult and pediatric patient populations with NAFLD ,69,70,71®) has shown accurate fibrosis staging ability in liver diseases of various etiologies including NAFLD [Vibration-controlled transient elastography  followed by a detailed noninvasive staging by the ELF test or VCTE. These sequential strategies are cost-effective as the high NPV of FIB-4 < 1.30 for advanced fibrosis allows researchers to reserve the costlier proprietary tests for those patients above this cut-off. In a recent individual patient data meta-analysis on 5735 patients from 37 studies, Mozes et al. validated the FIB-4 followed by VCTE algorithm, indicating high NPV for FIB-4 < 1.30 and reasonable PPV for FIB-4 > 2.67 or LSM > 10 kPa . ImportaUnfortunately, ALD is mostly detected in very advanced stages presenting with decompensated cirrhosis. Nevertheless, noninvasive fibrosis tests may be well-suited in screening for early ALD, e.g., during or shortly after alcohol detoxification treatment.Thiele et al. evaluated and compared 10 different noninvasive fibrosis tests in a Danish cohort of 289 patients with ALD attending an outpatient liver clinic or rehabilitation and demonstrated good to excellent diagnostic accuracy for the prediction of F3-4 stage on histology using FIB-4 (AUROC 0.85), ELF score (AUROC 0.92), and VCTE by FibroScan  [It should be noted that liver stiffness measured by VCTE depends not only on fibrosis but also inflammation, cholestasis, and/or congestion. During alcohol withdrawal, a rapid decline in LSM has been observed within 2 weeks after admission . In ordeAn individual patient data meta-analysis of 10 studies comprising the VCTE data of 1026 patients with ALD revealed an optimal LSM cut-off of 12.1 kPa for the prediction of F ≥ 3, which, however, varied from 8.8 kPa to 16.1 kPa depending on AST and bilirubin levels .Histological NASH is defined by the presence of >5% macrovesicular steatosis, lobular inflammation, and hepatocellular ballooning, typically with a predominantly centrilobular distribution . ActivitAccording to Yip et al., histological ASH is defined by the presence of steatosis (any degree), lobular inflammation, and hepatocellular ballooning . GradingAlongside the increasing burden of obesity and T2DM, alcohol consumption has become a major public health concern given that 55.5% of the world population, 76.5% of the European population, and 83.1% of the American population consume alcohol  ,93.Lifestyle modification resulting in weight loss currently represents the cornerstone of NAFLD treatment as its beneficial effects have been demonstrated in multiple studies. In a small randomized study, Promrat et al. tested the effect of intensive lifestyle intervention vs. structured education only on NASH and reported a significant improvement in ALT and a reduction in NAS on liver histology in parallel to weight loss of 9% in the intervention group . These fThe Western diet, containing high amounts of saturated fat and carbohydrates, should be replaced by Mediterranean diet rich in polyphenols and omega-3 polyunsaturated fatty acids . Low-fatAs the above-mentioned lifestyle changes are achieved in a minority of NAFLD patients only, effective drug therapy is highly desirable, especially in patients with active fibrotic NASH. Pharmacologic treatment of NASH aims to improve various pathophysiologic pathways, including metabolic , anti-inflammatory, and anti-fibrotic targets . To date2). A French study with serial liver biopsies at the time of surgery, 1 year later, and 5 years later has demonstrated resolution of NASH without worsening of fibrosis at 5 years in 84% and improvement of fibrosis by ≥1 stage at 5 years in 70% of patients [Bariatric surgery, using the Roux-en-Y gastric bypass or sleeve gastrectomy, is a very effective treatment option in patients with morbid obesity , psychosocial intervention, and pharmacological therapy . AlthougIn order to support abstinence, pharmacological treatment options in patients with ALD are limited. Baclofen is currently the only pharmacological treatment (off-label use) that has been tested in randomized-controlled trials in patients with AUD and advanced ALD, and its safety in patients with advanced liver disease has been supported by several additional observational studies ,107,108.Future treatment options for liver fibrosis in ALD might include rifaximin-α, which has recently been evaluated in an investigator-initiated, randomized, double-blind, placebo-controlled, single-center, phase 2 trial at Odense University Hospital in Denmark. The trial showed a lesser fibrosis increase after 18 months in the treatment arm . FindingAccording to the new nomenclature and definition, patients with MetALD present with features of both MASLD and ALD, including hepatic steatosis, ≥1 cardiometabolic risk factor out of 5, plus average daily alcohol consumption of 20–50 g  and 30–60 g , respectively . This neIt is well known that concurrent alcohol consumption worsens the prognosis of NAFLD/MASLD and ALD. However, the pathogenesis of NAFLD/MASLD and ALD is multifactorial, incompletely understood, and the true impact of alcohol consumption on the development and progression of SLD has remained unclear. Further research is needed to delineate the relative risk conferred by metabolic factors, various amounts (and type) of alcohol, and further pathomechanistic factors that might influence disease development, progression, as well as treatment targets. Importantly, current treatment of SLD should aim for both correction of cardiometabolic factors and alcohol abstinence. The designs of future clinical trials on the treatment of MASH, MetALD, and ALD need to consider characteristics of both MASLD and ALD.Understanding that there might be a huge overlap between NAFLD/MASLD and ALD, including the new entity of MetALD, requires further clarification of disease-contributing factors. Moving forward, a systematic screening for alcohol consumption and AUD as well as cardiometabolic risk factors is needed in patients presenting with (suspected) chronic liver disease. This should include the assessment of the amount of alcohol consumed with application of alcohol biomarkers to understand the true burden of disease, allow early diagnosis, and improve access to accurate treatment. Improving our understanding of the decisive drivers of NAFLD/MASLD and ALD, as well as their mutual interaction, will support further refinement of the MetALD definition and allow for a personalized treatment approach."}
{"text": "Non-Alcoholic Fatty Liver Disease (NAFLD) is a prevalent extraintestinal manifestation in patients with Inflammatory Bowel Disease (IBD). Liver fibrosis is the determinant long-term prognostic marker in patients with NAFLD because it is associated with progression to liver cirrhosis and hepatocellular carcinoma. In fact, NAFLD is already the most common liver disease worldwide and is becoming a leading cause of liver-related mortality. In this article, we systematically reviewed the available evidence regarding the prevalence and risk factors of liver fibrosis in patients with NAFLD and IBD, and we discussed the role and pathways of progression from liver fibrosis to hepatocellular carcinoma.The aim of the systematic review is to assess the prevalence and risk factors of liver fibrosis in patients with Inflammatory Bowel Disease (IBD) and Non-Alcoholic Fatty Liver Disease (NAFLD) and to discuss the role of liver fibrosis in the progression to hepatocellular carcinoma (HCC). We performed a structured search in PubMed, Web of Science, Embase, and Scopus up to 3 March 2023 to identify observational studies reporting liver fibrosis in patients with NAFLD and IBD. Quality of studies was assessed using the Newcastle-Ottawa Scale (NOS) score. A total of 23 studies met our inclusion criteria, including 629,781 patients. A total of 10 cross-sectional, 3 case-control, and 10 cohort studies were included. Fourteen studies had a NOS score ≥ 7 points. NAFLD was diagnosed in 2162/6332 (34.1%) IBD participants. However, NAFLD diagnosis was established in 924/2962 (31.2%) healthy individuals without IBD. Advanced liver fibrosis was found in 116 (11.6%) of 992 IBD patients with NAFLD. Most studies found an association between NAFLD and classic cardiovascular risk factors such as older age, male sex, higher BMI, diabetes, hypertension and dyslipidemia. In addition, metabolic syndrome features were also associated with an increased risk of significant and advanced liver fibrosis. Although no strong association between NAFLD and IBD therapy was reported, some studies associated NAFLD with IBD diagnosis, Crohn’s Disease, a complicated course of IBD, disease activity, and IBD duration. Advanced liver fibrosis was also associated with Crohn’s disease in several studies. In conclusion, NAFLD and advanced liver fibrosis are prevalent and clinically relevant extraintestinal manifestations, so its diagnosis and potential progression to HCC should be carefully considered in daily clinical practice. Non-Alcoholic Fatty Liver Disease (NAFLD) is defined as the presence of more than 5% of steatosic hepatocytes in patients without significant alcohol intake . Recentlp < 0.001) and the presence of IBD was a risk factor for developing MAFLD [The worldwide burden of NAFLD is increasing with a global prevalence of approximately 25% and it is becoming a leading cause of chronic liver disease and