Tribulus terrestris L. (TT) supplementation have been shown to enhance sports performance in many but not all studies. Moreover, data regarding the potential impact of TT supplementation on CrossFit® endurance is limited. This study aimed to determine whether TT supplementation improve body composition, hormonal response, and performance among CrossFit® athletes. In a randomized, single-blind, placebo-controlled trial, a total of 30 healthy CrossFit®-trained males were randomly allocated to receive either 770 mg of TT supplementation or a placebo daily for 6 weeks. Body mass, fat mass, fat composition, testosterone and cortisol levels, and CrossFit® performance  were assessed before and after intervention. There were no significant group x time interactions for the outcomes of the study except for testosterone levels and bench press performance (p < 0.05). TT supplementation did not impact enhance performance or body composition in CrossFit® male athletes. However, TT supplementation may act as a testosterone booster helping the recovery after physical loads and mitigating fatigue. Dietary supplements are substances that can be used to add nutrients to one’s diet and that have a potential nutritional or physiological effect . In 2020Tribulus terrestris L. (TT), originally from India. TT is a famous traditional Chinese medicine that has been widely used in many countries for thousands of years  [Total testosterone was determined by enzyme-linked immunosorbent assay (ELISA) . Cortisol concentration was assessed by enzyme-linked fluorescence assay (ELFA)-based technology by ready-to-use reagents with a compact multiparametric immunoanalyzer MinividasV)/100)] ,33. ® warm-up, strength and/or CrossFit® skill technique as well as a structured strength or conditioning workout between 10 to 30 minutes, followed by stretching and/or cool down. Each workout was performed in the participants’ own gym, supervised by a certified CrossFit® Degree 1- or 2-certified instructor (at each). All subjects completed the same physical activity routines to ensure that they performed the same prescribed workout during the study.During the 6 weeks of the study, training involved 3 weekly sessions on alternate days . Each session was one-hour long and was divided into a CrossFit® athletes were briefed on two diet-tracking systems  for CG and IG. Effect size among participants were calculated using eta-squared (η2p). The η2p is a measure of effector size association. Because this measure often overestimates effect size, values were interpreted as the following: where no effect is indicated, if 0 ≤ η2p < 0.05; a minimal effect is shown if 0.05 ≤ η2p < 0.26; a moderate effect is indicated if 0.26 ≤ η2p < 0.64; and a strong effect is indicated if η2p ≥ 0.64. The treatment was randomly assigned utilizing a sequence generated by date (or day) of admission in the study by Random Sequence Generator. Random Sequence Generator is an application, which was accessed through the Internet. The data were presented as means and standard deviations. Analyses was performed using SPSS version 4.0 software . Statistical significance was indicated when p > 0.05) for energy, proteins, fats, carbohydrates, micronutrients, and vitamins (There was no significant difference between groups (CG and IG) (vitamins .p < 0.05). However, the group-by-time interaction between CG and IG did not reveal significant differences (p > 0.05).In ® Total and Grace) for both GC and IG at the beginning and end of the study . Within the CrossFit® Total tests , only a significant increase (p < 0.05) was observed in the bench press in GI (T1: 69.17 ± 11.58 vs. T2: 80.00 ± 11.40). In addition, significant differences (p < 0.05) were only observed in group-time for the bench press . In both groups, a shorter execution time was observed, without statistical significance (p > 0.05), in the Grace test in both GI (T1: 28.00 ± 6.23 22.67 ± 5.92 vs. T2: 22.67 ± 5.92 28.00 ± 6.23) and CG (T1:30.17 ± 5.08 vs. T2: 24.33 ± 4.46). No significant group-time differences were observed for the Grace test .p > 0.05) were observed between baseline and at the end of the six-week study in testosterone, cortisol, and testosterone/cortisol ratio. Nor were there significant differences in group-by-time for cortisol  and ratio testosterone/cortisol ratio . Significant differences (p < 0.05) can be seen in the group-by-time for testosterone .In addition, p > 0.05) in the perceived fatigue between T1 vs. T2 in both groups. Additionally, there were no significant differences in the group-by-time for RPE .Finally, p < 0.05) in the bench press test between the IG vs. CG. There were no significant differences (p > 0.05) in dead lift (IG 4.64 ± 2.55 vs. CG 3.32 ± 4.17) and CrossFit® Total (IG 6.19 ± 3.19 vs. CG 5.07 ± 4.61), but there was an improving trend in the IG group. However, in the CG the percentage change was greater for squat than IG (CG 5.70 ± 8.303 vs. IG 3.01 ± 4.96). The results for the CrossFit® Total test were similar for both groups (CG −24.73 ± 10.58 vs. IG −25.00 ± 10.74). p < 0.05) were observed in the testosterone levels (IG −0.20 ± 13.63 vs. CG −21.85 ± 