liver transplantation in the United States ,6. The png MAFLD .Obese patients have an increased risk of developing NAFLD in the general population but also in IBD ,13,14. Np < 0.001) and IBD was an independent risk factor for advanced fibrosis [Beyond cardiovascular risk, NAFLD is a clinically relevant condition due to the potential progression to NASH, advanced fibrosis, cirrhosis, and HCC . About 1fibrosis . The potfibrosis .Due to the higher risk of NAFLD in IBD and the complex interaction between the two diseases, the aim of this systematic review is to assess the prevalence and risk factors of liver fibrosis in patients with IBD and NAFLD and to discuss the role of liver fibrosis in the progression to HCC.This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines . InstituWe performed a comprehensive literature search in PubMed, Web of Science, Scopus and Embase up to 3 March 2023. Only human studies were included but no language or time restrictions were applied. Database searches were performed independently by three authors  using the following terms: inflammatory bowel disease OR IBD OR ulcerative colitis OR UC OR Crohn’s disease OR CD. The Boolean operator “AND” was used to combine these terms with: NAFLD OR non alcoholic fatty liver disease OR NAFL OR non alcoholic fatty liver OR steatosis OR NASH OR steatohepatitis OR hepatosteatosis OR MAFLD OR metabolic associated fatty liver disease OR liver fibrosis OR liver cirrhosis. All references were imported into an EndNote 20.5 reference manager software file. Duplicate references were electronically removed and subsequently reviewed manually. Titles and abstracts were independently assessed by three authors . Later, full-text articles were reviewed for inclusion by three authors , and any disparities were discussed and resolved by consensus (F.G). Finally, references from eligible studies were also reviewed to identify potential studies missed by the electronic search.The following inclusion criteria were applied: (1) studies including patients with established diagnosis of IBD; (2) studies assessing prevalence and/or risk factors of liver fibrosis in patients with NAFLD or MAFLD; and (3) observational studies, with or without control group.Studies were excluded in case of: (1) absence of original data ; (2) conference communications; (3) non-human studies; (4) studies involving only non-IBD patients or when IBD population data could not be identified; (5) absence of data regarding liver fibrosis in participants with NAFLD or MAFLD; and (6) studies focusing on patients with IBD receiving hepatotoxic drugs.Each included study was critically reviewed by three authors  and disparities were resolved by consensus among all authors. The following data was extracted from the selected studies: first author, year of publication, study location, study design, sample size, age, gender, Body Mass Index (BMI), IBD type, IBD therapy, prevalence of NAFLD and liver fibrosis, and risk factors for NAFLD and liver fibrosis.NAFLD was defined as the presence of liver steatosis after exclusion of significant alcohol consumption and secondary causes of fat accumulation in the liver . MALFD wThe prevalence of NAFLD and liver fibrosis was calculated, including all studies with available information. Absolute frequencies were estimated when only relative frequencies were reported. Data from longitudinal studies were obtained from the beginning or end of follow-up depending on the study design and the available information  by three authors . Disagreements were solved thorough discussion among all authors. Studies were considered to have low risk of bias if they achieved at least 7 stars. NOS for cohort and case-control studies is compound by 8 items: 4 items for the selection of exposed/non-exposed group or cases/controls groups, 1 item for the comparability of the groups, and 3 items for outcome evaluation/ascertainment of exposure. A maximum of 1 star can be assigned to each item except for comparability, which can be assigned 2 stars  . NOS scaThe flow diagram of study retrieval for the systematic review is detailed in All the included studies were observational, including 10 43.5%) cross-sectional studies ,40,46,48.5% crossRegarding the diagnostic method for NAFLD, 3 performed a retrospective review of database records ,35,42, 22, except participants included by Bessisow 2016, Veltkamp 2022, and Yen 2021 [A total of 629,781 participants were included, 220,128 35%) with IBD and 409,653 (65%) without IBD at enrolment. CD was the most frequent IBD type with 218,162 (99.1%) participants, followed by UC with 1907 (0.9%) patients. Sample size ranged from 35 (0.01%) patients  to 405,4Yen 2021 ,48,49. DYen 2021 ,40. ThioYen 2021 ,45,46,48Yen 2021 ,39,45,48Yen 2021 ,35,37,41% with IBNOS scores for risk of bias assessment are shown in NAFLD was diagnosed in 2162 34.1%) patients of a total of 6332 IBD participants included in 18 studies .1% patie,47,48,49Data on liver fibrosis were reported heterogeneously in the different studies. Advanced liver fibrosis was found in 116 (11.6%) of 992 IBD patients with NAFLD included in six studies ,33,42. TMost studies found an association between NAFLD and classic cardiovascular risk factors such as older age, male sex, higher BMI, type 2 diabetes, arterial hypertension, and dyslipidemia. In fact, Spagnuolo (2019) demonstrated that weight gain is a predictor of steatosis worsening . RegardiIn parallel, metabolic syndrome features were also associated with an increased risk of significant liver fibrosis ,36,40,46This systematic review reported the prevalence and risk factors associated with NAFLD and liver fibrosis in patients with IBD. In the last few months, several articles focused on NAFLD and IBD ,38,46,48Due to the heterogeneous report of liver fibrosis status, we estimated a prevalence of advanced liver fibrosis of 11.6% including information from six studies ,32,33,42Unfortunately, we did not find studies reporting the prevalence and/or risk factors of HCC specifically performed in the IBD population. However, a recent study assessed the effect of immune checkpoint in patients with pre-existing IBD, finding that approximately 40% patients experienced relapse of IBD with a rate of discontinuation of 35% . ConversMany studies have investigated the elements involved in fibrosis progression . In thisGenetic factors can also influence the risk of disease progression, fibrosis and HCC. The polymorphism p.I148M of the PNPLA3 gene has been linked to an increased risk of NAFLD, NASH, HCC, and liver-related mortality . It is oThe development of HCC in non-cirrhotic patients with NASH suggest that other pathogenic ways independent of fibrosis can lead to carcinogenesis, so some studies have investigated the role of the microbiota, metabolic changes, and immune and fibrotic cells . There iBile acids are produced in the liver through cholesterol oxidation, secreted into the duodenum and conjugated by gut microbiota in order to facilitate the absorption of fatty acids and also to activate the Farnesoid X Receptor (FXR) of the enterocytes, which regulates the enterohepatic circulation and play an important role in the glucose metabolism . DysbiosConcerning the role of metabolic changes, the development of NASH is secondary to the accumulation of free fatty acids in the liver with an increase in the gluconeogenesis, which leads to higher levels of glucose and more conversion to fatty acids ,71. WhenInnate but also adaptive immunity seem to play an important role in the development of fibrosis and HCC, since in NAFLD the continuous inflammatory response ends up producing dysregulation of the immune system with immunosuppression and tumor tolerance. Monocyte-derived macrophages form complexes with dead steatotic hepatocytes triggering a pro-fibrotic response, which could be involved in carcinogenesis . NeutropIn conclusion, liver fibrosis is an independent risk factor for HCC but NAFLD HCC can also occur in absence of cirrhosis, so other elements should be considered in this population ,76,77. MFinally, we acknowledge the following limitations of our study. First, the different study designs of included studies and the high rate of uncontrolled studies. Second, the heterogeneity of diagnostic methods and cut-offs used for NAFLD and liver fibrosis diagnosis. Third, the variability of data reported by each study, especially for liver fibrosis. Fourth, the absence of solid evidence on the prevalence and specific risk factors of HCC in patients with concomitant IBD and NAFLD. Nevertheless, the strengths of the study are its large sample size, the high rate of recently published articles and that, on average, the quality of the included studies was high. Our findings are clinically relevant as they reveal the magnitude of a comorbidity, whose adequate prevention and treatment could improve the prognosis and quality of life of the IBD population.NAFLD is a prevalent extraintestinal manifestation in patients with IBD and it can promote HCC thorough liver fibrosis development, although not exclusively. The presence of advanced liver fibrosis is not rare in the IBD population with NAFLD, especially in those suffering from Crohn´s Disease. Therefore, the presence of NALFD should be considered in patients with IBD, especially in association with additional risk factors. After diagnosis, hygienic-dietary measures should be initiated and evolution should be monitored to prevent complications, such as cirrhosis or HCC. Future studies are needed to evaluate the prevalence and specific risk factors of HCC in IBD population with NAFLD."}
{"text": "Nonalcoholic fatty liver disease (NAFLD), the most widespread chronic liver disease worldwide, confers a significant burden on health systems and leads to increased mortality and morbidity through several extrahepatic complications. NAFLD comprises a broad spectrum of liver-related disorders, including steatosis, cirrhosis, and hepatocellular carcinoma. It affects almost 30% of adults in the general population and up to 70% of people with type 2 diabetes (T2DM), sharing common pathogenetic pathways with the latter. In addition, NAFLD is closely related to obesity, which acts in synergy with other predisposing conditions, including alcohol consumption, provoking progressive and insidious liver damage. Among the most potent risk factors for accelerating the progression of NAFLD to fibrosis or cirrhosis, diabetes stands out. Despite the rapid rise in NAFLD rates, identifying the optimal treatment remains a challenge. Interestingly, NAFLD amelioration or remission appears to be associated with a lower risk of T2DM, indicating that liver-centric therapies could reduce the risk of developing T2DM and vice versa. Consequently, assessing NAFLD requires a multidisciplinary approach to identify and manage this multisystemic clinical entity early. With the continuously emerging new evidence, innovative therapeutic strategies are being developed for the treatment of NAFLD, prioritizing a combination of lifestyle changes and glucose-lowering medications. Based on recent evidence, this review scrutinizes all practical and sustainable interventions to achieve a resolution of NAFLD through a multimodal approach. Before the middle of the last decade, the worldwide prevalence of non-alcohol fatty liver disease (NAFLD) was approximately 25% in adult individuals. NAFLD is currently defined as an ectopic accumulation of lipids in the liver in the absence of secondary causes or other etiologies of liver disease ,2. It isIn the 21st century, among other metabolic diseases, diabetes mellitus has become the main concern for societies and health care systems. In particular, T2DM is mentioned to affect 1 in 11 adults and up to 463 million people worldwide . This tyThe development of NAFLD is an intricate process and is not completely understood. The liver, as is widely known, promotes many functions through the mobilization, regulation, and storage of nutrients. Hepatocytes play an essential role as regulators of amino acid, triglyceride and lipoprotein metabolism, gluconeogenesis, and ketogenesis . InteresIn a small percentage of patients, NAFLD is associated with infectious pathologies that can lead to the appearance of liver steatosis, such as hepatitis C and the human immunodeficiency virus (HIV). In some cases, it is related to medications  and specific toxins or acquired/inherited metabolic diseases such as cachexia, lipodystrophy, or even gastrointestinal surgery ,18.The development of NASH has been reported to be divided into two phases. The first is fatty deposition in the liver combined with an increase in insulin resistance. The second phase is related to cellular and molecular changes, including primarily oxidative stress and oxidation of fatty acids through lipid peroxidation, hyperinsulinemia, energy homeostasis, variation in the extracellular matrix, and changes in immune system function ,20. ConsRobust evidence suggests that there is a strong and bidirectional relationship between the progression of NAFLD and T2DM. High levels of diacylglycerol or ceramides can affect liver–insulin signaling, leading to an abnormal increase in liver insulin resistance . FurtherIt should be noted that several studies have shown that improvements in insulin sensitivity have been positively associated with histological improvements in NAFLD/NASH and regression of fibrosis . In 2013The purpose of this narrative review is to summarize the current literature on modern therapeutic approaches to NAFLD in patients with and without diabetes, focusing on the targeting of metabolic disturbances. Another purpose is to discuss future potential treatments and knowledge gaps in the therapy of NAFLD.The therapeutic quiver of NAFLD consists of several levels, of which lifestyle, pharmaceutical, and surgical approaches are the main treatments. A multimodal intervention with multiple aspects, such as lifestyle modification, weight loss, specific diets, and medication, is the most appropriate and holistic approach for most people with NAFLD.Robust evidence supports the crucial role of lifestyle changes as primary options for the treatment of NAFLD. These approaches, which include diet, exercise, or physical activity, mainly aim to control metabolic status . LookingWeight loss is a gold standard therapy for most patients with NAFLD and can regress liver disease, along with the reduction of cardiovascular disease and the risk of T2DM . Some reA clinical trial by Holmer et al., which recruited 74 patients with NAFLD, indicated that intermittent calorie restriction and a low carbohydrate high fat diet (LCHF) are more effective in reducing liver steatosis and body weight compared to general lifestyle modification. Participants were randomized into 3 groups: intermittent calorie restriction, including 500 kcal/day for women and 600 kcal/day for men; LCHF, with an average daily calorie intake of 1600 kcal/day for women and 1900 kcal/day for men; and general lifestyle advice . FurtherIn addition to the above, it should be noted that some studies reported the beneficial effect of diabetes remission on NAFLD and pancreatic morphology. In 2020, a post hoc analysis of the DiRECT trial showed changes in the gross morphology of the pancreas 2 years post T2DM remission. The size of the pancreas had increased in patients who achieved remission and weight loss, compared to those who did not respond to the weight loss intervention. Intrahepatic fat and levels of FGF-21 and FGF-19 also decreased. However, it is notable that there is no significant increase in pancreas volume after 6 months of reversal of type 2 diabetes . AdditioNumerous studies have corroborated the pivotal role of certain macronutrients in the initiation and progression of NAFLD, regardless of caloric intake. In particular, macronutrients such as saturated fatty acids (SFA), trans fats, simple sugars such as sucrose and fructose, and animal proteins are known to inflict damage on the liver through the accumulation of triglycerides and impaired antioxidant activity, compromising insulin sensitivity and postprandial triglyceride metabolism . In contFurthermore, Halima et al. conducted a study investigating the impact of apple cider vinegar on rats with diabetes and demonstrated that in addition to its potent antihyperglycemic properties, it also exhibited a crucial hepatoprotective effect. In particular, indicators of liver toxicity, namely ALT, AST, total and direct bilirubin, as well as levels of TC, TG, and LDL-c, demonstrated a significant reduction, which was particularly prominent after four weeks of treatment, together with an elevation in HDL-c . These f2) [The potential effects of bariatric surgery on liver fat disease may extend beyond weight loss. In fact, serum concentrations of glucagon-like peptide-1 (GLP-1) increase after metabolic surgery, leading to decreased appetite, slower gastric emptying, and improved insulin sensitivity . Further2) ,79. Alth2) .The most common bariatric surgery procedures include adjustable gastric band (AGB), biliopancreatic diversion (BPD), vertical sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). Consequently, different methods might induce variable biological effects depending on the surgical procedure. The most common metabolic operations are SG and RYGB. In the first, about 80% of the stomach portion is removed along the gastric greater curvature and the small dimensions of the stomach, along with the changes in the hormonal environment, reduce hunger and delay gastric emptiness. In the second procedure, the stomach is separated into a smaller pouch in the smaller curvature (through stapling) and anastomosed with the jejunum ,82. In fIt is well established that fatty liver and T2DM are the two sides of the same coin, sharing common pathogenic pathways and factors such as insulin resistance. Although the coexistence of fatty liver and T2DM is increasing, the treatment is not adequate. Due to this close link between T2DM and NAFLD, various glucose-lowering drugs have been used as NAFLD therapeutics. Numerous clinical trials have shown the beneficial effects of GLP-1 receptor agonists, insulin-sensitizing thiazolidinediones, and sodium-glucose cotransporter 2 (SGLT2) inhibitors on liver fat content. GLP-1 binds to a specific GLP-1 receptor, whose activation can promote the reduction of liver steatosis by improving insulin signaling pathways, hepatocyte lipotoxicity, and mitochondrial function ,96. On tIt is significant to mention that a multitude of studies have recently elucidated the efficacy of SGLT2 inhibitors in alleviating liver steatosis. Of particular interest, in 2018, Shibuya et al. revealed, for the first time, the statistically significant and advantageous impact of luseogliflozin, compared