11.10). There were no significant differences (p > 0.05) in cortisol (IG 4.64 ± 2.55 vs. CG 28.65 ± 43.19) and testosterone/cortisol ratio (IG 22.43 ± 69.60 vs. CG −29.07 ± 37.38), but there was an improving trend in the IG group. In addition, −1) on body composition, hormonal response, RPE, and CrossFit® performance in CrossFit®-trained males. We hypothesized that TT supplementation would improve CrossFit® performance and testosterone levels since previous studies in athletes demonstrated that TT enhanced sports performance [® Total and Grace test), body composition parameters , cortisol, and testosterone/cortisol ratio.The purpose of our investigation was to evaluate the impact of six-weeks TT supplementation  and with no significant difference in the group-time interaction, indicating that the CrossFitathletes ,27,36,37athletes . Howeverathletes . ® training program, and/or the absence of TT could have led to a lower level of adaptation in CG. Therefore, the information from bodybuilding and fitness websites on the activity of TT compound supplements in decreasing body fat and increasing lean muscle mass is questionable [These results would suggest that the increase in bench press performance in IG was not mediated by changes in body composition or increased muscle mass. This could be because of the adequate adaptability of the IG athletes to the CrossFittionable ,11. −1 of TT [−1 for 8 weeks) [® Total exercise, squat and dead lift showed no significant gains. The squat showed a slightly better percentage improvement in IG than in CG. However, in dead lift, the opposite was true. Squat and dead lift employ muscle groups, mainly of the posterior chain, involved in many other CrossFit® movements . This could indicate that habitual training is responsible for the individual adaptations of each athlete [We did not observe significant improvements in WODs, which evaluates global strength by the sum of the measurement of 1 repetition maximum (RM) of bench press, squat, and dead lift. These results are similar to those reported in a study conducted in rugby players, where no significant improvement in total strength was observed after supplementing for five weeks with 450 mg · day−1 of TT . In addi8 weeks) , researc athlete , which i athlete ,18. Alternatively, the bench press showed significant improvements in the IG between T1 and T2, twice as much as in the CG. Our results are similar to those obtained in 1-RM bench press after three weeks of TT supplementation in powerlifters ,21. The TT-mediated inhibition of cyclooxygenase-2 (COX-2)  could alTT is recommended as an herbal supplement that optimizes and increases mood ,41, prob−1 · day−1) in young adults [−1) for three months in adults with idiopathic infertility [Continuous, intense exercise directly influences an inadequate functioning of the hypothalamus-pituitary-testes axis . This alg adults . Similarertility . To our ertility . In thisertility ,26. Addiertility . Other mertility , and theertility .−1 increased, but this change was not statistically significant [® trained vs. athletes belonging to a physical education program). These results could indicate that the possible efficacy of TT on hormonal status would be conditioned in relation to the sport modality and/or the physical condition of the athletes.Cortisol is released from the adrenal cortex in response to stressors linked to exercise and catabolic actions; it is also directly influenced by duration and intensity . The cornificant . Althougp > 0.05) in the testosterone/cortisol ratio concerning CG at the end of the study, which could translate into a potential better response to training load and volume and establish a positive trend in the recovery process in athletes. In this way, improvements in muscle recovery after high-intensity endurance exercises by reduction of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) have been reported after supplementation with 500 mg · day of TT [Testosterone/cortisol ratio is indicative of the anabolism/catabolism ratio in the body due to their role in protein synthesis and degradation. It can be used as a biological indicator to supervise fatigue and establish optimal training loads for athletes . Testostay of TT . Nutritional supplements contaminated with banned substances have been reported . TaintedThe present study has certain limitations. The sample size is small; a larger one would produce more robust results. Moreover, the realization in professional athletes of different sports modalities would also give us the accurate dimension of the potential properties of TT supplementation because the nature of each sport has demands that differ between disciplines, such as aerobic or anaerobic capacity, the type of strength used, the predominant energy systems, and other factors that would justify the individualization of TT testing. Additionally, the design exclusively focuses on males, which may reduce generalizability. Nor were biochemical tests included, especially determination of muscle damage marker levels, such as lactate dehydrogenase, creatine kinase, and myoglobin. Thus, it would be necessary to include such as analysis in future studies.