to metformin, on NAFLD and weight loss . The resThiazolidinediones increase peripheral insulin sensitivity by stimulating adipokines, promoting triglyceride storage in adipose tissue, and improving the suppressive action of insulin on lipolysis . In thisRecently, semaglutide and tirzepatide have been added to the therapeutic quiver against T2DM and obesity. Semaglutide is a GLP-1 analogue, and tirzepatide is a dual analogue of GLP-1 and GIP (glucose-dependent insulinotropic polypeptide) . Both deRealizing that NALFD is strongly related to metabolic syndrome, there is a need for an integrated approach for individuals with a high liver fat content. The definition of metabolic syndrome includes the following criteria: abdominal obesity, with waist circumference of ≥90 cm in men or ≥85 cm in women; low high-density lipoprotein (HDL) cholesterol with HDL-cholesterol of <40 mg/dL in men and <50 mg/dL in women; hypertriglyceridemia with triglyceride (TG) of ≥150 mg/dL; high systolic blood pressure (BP), with systolic BP of ≥130 mmHg and/or diastolic BP of ≥85 mmHg; or hyperglycemia, with fasting plasma glucose (FPG) of >100 mg/dL ,126. ConFurthermore, the category of omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as a-linolenic acid (a-ALA), stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), has a beneficial effect on peripheral insulin sensitivity and on triglycerides levels, leading to a lower deposition of liver fat . A randoAlthough statins have long been used as a widely accepted method for reducing cholesterol and minimizing the risk and mortality associated with cardiovascular disease, recent years have seen increasing scrutiny of their potential side effects. It should be noted, in particular, the increasing evidence linking prolonged statin use with diabetes progression and ectopic fat deposition, particularly in the kidneys of patients with diabetic nephropathy . RecentlMore recently, new potential drugs for NAFLD have been studied. The most common treatment targets are the farnesoid X receptor (FXR), a nuclear receptor, and obeticholic acid (OCA), which is a synthetically modified analogue of chenodeoxycholic acid . These a2 or >27 kg/m2 in the presence of at least one metabolic comorbidity, because it can lead to the remission of diabetes and improves the progression of fatty liver disease [During recent years, weight loss pharmacotherapy has been an attractive option for patients with NAFLD, with or without T2DM and with a BMI > 30 kg/m disease ,146. Τhe disease . In 2005 disease . In cont disease .As previously stated, two recently approved drugs, tirzepatide, a dual analogue of GIP/GLP-1, and semaglutide, a GLP-1 analogue, were added to the therapeutic arsenal of NAFLD . A phaseTirzepatide is associated with a significant reduction in liver fat content (LFC), subcutaneous abdominal adipose tissue (ASAT), and volume of visceral adipose tissue (VAT) ,153. A mIn 2020, Hartman et al. determined the result of tirzepatide on the biomarkers of NASH and fibrosis in people with diabetes. A reduction in ALT, AST, keratin-18 (K-18), and N-terminal pro-peptide of type III collagen (Pro-C3) levels was revealed, combined with a significant increase in adiponectin levels that was dose-dependent . It is aSemaglutide has also been effective in improving liver biochemistry. A prospective study by Volpe et al. showed a significant decrease in glucometabolic parameters, liver enzymes, and laboratory indicators of liver steatosis during therapy, as well as a reduction in fat mass and VAT. Interestingly, liver steatosis improved in 70% of participants after 52 weeks of treatment . FurtherRecently, an open-label phase 2 trial by Alkhouri et al. reported that the combination of semaglutide with cilofexor and firsocostat achieved greater improvements in liver steatosis and biochemistry profile than monotherapy . Based oFuture studies are expected to add additional evidence on the effects of new antidiabetic agents on NAFLD progression and clarify whether the benefits are driven primarily by weight loss or by the pleiotropic actions of the drugs.The review of available evidence supports the notion that NAFLD is an important risk factor for the development of T2DM, but also underlines the bidirectional relationship between these conditions. Despite many advances in our knowledge on the epidemiology and pathogenesis of NAFLD, the most established treatment so far is intensive weight loss. The importance of weight reduction is highlighted in people with NASH, where weight loss greater than 7% is associated with a clinically significant regression of disease status . ExercisToday, the question is no longer whether lifestyle modification is an effective clinical therapy, but how we implement lifestyle as a therapy in daily clinical care. However, most studies indicate several barriers to maintaining a healthy lifestyle in the long term and that weight regain is very common among people with diabetes and/or obesity, making the use of pharmacotherapy a necessity in most cases. In this context, a better understanding of the mechanisms linking NAFLD and T2DM will help design targeted therapies that can stop or reverse disease progression."}
{"text": "A characteristic of nonalcoholic fatty liver disease (NAFLD) is the buildup of excess fat in the liver which encompasses various clinical phases, including steatosis, inflammation, ballooning, fibrosis, and liver cirrhosis. Nonalcoholic steatohepatitis (NASH) represents a severe form of NAFLD. The prevalence of NAFLD, particularly NASH, is notably high among Hispanics and those with morbid obesity. Diabetes, obesity, and dyslipidemia are significant risk factors in patients with NAFLD. The pathogenesis of NAFLD involves complex interactions between hormonal, nutritional, and genetic factors. Different clinical trials have been conducted to determine if there are any supplements that could help patients with NASH. Evidence has shown that vitamin E decreased the NAFLD activity score but not fibrosis. Our review summarizes the influence of supplementation on patients with NAFLD and NASH, focusing on the use of different clinical trials, systematic reviews, and meta-analyses. In the future, patients and physicians will play crucial roles in exploring diverse approaches and finding effective solutions to address this growing issue. Nonalcoholic fatty liver disease (NAFLD) is a condition in which excess fat is stored in the liver . The livObesity and related ailments, such as cardiovascular diseases and type 2 diabetes mellitus (T2DM), have been linked to oxidative stress, which has been recognized as a major contributing factor in NAFLD . ThroughSearch strategyUsing NAFLD, NASH, supplement use in NASH and NAFLD, epidemiology, risk factors, and pathophysiology of NASH and NAFLD as research keywords, articles were reviewed to illustrate and analyze the role of supplements in the improvement of NAFLD, NASH, and liver function tests. Studies published between 2011 and 2023 were considered for inclusion in this review. Medline was used as the primary database. Data were collected from literature reviews, systematic reviews, meta-analyses, and several types of clinical trials, cohort studies, and retrospective studies. There were no age or ethnicity limitations to the search. Only articles published in the English language were included in this review.This section will discuss the epidemiology, risk factors, and pathophysiology of NAFLD and NASH; the role of multiple kinds of supplements in both diseases; and how supplements can benefit NASH patients and improve their liver function tests.Epidemiology of NAFLDThe incidence and prevalence of NAFLD are increasing quickly around the globe, ranging from 13% in Africa between 1989 and 2015 to 40% in Southeast Asia between 1999 and 2019 . The preRisk factors of NAFLDDiabetes and obesity are major risk factors in patients with NAFLD . T2DM haPathophysiology of NAFLD and NASHThe pathogenesis of NAFLD and NASH is multifactorial, and many mechanisms have been proposed as possible causes of fatty liver infiltration . In receRole of supplements in NAFLD and NASHSilybum marianum), can mitigate lipid peroxidation and free radical injury [Curcuma longa Linn, has reportedly been effective in reducing oxidative stress and inflammatory cascades [Vitamin E is a naturally occurring fat-soluble vitamin with the reputation of being a crucial antioxidant . The PIVl injury . Five mel injury . Curcumicascades . In a plcascades . Hepaticcascades . Omega-3cascades . A meta-cascades . In a 12cascades . Improvecascades .Synbiotics are formulas containing probiotics plus prebiotics . A probiNASH is rapidly rising worldwide and is the fastest-growing cause of liver cancer death globally, especially in the Spanish community. Lifestyle plays a significant role according to new data. Therefore, it is important to study the role of supplements and how they benefit patients with NAFLD and NASH. According to numerous studies published in the last 10 years, supplementation plays a role in helping NAFLD and NASH patients by improving liver function tests and other factors; however, no significant improvement has been reported in the level of fat in the liver or improvement in any scarring tissue. Both physicians and patients should be educated about the disease and lifestyle changes, encouraging patients to participate in ongoing pharmaceutical clinical trials worldwide to help find a new solution."}
{"text": "Nonalcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD) affects ~70% of patients with type 2 diabetes (T2D), with ~20% showing signs of advanced liver fibrosis. Patients with T2D are at an increased risk of developing cirrhosis, liver failure, and hepatocellular carcinoma and their liver-related mortality is doubled compared with non-diabetic individuals. Nonetheless, the condition is frequently overlooked and disease awareness is limited both among patients and among physicians. Given recent epidemiological evidence, clinical practice guidelines recommend screening for NAFLD/MASLD and advanced liver fibrosis in patients with T2D. While many drugs are currently being tested for the treatment of NAFLD/MASLD, none of them have yet received formal approval from regulatory agencies. However, several classes of antidiabetic drugs  have shown favorable effects in terms of liver enzymes, liver fat content and, in some occasions, on histologic features such as inflammation and fibrosis. Therefore, diabetologists have the opportunity to actively treat NAFLD/MASLD, with a concrete possibility of changing the natural history of the disease. In the present narrative review, we summarize evidence and clinical recommendations for NAFLD/MAFLD screening in the setting of T2D, as well as on the effect of currently available glucose-lowering drugs on hepatic endpoints. Nonalcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated fatty liver disease (MAFLD)  or metabIn a recent study in patients with NAFLD/MASLD studied with paired liver biopsies, even after adjustment for potential confounders, the presence of T2D was associated with a 70% increase in the relative risk of fibrosis progression . While pImportantly, the degree of liver fibrosis is the most important histologic predictor of future development of liver-related events, as shown by several cohort studies and meta-analyses ,17. The In the present narrative review, we will provide the reader with a practical summary of available evidence and guideline recommendations on screening, diagnosis, and treatment of NAFLD/MASLD in patients with T2D. We will also focus on the effect of currently available and future antidiabetic medication on liver-related endpoints.Liver biopsy remains the established and most reliable method for assessing the severity of NAFLD/MASLD as it allows for a comprehensive evaluation of the three histologic aspects of the disease: steatosis, inflammation, and fibrosis. Nonetheless, this procedure is invasive and not well-received by patients  and it cInternational guidelines recommend the use of a conventional liver ultrasound as a first-line diagnostic technique to be applied in clinical practice for the diagnosis of steatosis (and therefore NAFLD/MASLD itself) and suggest that all patients with T2D, independently from liver enzymes levels, should be screened for steatosis with an ultrasound examination ,27,28. EWhile magnetic resonance spectroscopy (MRS) or magnetic resonance imaging-Proton Density Fat Fraction (MRI-PDFF) have higher accuracy in identifying and quantifying intrahepatic fat compared with ultrasound , their uFinally, Controlled Attenuation Parameter (CAP), a measure that can be obtained concomitantly with the liver stiffness measurement (LSM) using vibration-controlled transient elastography (VCTE), has gained attention as a rapid and inexpensive technique to detect and quantify liver fat . The CAPGiven the prominent role of liver fibrosis in determining the prognosis of patients with NAFLD/MASLD and the difficulties encountered in the development of accurate biomarkers of inflammation, efforts have been made to identify accurate and readily available non-invasive methods to detect and stage liver fibrosis. Ideally, biomarkers should possess more than just diagnostic capabilities when compared to the current gold standard technique of liver biopsy. They should also have the ability to predict future liver-related events, facilitate disease monitoring over time, and indicate the effectiveness of a specific intervention . RegrettNonproprietary algorithms utilizing common clinical and biochemical variables (indirect markers of fibrosis) are simple blood tests easily accessible in routine clinical practice, and they are correlated with histologic identification of advanced fibrosis. The most validated and applied are the FIB-4  (based oProprietary blood tests are designed with specific markers directly related to various elements of the extracellular matrix (ECM). In contrast to simple blood tests, these proprietary tests provide a more accurate representation of the dynamic processes involved in ECM deposition (fibrogenesis) and reorganization (fibrolysis). Many of these scores , commercially available as Fibroscan by Echosens, was the first to be introduced and is currently the most extensively validated . AlthougFollowing that, elastography methods have been integrated into ultrasound devices using distinct technologies, such as point shear wave elastography (p-SWE) and two-dimensional shear wave elastography (2D-SWE) . DespiteMost of these limitations can be addressed through the application of magnetic resonance elastography (MRE). MRE employs specific hardware to generate a pulse sequence, and the acquired data are then processed by dedicated software to create a color elastogram of the entire liver . Althougn = 3202) [Several studies investigated whether combining either simple scores with patented ones or simple scores with imaging modalities could increase the accuracy in identifying the minority of patients with NAFLD/MASLD and advanced liver fibrosis, in order to facilitate referral to hepatologists for adequate management. For instance, a baseline analysis of the STELLAR trials, which evaluated the efficacy of selonsertib in patients with NASH, evaluated possible concomitant or sequential combinations of several noninvasive methods in a large population of patients evaluated by liver biopsy ( = 3202) . The autThe first guidelines to recommend generalized screening for NAFLD/MAFLD in patients with T2D were published in 2016 by EASL, the European Association for the Study of Diabetes (EASD) and the European Association for the Study of Obesity (EASO) . This muWhile these guidelines represent a fundamental effort to achieve a systematic approach to the problem, several reports have underlined a potential for over-referral of patients in the setting of T2D or obesity clinics ,57. MoreIn the following years, data have accumulated on the performance of combining different methodologies and on the high prevalence of advanced fibrosis and cirrhosis in patients with T2D ,8,44,56.The first step is FIB-4 calculation, and if a value ≥ 1.3 is obtained, VCTE should be performed. If the LSM value is ≥8 kPa, the patient should be referred to the hepatologist, while advanced fibrosis can safely be excluded if a lower value is obtained .This strategy has generally been implemented in other recent guidelines from several international hepatologic and endocrinologic societies. In 2023, AASLD published new guidelines in which they recommended screening for advanced fibrosis in all patients with T2D, which was considered a condition facilitating progression towards cirrhosis . SimilarAs a summary, agreement on the need for systematic screening for NAFLD/MASLD, but most importantly for advanced liver fibrosis, has increased progressively in the last decade. Most international societies also recommend similar two-step algorithms in which a simple inexpensive blood test is performed first and more specialized tests are only performed in the case of uncertainty on the first. Our data on patients with T2D suggest that FIB-4 excludes advanced fibrosis in 55–60% of patients , leavingThe important favorable effect of lifestyle interventions and bariatric surgery on histologic features of NAFLD/MASLD were demonstrated in multiple observational studies . As a coNonetheless, diabetologists are in a position of favor, as some drugs currently used to treat T2D itself have shown some efficacy on liver disease endpoints as well. Here, we report results with drug classes currently used for the treatment of T2D that were studied in RCTs on different NAFLD/MASLD endpoints. A summary of current evidence is also provided in The rationale for employing pioglitazone is based on its activation of peroxisome proliferator-activated receptor gamma (PPARγ), which can exert a significant impact on the pathophysiology of NAFLD/MASLD. By ameliorating insulin resistance, modulating lipid and glucose metabolism in a favorable manner, and reducing hepatic and gastrointestinal inflammation, pioglitazone contributes to a reduction in portal hypertension, splanchnic inflammation, angiogenesis, and porto-systemic shunts .Based on a post-hoc analysis involving 55 patients with biopsy-proven NASH, treatment with pioglitazone appears to result in changes in body fat distribution, specifically a decrease in the visceral-to-subcutaneous fat ratio, and biochemical alterations, including an increase in plasma adiponectin levels, when compared to the control group. These changes reflect the mechanism by which this drug contributes to the reduction of steatosis and necroinflammation in NASH patients .In the literature there are several randomized controlled