−1 of TT used in CrossFit®-trained men does not appear to influence body composition, RPE, total strength, and anaerobic performance. However, our results suggest that it could potentially intervene in the recovery process by a tendency, although not significant, to increase serum testosterone and maintain a favorable trend in the testosterone/cortisol ratio, which would mean that it could potentially influence less fatigue and catabolism associated with exercise. The dose 770 mg · dayTT supplementation could be recommended for athletes playing sports at levels where the competition rules prevent adequate recovery, such as play-offs in team sports  or tournaments in individual sports  and in training stages characterized by high loads and volumes. In addition, since the beginning of the COVID-19 pandemic, more males have been affected as well as a higher rate of intensive care unit admissions and deaths. Perhaps a lower bioavailability of testosterone or a decrease in its receptors could be the cause. In this regard, low total testosterone levels are associated with a hyperinflammatory state in hospitalized men with COVID-19 and represent a risk factor for hospital mortality in COVID-19 patients. Therefore, inadequate total testosterone levels may serve as markers of poor prognosis in COVID-19 . Thus, g


According to the Standards of the World Marrow Donor Association (WMDA) 2020 [1] unrelated stem cell donor registries are responsible for compliance of their donor centres with these Standards. To ensure high stem cell product quality and high standards for safety and satisfaction of voluntary unrelated stem cell donors, we present here guidelines for audits of donor centres (DC) that can be used by new and established donor registries. They have been developed for registries relying on independent national or international DCs for the recruitment and management of Unrelated Donors (UD) for verification typing (VT)/extended tying (ET), work up processes and Hemopoietic Progenitor Cell (HPC) donation. The main goal of these guidelines is to support registries in verifying and auditing their affiliated DCs to ensure they are compliant with the WMDA Standards, as well as WMDA recommendations. We define the general requirements and recommendations for collaboration with the DC and guidelines to manage the UD, step by step from recruitment to follow-up. We also provide a checklist, intended to serve as a resource for auditors performing an audit at a DC. This document was prepared by the World Marrow Donor Association (WMDA) Quality and Regulatory Working Group (QRWG) and approved by the WMDA board. It contains the guidelines for audits of (DCs) by unrelated Donor Registries (DR).https://wmda.info/professionals/quality-and-accreditation/wmda-standards/] “If a registry relies on an independent donor centre or cord blood bank to recruit and characterise donors/umbilical cord blood units, the registry must ensure that the donor centre/cord blood bank complies with relevant WMDA Standards. The nature of these affiliations and the duties and responsibilities must be documented in an agreement.”WMDA ed. 2020 Standard 1.06 states: .By setting KPIs for participating institutions (registries and DCs), the WMDA aims to improve the quality of processes. There should be procedures in place for establishing and evaluating the institution’s KPIs in relation to the WMDA recommendations, as well as for analysing associated procedures if shortfalls are identified, to determine a suitable action plan. The institution’s KPIs can also be compared to the data of other institutions of similar size (see WMDA report). Results and evaluations should be documented. Monitoring of KPIs should be on an annual basis since results are collected and published by the WMDA annually. Further information can be found on the WMDA website , , , 3, as pThe focus of the audit is, therefore, to assess that the DC conveys required information at the time of recruitment to ensure informed and committed consent.This can be done by review of selected consent forms completed by donors recruited in the last 4 years.A policy should be available which outlines information to be collected from a donor at the time of registration and this must include donor age and gender as per WMDA requirements. Tools such as a medical health questionnaire or checklist may also be used to capture this information. DC staff should have access to guidance material which defines and clarifies all medical criteria that would exclude an individual from joining the registry based upon safety considerations for the donor, as well as considering safety of the patient. The WMDA provides minimum standards for which potential donors should be assessed .Additionally, there should be a policy in place describing the circumstances for when a donor may receive the results of their health screening and the method of communicating these results to the donor must be appropriate to the situation based on privacy, regulatory and/or legal requirements where applicable.Each country has its own laws and regulations relating to