trials (RCTs) conducted in different parts of the world  which involve patients with or without T2D and biopsy-proven NAFLD/MASLD. A phase 2 meta-analysis published by Musso et al. encompasses eight RCTs, involving approximately 500 patients with biopsy-proven NASH, who were treated with thiazolidinediones . These tAccording to the European guidelines (EASL-EASD-EASO), pioglitazone may be used in the treatment of NAFLD/MASLD in individuals with type 2 diabetes (off-label in those without type 2 diabetes). On the other hand, the American guidelines (AASLD), in line with UK guidelines (NICE), suggest the use of pioglitazone only in diabetic individuals with NAFLD/MASLD . The recThe GLP-1 receptor agonists (GLP-1 RAs) play a direct and indirect role in the pathophysiology of NAFLD/MASLD. They increase insulin levels, thereby reducing hepatic gluconeogenesis. Additionally, GLP-1 RAs decrease lipolysis and the influx of free fatty acids in the liver. These agents may also contribute to reducing inflammation and apoptosis, promoting tissue remodeling, and increasing adiponectin levels. However, most importantly, they induce weight loss .p = 0.019). Moreover, two (9%) of twenty-three patients in the liraglutide group versus eight (36%) of twenty-two patients in the placebo group had progression of fibrosis  [The first RCT conducted to evaluate the effect of a GLP1-RA on histologic liver endpoints was the Phase 2 LEAN trial, in which 26 patients were randomly assigned to receive liraglutide and 26 to placebo. This relatively small study showed positive results as nine (39%) of twenty-three patients who received liraglutide had NASH resolution, compared with two (9%) of twenty-two in the placebo group  .2 value of 0.791. This supports the idea that the effect of GLP-1 RAs on NAFLD/MASLD endpoints is mostly driven by their capacity to promote weight loss [The effectiveness of GLP-1 RAs has been recently summarized in a meta-analysis that included 11 placebo-controlled or active-controlled phase-2 RCTs involving a total of 936 middle-aged individuals . These tght loss .n = 3) or vildagliptin (n = 1) to specifically treat NAFLD/MASLD [The role of DPP4 inhibitors (DPP4-i) in NAFLD/MASLD is considered minimal. Although these drugs can reduce HbA1c levels, they have a neutral effect on weight. There are four placebo-controlled or active-controlled RCTs that used either sitagliptin (LD/MASLD . These RSGLT2 inhibitors (SGLT2-I) induce a series of modifications that have the potential to improve the liver condition in individuals with NAFLD/MASLD: they improve glycemic profile, reduce visceral adipose tissue, increase plasma adiponectin levels, and decrease uric acid levels; they also diminish oxidative stress and systemic inflammation and increase glucacon levels .n = 6 RCTs), empagliflozin (n = 3 RCTs), ipragliflozin (n = 2 RCTs), and canagliflozin (n = 1 RCT), administered for a median period of 24 weeks. The subjects included in these studies were predominantly diabetic (≃90%), and NAFLD/MASLD was diagnosed through imaging. From the meta-analysis of these studies, it is evident that SGLT2-I, compared to the control group (placebo or reference therapy), led to a significant reduction in the percentage of hepatic fat as evaluated by MRI. Currently, there are no published RCTs that utilize histological hepatic endpoints. According to most international guidelines, SGLT2-I are considered safe for patients with liver diseases, but their specific use for the treatment of NAFLD/MASLD is not yet recommended.Twelve RCTs involving the use of these drugs for the specific treatment of NAFLD/MASLD were conducted in different parts of the world  . The SGLMetformin is the first-line drug in the treatment of diabetes and can reduce gluconeogenesis and liponeogenesis, lower systemic inflammation, increase GLP1 levels, and modify intestinal microbiota, all of which have potential beneficial effects on the liver .In the literature, there are trials conducted in various parts of the world that involve the use of metformin in diabetic, non-diabetic, or prediabetic patients with NAFLD/MASLD diagnosed through biopsy or imaging. It has been observed that metformin can reduce liver steatosis, leading to a significant decrease in transaminase levels . However, it does not have a significant effect on fibrosis and resolution of NASH . The AISTirzepatide, a dual glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 receptor agonist, has demonstrated superiority compared with GLP1-RA on both glycemic control and weight loss, and achieved favorable effects on hepatic endpoints. In a substudy of the SURPASS-3 trial, which enrolled exclusively patients with T2D, tirzepatide showed a significantly greater reduction in liver fat content (LFC), volume of visceral adipose tissue (VAT), and abdominal subcutaneous adipose tissue (ASAT) compared to insulin degludec . These rRetatrutide (RETA), a once-weekly injectable triple hormone agonist of the GIP, GLP-1, and glucagon receptors, has also shown promise in the treatment of obesity. In a phase 2 obesity trial, RETA treatment resulted in substantial reductions in body weight (up to 24%) . In a suRecommendations of international societies on screening and pharmacological treatment of NAFLD/MASLD in patients with T2DM are summarized in NAFLD/MAFLD and T2D are tightly linked in a strong, bidirectional relationship. Given the extremely high disease prevalence, referral of all affected patients to hepatologists is not feasible or cost-effective. Diabetologists are therefore called to actively screen and risk-stratify patients to identify those that are at higher risk of clinically relevant liver-related outcomes. Current guidelines recommend a two-step strategy in which a simple blood-based score such as FIB-4 is followed (if advanced fibrosis cannot directly be excluded) by an imaging technique (most commonly VCTE). This screening procedure does not only aim to identify patients to refer to the hepatology clinic, but also to inform treatment. Indeed, while no specific agent has been approved with the indication to improve NAFLD/MASLD outcomes, several glucose-lowering agents showed some efficacy on hepatic endpoints in dedicated RCTs. In particular recent guidelines recommend pioglitazone or GLP1-RA in patients with T2D and biopsy-proven NASH or those at a high risk of advanced liver fibrosis. We believe that diabetologists are currently in a privileged position to actively treat patients with T2D not only to reduce their risk of developing micro- and macro-vascular complications, but also to reduce the disease burden associated with cirrhosis and hepatocellular carcinoma."}
{"text": "Over the past 100 years, cardiovascular disease (CVD) has become a leading cause of mortality and morbidity in developed countries, and similar trends have occurred for chronic liver disease. Subsequent research also indicated that people with non-alcoholic fatty liver disease (NAFLD) had a twofold increased risk of CV events and that this risk was doubled in those with liver fibrosis. However, no validated CVD risk score specific for NAFLD patients has yet been validated, as traditional risk scores tend to underestimate the CV risk in NAFLD patients. From a practical perspective, identifying NAFLD patients and assessing severity of liver fibrosis when concurrent atherosclerotic risk factors are already established may serve as an important criterion in new CV risk scores. The current review aims to assess current risk scores and their utility for the prediction of CV events among patients with NAFLD. Obesity is a multifactorial relapsing disease, as defined by the International Classification of Disease . AccordiBeing overweight or obese is among the main risk factors for developing non-alcoholic fatty liver disease (NAFLD), which is considered to be the most prevalent chronic liver disease worldwide . NAFLD iHowever, a new concept is emerging that is known as metabolic-associated fatty liver disease (MAFLD), which is a heterogenous clinical entity. The diagnostic criteria of MAFLD requires the presence of metabolic risk factors in the setting of hepatic steatosis, includes individuals with other concomitant liver diseases, and excludes those with hepatic steatosis who do not fulfill the metabolic risk criteria . Large cThe majority of people with NAFLD will not develop liver fibrosis or cirrhosis, yet some studies argue that by 2030, NASH will become the leading cause of liver transplant in the USA . An analAs previously reported, chronic liver disease (CLD) and cardiovascular diseases (CVDs) are stealing the spotlight in current epidemiological trends concerning morbidity and mortality rates. CVDs, which include ischemic heart disease, acute myocardial infarction, and stroke, are the most common non-communicable diseases globally, responsible for an estimated 17.8 million deaths in 2017 ,17. As tIn this manuscript, we aimed to review current risk scores and their utility for the prediction of cardiovascular events among patients with NAFLD. In light of more recent discoveries, we performed a literature search between January 2012 and January 2023 on MEDLINE, EMBASE, Web of Science, Scopus, DDW.org, and ClinicalTrials.gov for English articles. The search terms used included, but were not limited to, “NAFLD”, “non-alcoholic fatty liver disease”, “risk scores”, “cardiovascular risk