privacy, confidentiality, and the protection of personal data. Policies and procedures regarding information management should be in place which indicate how data are captured/entered by DC staff and how the data are verified, saved, and transferred to the registry database.It should also be confirmed that access to electronic data is limited to authorised individuals. Since the search for an unrelated donor is an anonymous process, policies and procedures must be in place to ensure donor confidentiality, including the assignment of an anonymous donor identifier.Recruiters are often the first point of contact for potential new registrants and have an important role in educating potential registrants as well as ensuring availability and commitment of registered donors. Due to the important work involved with stem cell recruitment, a rigorous training programme should be in place for recruiters and recruitment supervisors. The WMDA has developed recommendations  for minihttps://share.wmda.info/display/DMSR/WMDA+Donor+Medical+Suitability+Recommendations+Main+page], U.S. Food and Drug Administration regulations, AABB (formerly known as American Association of Blood Banks) and FACT-JACIE International Standards.Auditing of the VT process should include all aspects of the process: from maintenance of appropriate policies on suitable donor counselling, maintaining confidentiality, adherence to the respective health screening and testing requirements, correct sample collection, labelling and shipment procedures, as well as timely and accurate processing of requests and transmission of results. Donor screening and testing, as well as questionnaires designed to detect and evaluate conditions that increase risk of donation, can be found in WMDA guidelines and the donor medical suitability website . The geographical location or other local factors may require additional testing according to national requirements as well. WMDA Donor Medical Suitability [https://share.wmda.info/display/DMSR/WMDA+Donor+Medical+Suitability+Recommendations+Main+page] provides additional information on testing at VT.The purpose of IDM testing is to assess the donor’s exposure to infectious diseases and the likelihood of transmitting a disease to the patient. It indicates if an individual currently has, or previously had, an infectious disease that could be transferred to another person. Routine health screening and personal history and physical examinations are not sufficiently comprehensive evaluations for hematopoietic cell donations. There are numerous infectious agents that, if present in the donor, pose definite risk to the transplant patient and are only detected via IDM testing. Testing must be performed to meet the minimum requirements of the WMDA . The DC/CC must have a procedure in place to inform the TC immediately regarding medical ineligibility of the UD. Additionally, this information must be forwarded to the registry in writing using WMDA forms or equivalent.A policy must be in place which clearly states what to do in case of medical ineligibility of the UD for the requested collection procedure. The registry standards and national regulations must be followed; additionally, WMDA donor suitability as well as registry-specific medical eligibility criteria can be consulted for general recommendations [When a potential risk for the patient is identified, there may be a need to declare the UD as ineligible for the patient according to national requirements and laws. The TC is to be informed via the responsible registry according to the applicable data protection laws and requirements using the WMDA form .Additionally, some country-specific requirements must be fulfilled e.g. declaration of medical need, declaration of ineligibility. These documents must be included in the donor records and provided to the responsible registry. UD eligibility can only be confirmed once the TC has evaluated the risk and deemed acceptable to proceed. Additional information about the health of the UD that might be relevant for the TC but does not have a direct impact on the UD’s eligibility, can be included as comments on the WMDA form or equivalent.It’s important to ensure information is provided to the donor about expenses and disability and death benefits for volunteer donors. The DC should have a policy in place to inform the donor regarding extra expenses and insurance coverageThe registry must have a process in place to provide donors a method to report post donation related medical concerns. Post donation, it’s recommended the registry follows the donor a minimum of 10 years to monitor for potential medical issues.Donors are healthy persons who voluntarily donate for a patient and are accepting some small risks in order to help others. Therefore, although the risks for both parties are clarified before donation/transplantation and are generally minor, it is still important to monitor the health and well-being of the donor not only during the collection procedure, but also afterwards.The intent of WMDA Standards is to ensure that measures are in place to assist in detection and reporting of the occurrence of a SAE and a SAR, and that should a SAE or SAR occur, that the donor can be monitored and treated appropriately, and that all parties involved are suitably informed so that the TC can also care for the recipient appropriately, if necessary.Additionally, the WMDA reporting system (S(P)EAR) can assimilate long-term data on possible risks associated with the donation process, as well as assist in the (rapid) distribution of SAE-/SAR-related information to registries and their partners to assist in the prevention of the recurrence of such events.The focus of the audit is, therefore, to ensure procedures exist for the recognition, documentation, and appropriate action plans in relation to SAEs and SARs, examples of documentation and investigation of SAR/SAE, and initiation of necessary remedial and/or corrective actionBoth SAEs affecting a cellular product intended for a specific patient as well as SARs affecting donors undergoing collection of HPC and/or cellular product must be identified, documented, investigated and remedial and/or corrective action taken.Procedures should also include the system of documentation of cases and corrective action and may also describe who processes and reviews cases. DC must have a procedure in place, which describes how and when cases are reported to the registry or directly to the WMDA.The checklist in the appendix is intended to serve as a resource for auditors performing an audit at a DC. It is intended to enable the auditor to perform an audit using the checklist only. The checklist mentions the relevant WMDA Standards and refers to the chapter that should be covered by the audit. The auditor is also provided with clear information on which files should be inspected and which processes should be verified. Depending of the type of the UD’s affiliation ; the checklist may be used on its own, combined with the addendum checklist with specific points for cross border DCs.The DR is ultimately responsible for ensuring its affiliated DCs, whether local or international are compliant with the WMDA Standards; this is to ensure the safety of unrelated adult donors and to make their donation journey the best experience possible.It is strongly recommended that DRs and their affiliated DCs have well established policies and procedures, using the recommendations of the WMDA and outlining the roles and responsibilities of each party, to support unrelated adult donors from the recruitment process, donor selection and collection to donor follow up as well as incident reporting. DRs need to ensure DCs are well equipped to carry out these activities.When collaborating with cross border DCs, DRs should be aware of laws and regulations governing the country of each DC.Supplementary Data 1Supplementary Data 2


The current audit aims to identify the gaps in the practice of administering prn buccal midazolam, for management of epilepsy in people with intellectual disability and to review training needs, with a view to improve patient care.Convulsive status epilepticus is a medical emergency requiring admission to hospital and has a mortality as high as 20% (SUDEP -Sudden Unexpected Death in Epilepsy). It is imperative that the carers are fully aware of the risks associated with the epileptic attacks, are able to recognize the attacks and offer rescue medication to the patient in a timely and effective manner. National guidelines have been drafted jointly by ESNA  and ILAE  for prn administration of buccal midazolam. The use of rescue medication by trained carers can significantly improve the outcome and reducing the risk of hospital administration and chances of SUDEP. Buccal midazolam is widely used to manage prolonged seizures. Administration should be undertaken only by people who have received both epilepsy awareness and buccal midazolam training.All patients with intellectual disability with epilepsy were studied and patients who were prescribed prn midazolam have been shortlisted shortlisted for data analysis. A template was designed and data are being collected from the carers, community nurses and the prescribing clinicians. Data are categorized under headings of background information about epilepsy, recognizing complications during a seizure and the ability to administer buccal midazolam. Further information about the type and the frequency of training courses (including the refresher courses) being offered to the carers for administration of buccal midazolam would also be documented. There is also data collection pertaining to the timely review of the care plan by the prescribing clinicians and the about the licenced or unlicensed used of midazolam. The quality of epilepsy awareness training is being explored in depth and also the competency of the county council nurses offering the training to the carers shall be analysed under different headings.Data are currently being collected in Hertfordshire and Essex mental health services.After the data analysis, the short comings shall be addressed and effective measures shall be put in place to improve the quality of training being provided to the carers and to standardise the practice for prn midazolam prescription.