scores”, “cardiovascular risk factors”, “mortality prediction scores”, “cardiovascular risk assessment”, and “prediction models”. The cited articles were selected based on their relevance to the review objective.Whether liver disease in NAFLD confers any additional CV risk, or whether an increase in CV risk in NAFLD is due to associated CVD risk factors, is still a matter of avid discussion. Understanding whether the liver disease in NAFLD contributes to additional CVD risk is important, as it is plausible that treatment of the liver disease may reduce the overall risk of CVD above the treatment of NAFLD-associated risk factors . Recent Risk factors for NAFLD are quite diverse. Beginning with alterations of hepatic microvasculature, increased arterial stiffness, and distortion of sinusoidal patterns which leads to intrahepatic endothelial dysfunction  and activation of angiogenesis and vascular remodeling via VEGF, we can better understand the complex pathophysiological mechanisms behind an otherwise benign affliction . SystemiFurthermore, mitochondrial malfunction and disruption is one of the main mechanisms of liver and heart damage. This should not come as a surprise since mitochondria are crucial organelles involved in cellular metabolism and homeostasis of energy . Many elThere are multiple intricate underlying mechanisms by which NAFLD increases the risk of CVD . NAFLD iSystemic inflammation, endothelial dysfunction, plaque instability, IR, oxidative stress, and altered lipid metabolism are some of the mechanisms involved in the complex pathogenesis of NAFLD-CVD . The livVisceral adipose fat appears to have a strong independent positive correlation with liver fat. However, the mechanism linking visceral fat to CVD is related to IR, which is also associated with an increased CV risk and atherosclerosis . BesidesApolipoprotein-B-containing lipoproteins operate as damage-associated molecular patterns (DAMPs) that activate Toll-like receptors (TLRs) after being transported and oxidized within the subendothelial vascular wall . TriglycAs previously reported, vascular abnormalities and endothelial dysfunction are recognized as initial steps in disease progression in both CLD and CHD. Endothelial dysfunction is a complex process and is the foundation for atherosclerosis before plaque inflammation ,43. In aIR is another key metabolic feature of both cardiovascular and hepatic injury. Patients who have IR have a suppression of insulin’s effects, which causes the liver to continuously produce glucose . AdiposeThe genetic profile also contributes to disease outcome. Changes in the polymorphism of patatin-like phospholipase domain-containing 3 and transmembrane 6 superfamily member 2 are linked to an increased risk of liver steatosis, fibrosis, and cancer ,54. HoweWhile some cohort studies revealed that NAFLD patients may have increased cardiovascular mortality, other research was unable to support this idea. For instance, a recent meta-analysis by Targher et al. revealed that people with NAFLD had a 64% higher likelihood than those without NAFLD of having fatal or nonfatal cardiovascular events. Non-fatal events included myocardial infarctions requiring coronary revascularization, strokes, and angina pectoris. The findings of Wu et al. differ from those of Tagher et al. in that they did not find a clear connection between the presence of NAFLD and an increase in cardiovascular mortality .A distinct risk group can be identified in patients with NAFLD who also have concurrent T2DM, NASH, or advanced fibrosis (F3–F4) . NAFLD pHs-CRP is a non-specific and sensitive marker for detecting inflammation that was proposed for its potential to add predictive value in several chronic diseases . A folloThe prevalence of arterial hypertension in NAFLD patients ranges from 40 to 70%, and recent research has demonstrated that NAFLD is closely linked to an elevated risk of incident prehypertension and hypertension . People Patients with NAFLD are more likely to develop both clinical and subclinical atherosclerosis. Patients with NAFLD may also be more susceptible to the development of atherosclerotic CVD due to an imbalance in procoagulant factors . In addiPatients with NAFLD/NASH are more likely to have subclinical CVD and several other cardiovascular risk factors . A summaBecause of the inherent limitations of using risk scores based on conventional CV risk-factor-derived multivariable statistical models to identify at-risk patients, the current global risk prediction studies are imperfect even though they may contribute to describing, at least in part, the relationship between NAFLD and increased CV risk ,31. AddiWith varying degrees of severity in each stage of simple steatosis and NASH, NAFLD progresses differently . Except for more severe cases of NASH+ fibrosis, these stages are typically reversible ,16,19. CTo diagnose the NAFLD stage and the degree of NASH and fibrosis by histological examination, as well as to track the course of the illness, liver biopsy is currently the “gold standard” (accounting for possible errors caused by sampling variability) . CurrentNAFLD can develop in individuals who are not overweight. In recent years, emerging evidence has demonstrated that lean NAFLD is a progressive condition and that predominantly male patients with lean NAFLD exhibited some features of MetS and might have worse outcomes than obese-NAFLD . WhetherThe NAFLD Liver Fat Score has its origins in a Finnish population . InsulinThe fatty liver index is based on BMI, the AST/ALT ratio, and the existence of T2DM, and this measure evaluates MAFLD/NAFLD ,85,86,87The APRI (AST-to-Platelet Ratio Index) is another easy-to-use tool to quickly assess the presence of fibrosis. It has been validated for the identification of NAFLD and can identify advanced fibrosis in those with viral infection. APRI is regarded as a good predictor of progressive fibrosis in patients with NAFLD . NeverthAnother widely used score is the NAFLD fibrosis score. Age, glucose levels, BMI, albumin and platelet count, and AST/ALT ratio are the parameters used. This score is widely used to predict advanced fibrosis. Particularly, patients with NAFLD have reduced levels of albumin and albumin-binding function. A high score (>0.68) significantly increased the probability of death in MAFLD/NAFLD patients by four times . Even thIn addition to risk-stratifying methods based on fibrosis for the MAFLD/NAFLD spectrum, many authors have proposed non-invasive scoring systems as screening tools for liver disease. However, some healthcare experts continue to emphasize liver biopsy as the gold standard for diagnosis while arguing for a thorough characterization of biopsy indications. However, fatty liver index, APRI, fibrosis-4 index, and NAFLD fibrosis score, in contrast to other available risk scores, depend on commonly requested parameters, making them easier to utilize in daily clinical practice . AdditioAlthough NAFLD is a common disease, there are no approved therapies. As previously reported, the leading causes of death in patients with NAFLD are complications of cardiometabolic disease. Therefore, efforts are directed toward developing drugs that target both NAFLD and cardiometabolic risk factors. Currently, several therapeutic agents have been proposed and are under assessment . Given tThe causal bidirectional relationship between CVD and NAFLD is more tangible nowadays. Early clinical intervention in at risk patients could alter the disease course. Moreover, patients who share multiple cardiometabolic risk factors who are at risk for developing CV events should benefit from early clinical and therapeutic intervention. Current guidelines do not recognize NAFLD as an independent risk factor for CVD, despite recent research indicating NAFLD’s involvement in incident CVD. Improving patient safety requires a greater understanding of the biopsy-free scores that are available for staging. Both NAFLD and CVD are silent pandemics, which are constantly expanding, and they compel clinicians to look for alternate methods of screening, early detection, and follow-up. Prospective long-term studies with homogeneous diagnostic criteria, considering not only the presence but also the severity of NALFD, are necessary to test if this diagnosis can improve cardiovascular disease risk stratification. New and inclusive risk scores are needed to address complex patients who are at risk of developing either CV events or progression toward more advanced and irreversible liver disease with overall poor outcome. Emerging new therapies could change the face of NAFLD patients."}