Increased dietary fiber consumption has been shown to increase human gut microbial diversity, but the mechanisms driving this effect remain unclear. One possible explanation is that microbes are able to divide metabolic labor in consumption of complex carbohydrates, which are composed of diverse glycosidic linkages that require specific cognate enzymes for degradation. However, as naturally derived fibers vary in both sugar composition and linkage structure, it is challenging to separate out the impact of each of these variables. We hypothesized that fine differences in carbohydrate linkage structure would govern microbial community structure and function independently of variation in glycosyl residue composition. To test this hypothesis, we fermented commercially available soluble resistant glucans, which are uniformly composed of glucose linked in different structural arrangements, in vitro with fecal inocula from each of three individuals. We measured metabolic outputs  and community structure via 16S rRNA amplicon sequencing. We determined that community metabolic outputs from identical glucans were highly individual, emerging from divergent initial microbiome structures. However, specific operational taxonomic units (OTUs) responded similarly in growth responses across individuals’ microbiota, though in context-dependent ways; these data suggested that certain taxa were more efficient in competing for some structures than others. Together, these data support the hypothesis that variation in linkage structure, independent of sugar composition, governs compositional and functional responses of microbiota.  Although diet is increasingly understood to play a major role in modulating the gut microbiome ,2, the mThe gut microbiome has recently been shown to be very sensitive to even subtle structural differences in both insoluble  and soluHere, we aimed to test the related hypotheses that (1) complex carbohydrates identical in composition  but varying in structure  would select for distinct microbiota; and (2) that the same microbes would be selected by identical structures across different individuals’ microbiota. To test this hypothesis, we used commercially available glucans, generated either by modification of starches  or polymerization of glucose (polydextroses) by heating or enzymatic catalysis to be at least partially resistant to degradation by human enzymes . Resistant glucans are made by multiple companies with varying sugar and starch sources and processes, and are commonly added to food products in order to increase dietary fiber content to improve health outcomes ,18,19,20The resistant glucans and one fructan used in this study were gifts from several companies . These glucans ranged in moisture content from 2–8% (dry weight), which was measured by a Mettler Toledo Moisture Analyzer HE53 (115V) . Masses added for all analytical procedures and microbial cultures were adjusted for moisture content to ensure equivalent carbohydrate loading.3I and hydrolyzed with 2M TFA at 121 °C. Myo-inositol (20 μL of 2.5 mg per mL 2M TFA) was added as an internal standard. Prior to the reduction of the samples with acetic acid, they were treated with 2M NH4OH and 1M freshly prepared NaBD4 in 2M NH4OH. Acetylation was performed with the addition of acetic anhydrate, followed by dichloromethane and multiple ddH2O washes, and the residue was dissolved in acetone. The GC-MS conditions were as follows: injector volume, 1 μL; injector temperature, 240 °C; detector temperature, 300 °C; carrier gas, helium: 1.9 m/second; split ratio, 100:1 and the temperature program, 100 °C for 2 min, 8 °C/min to 240 °C for 20 min.The glycosidic linkage composition of the glucans was determined via the partially methylated alditol acetate approach and measured using gas chromatography  with a fused capillary column  coupled with a mass spectrometer  as previously described by Pettolino et al.  and Xu e2 < BMI < 30 kg/m2) who had not experienced significant dietary change in the past two weeks and who resided within 30 miles of Purdue University. Donors that were pregnant or lactating, had a history of gastrointestinal or chronic metabolic disease, had had major surgery of the gastrointestinal tract, or had taken prebiotic or probiotic supplements in the prior three months or antibiotics in the prior six months were excluded. All selected donors were male with an omnivorous diet and reported distinct dietary patterns. Donor 1 was a 25-year-old with a BMI of 31.7 and reported frequent consumption of high-fat and spicy foods. Donor 2 was a 22-year-old with a BMI of 24.9 and reported a diet with elevated consumption of fruits and vegetables. Donor 3 was a 22-year-old male with a BMI of 25.4 and reported a diet with high fermented dairy consumption. Involvement of human subjects in this study was reviewed and approved by Purdue University under the IRB Protocol #: 1701018645.Feces from three healthy donors were collected for inocula. Donors were selected from healthy adults 18–65 years of age, of normal or overweight BMI  and filtrated through four layers of cheese cloth to make a slurry. Glucans were dissolved in sterile water, passed through a 0.22 μm Millipore syringe filter and mixed 1:1 with anoxic 2X phosphate buffer to a concentration of 1.25% w/v. This solution was then mixed with the fecal slurry to a 4:1 ratio into Balch tubes for each time point in triplicate, with a total carbohydrate concentration of 1% w/v. Tubes were closed with butyl rubber stoppers and aluminum seals and incubated outside the chamber in an incubator at 37 °C shaking at 150 rpm. Culture tubes were