{"text": "Non-alcoholic fatty liver disease affects up to a quarter of the adult population in many developed and developing countries. This disease has become the fastest-growing cause of hepatocellular carcinoma (HCC) in industrialized countries. The annual incidence of hepatocarcinoma varies between patients with cirrhosis and noncirrhotic patients. In this review, HCC associated with non-alcoholic fatty liver disease will be described, considering its epidemiology, risk factors, and management, including preventive strategies and therapeutic approaches.Non-alcoholic fatty liver disease (NAFLD) affects up to a quarter of the adult population in many developed and developing countries. This spectrum of liver disease ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) and cirrhosis. The incidence of NASH is projected to increase by up to 56% over the next 10 years. There is growing epidemiological evidence that NAFLD has become the fastest-growing cause of hepatocellular carcinoma (HCC) in industrialized countries. The annual incidence of HCC varies between patients with NASH cirrhosis and patients with noncirrhotic NAFLD. In this review, NAFLD/NASH-associated HCC will be described, including its epidemiology, risk factors promoting hepatocarcinogenesis, and management of HCC in patients with obesity and associated metabolic comorbidities, including preventive strategies and therapeutic approaches to address this growing problem. Non-alcoholic fatty liver disease (NAFLD) is becoming the most common cause of chronic liver disease and cirrhosis. The condition encompasses a spectrum of conditions ranging from non-alcoholic fatty liver (NAFL) or simple steatosis, characterized by hepatic triglyceride accumulation in the hepatocytes without inflammation, to non-alcoholic steatohepatitis (NASH), which is represented by steatosis plus liver inflammation, and finally hepatic fibrosis and cirrhosis and/or hepatocellular carcinoma (HCC) .The incidence and prevalence of NAFLD have had a rapid and significant rise in recent decades worldwide ,3. A recRegarding the incidence of NAFLD, even if the data on it are more scarce, because of the lack of methodologically correct studies on this matter, the abovementioned meta-analysis reported an increase of about 58% from 1994–2006 to 2010–2014, with an estimation of 28.9 cases per 1000 person-years, with a strong correlation with older age (>50 years) and obesity, which is closely related to the increased incidence of insulin resistance, metabolic syndrome, and type 2 diabetes (T2DM) in the general population  and incidence [incidence rate ratio of 0.28 (95% CI: 0.18–0.42)] with respect to those (in matched cohorts) who did not undergo this procedure [Patients with NAFLD and a BMI ≥ 35 kg/m surgery . These r surgery . Moreoverocedure When lifestyle changes are not sufficient, pharmacological therapy should be indicated. Several drugs have been proposed, targeting directly or indirectly the major components of the pathophysiology of inflammation and fibrosis in NAFLD . A comprStatins, HMG-CoA reductase inhibitors, are the major lipid-lowering drugs prescribed; they act by reducing the endogenous production of cholesterol with partial resolution of the liver histology, but also by their anti-inflammatory, anti-proliferative, and anti-angiogenic effects . A recenAmong antidiabetic drugs, thiazolidinediones, and Pioglitazone in particular, showed good results in terms of liver histology of diabetic patients with NAFLD . They acRecent studies are evaluating the effects of GLP-1 agonists on the liver. Liraglutide appears to be able to inhibit de novo lipogenesis in the liver and to improve the sensitivity of cells to insulin ,100. SimOther studies have demonstrated a reduction in necroinflammation and ballooning degeneration, with resolution of NASH in a proportion of cases after the intake of high doses (800 IU/day) of vitamin E, which has an antioxidant action ,103. HowFurthermore, recent studies in mouse models on Nicotinamide N-methyltransferase, an enzyme mainly expressed in the liver , report Finally, very recently, other studies have reported on a new class of drugs which is the “dual agonist drugs”, such as Efinopegdutide and Tirzepatide, in patients with NAFLD, obesity, and/or type 2 diabetes ,115. ThiObeticholic acid, a farnesoid X receptor agonist, is the only drug shown to improve fibrosis without worsening NASH; however, it is necessary to point out that its use has also been related to the side effect of LDL hypercholesterolemia which further increases the cardiovascular risk of these patients . HoweverCenicriviroc, a dual antagonist of the chemokine receptors CCR2 and CCR5, localized on stellate and Kuppfer’s cells, has been tested in NASH subjects. The pathophysiological basis was that the antagonization of these receptors blocks hyperinflammation and fibrogenesis . AccordiIn conclusion, at the time of this review, no pharmacological therapy has been approved precisely for the cure of NAFLD/NASH, and, whereas several molecules are at various levels of experimental development, just as many promising therapies have failed at the interim analyses of the phase 3 studies by not achieving the primary endpoints proposed by FDA and EMA for considering them efficacious in NAFLD/NASH: (i) resolution of NASH by histology without worsening of fibrosis and/or (ii) improvement in fibrosis without worsening of NASH. The causes of these failures are complex and multifaceted and are related to the complexity of the pathophysiology of NAFLD itself, together with the uncertainties on the assessment of its diagnosis and progression/regression. These aspects make difficult to identify the right therapy for the right patients, thus dooming potentially promising drugs to failure .Treatment options for HCC can be classified into surgical resection and non-surgical therapies . As descIn a such scenario, in which any novel therapy for NAFLD has failed the abovementioned endpoints on fibrosis and/or steatohepatitis or it is still in a preliminary phase of study, it is difficult to report on any noticeable effect on HCC occurrence of these drugs. However, it is logical to infer, even if without any evidence so far, that the development of any therapy that could be efficacious in improving steatohepatitis and/or fibrosis progression could lead to a reduction in HCC incidence, also because of several pathophysiological mechanisms they have in common .As a proof of this fact, Metformin, the drug used in the first line in the pharmacological therapy of TD2M, has not demonstrated benefits on liver histology; however, this drug would potentially be able to reduce the incidence of HCC in patients with NASH as, through AMP, it activates the protein kinase that downregulates c-Myc as demonstrated in a mouse model . Even ifWith regard specifically to HCC pharmacological chemotherapy, recent findings suggest that immunotherapy is promising to reduce the recurrence of HCC and provides treatment options for advanced HCC that is not suitable for surgical resection. The U.S. Food and Drug Administration (FDA) in recent years has approved several drugs for systemic HCC treatment, such as Sorafenib, a multi-kinase inhibitor , LenvatiTo date, the main management strategy in the case of cirrhosis and fibrosis is identical to the chronic liver diseases of other etiologies. In particular, it is aimed at controlling the underlying condition and secondary prevention of complications. Patients with cirrhosis will have to undergo periodic control esophagogastroduodenoscopy (EGDS) to evaluate esophageal varices and to eventually proceed with their ligation; in these subjects, the use of non-selective β-blockers to prevent their enlargement is also indicated . In the International guidelines suggest that, in the presence of liver cirrhosis of any etiology, which is considered the major driver of hepatocarcinogenesis, an active surveillance ultrasound is indicated every 4–6 months, aimed at the early identification of potentially neoplastic nodules. Nodules smaller than 1 cm should be followed with a closer ultrasound check (every 3 months) to monitor the growth more accurately. If the nodule is larger than 1 cm, more in-depth examinations such as contrast enhanced ultrasonography (CEUS) or CT/MRI with contrast are indicated. For a definitive diagnosis, liver biopsy is indicated ,137. HowThe BCLC (Barcelona Clinic Liver Cancer) system used for staging liver cancer, takes into account the number and size of the tumor, the stage of the underlying liver disease  and the patient’s condition, dividing them into five stages: stages 0 and A, B, C, and D .In stages 0 and A, it is possible to conduct therapy with curative intent by resection and ablation. Liver transplantation is the best option for patients who meet the Milan criteria: single tumor <5 cm or less than three tumors each <3 cm. In stage B, transarterial chemoembolization (TACE) is indicated; it blocks the hepatic artery to deliver massive doses of chemotherapy with minimal systemic toxicity; it is not applicable in the presence of cirrhosis and portal hypertension due to the vascular consequences induced by these stages. In the advanced stage (BCLC C), systemic therapy with VEGF inhibitors and, recently, with immune checkpoint inhibitors is indicated. However, there are several concerns regarding the literature data that seem to indicate that precisely NAFLD-related HCC is less chemosensitive to these new treatments with respect to those related to viral etiologies . TerminaIn conclusion, the fat accumulation in the hepatocytes (namely liver steatosis) associated with lipid and sugar metabolism derangements, a condition which went under the name of non-alcoholic fatty liver disease (NAFLD) and now has been categorized as metabolic-associated steatotic liver disease (MASLD), represents a continuously increasing challenge for physicians and researchers who are facing (and more and more in future they will) several issues related to its increasing epidemiology and diffusion, diagnostic and management issues, therapeutical problems, and, finally, the problematic HCC surveillance, diagnosis, and management."}