taken out of the incubator at each time point  and pH and gas measurements were taken as previously described [For each donor’s microbiota, collected samples were immediately placed into ice post-collection and rapidly transferred to an anaerobic chamber , where they were processed as previously described . The basg rpm. 400 μL of supernatants were mixed with 100 μL of an internal standard . The external standards used were acetic, propionic, butyric, isobutyric, and isovaleric acids mixed in equimolar proportions.Short-chain fatty acids were analyzed by gas chromatography with a flame ionization detector  with a fused silica capillary column  as previously described  and undeDNA was extracted using the FastDNA SPIN extraction kit . The aliquots taken at 48 h were thawed, centrifuged, and the pellet was re-suspended. The process was followed as the FastPrep 24 protocol indicates with the following modifications: bead beating time (twice for 40 s at 6.0 m/s) and triplicating shaking time of the sample and binding matrix (shake 15 min instead of 5 min). The extracted genomic DNA was stored at −20 °C until further sequencing analysis.DNA was quantified and normalized to 100 ng/μL. The V4–V5 region of the 16S rRNA gene was amplified by PCR using the universal bacterial primers: 515-FB forward (GTGYCAGCMGCCGCGGTAA) and 926-R reverse (CCGYCAATTYMTTTRAGTTT)  and KAPAThe following thermal cycler conditions: initial denaturation, 95 °C for 5 min, 20 cycles of denaturation , annealing (60 °C for 15 s), and extension (72 °C for 30 s), and a final extension  followed by an infinite hold at 4 °C. PCR products were purified and prepared as previously described by Tuncil et al. ; brieflyhttps://mothur.org/wiki/miseq_sop/ (accessed on 24 August 2021)) with the following modifications. Sequences were screened for a maximum length of 411 nt, zero maximum ambiguous bases and a maximum homopolymer length of 9 nt, then aligned to the mothur-formatted SILVA reference alignment across positions 13,862 to 27,654. Sequences were classified based on the Ribosomal Database Project reference training set with an 80% cutoff. Chloroplast, Mitochondria, Archaea, Eukaryota and unknown classifications were removed from further processing. OTU classifications at the species levels are reported as percentage of reads based on a 97% similarity. Ecological metrics were calculated within mothur: α-diversity metrics were calculated using the simpsoneven, chao, invsimpson and sobs calculators and β-diversity metrics were calculated using the jaccard and thetayc calculators as implemented in mothur. Distance matrices for β-diversity metrics based on the pcoa command in mothur were plotted for visualization using R. Abundance changes were log2-transformed for the most-abundant 33 OTUs.Sequences were processed with mothur (v.1.39.3)  followinp < 0.0001 with a Kruskal-Wallis test for multiple comparisons. Linear discriminant effect size (LEfSe) analysis was conducted using LEfSe v. 3.12 [All analyses were performed in triplicate. SCFA data are presented as means and standard deviation. Tukey HSD at α = 0.05 was used to compare mean differences. Analysis of molecular variance (AMOVA) tests command in mothur were also computed between glucan classes to determine whether centroids were significantly distinct. Alpha-diversity was determined by One-way ANOVA  v. 3.12 . OTUs coGlucans provided for this study came from different commercial sources and were categorized into three different groups based upon their source and structural characteristics. Significant differences in glucan structure were revealed by carbohydrate linkage analysis . These gWe tested multiple glucans with different configurations to determine how similar or different metabolic outputs would be in fermentation by fecal microbiota from each of three different healthy donors. Throughout in vitro batch fermentation, we measured pH, gas production, and concentrations of the most common short-chain fatty acids  at intervals over a 48 h time course.As expected, we observed significant gas and acid production from all glucans, although the magnitude varied across glucan classes and individual donor microbiota . AlthougDespite similarity in overall gas and acid production for most glucans, patterns of SCFA production from different structures varied substantially across donors’ microbiota . Althougp < 0.05). Together, these data suggested that, although distinct glucan structures were differently metabolized by different microbiota, which produced different metabolic outputs at equivalent total carbohydrate loadings, the eventual fate of glucan metabolism emerged largely from initial microbial community structure.Despite these idiosyncrasies, we observed some general tendencies in fermentation of different glucan classes. Across donors, the resistant maltodextrin category produced the most butyrate, whereas the mixed linkage α-glucans produced the least butyrate or propionate but the most acetate (p < 0.0001) as determined using the inverse Simpson index, and donor 2 and donor 3 microbiota were not significantly different (p = 0.09). However, donor 2’s initial community was significantly higher in richness compared to the other donors (p < 0.0001). Donor 1’s initial community was significantly less even when compared with the other two donors (p < 0.0001), however donor 2 and donor 3 did not significantly differ in evenness (p < 0.05)  and evenness (Simpson’s evenness) components. Most notably, donor 1 microbiota displayed significantly lower overall diversity compared with the other two donors (evenness . By comp < 0.05) . No signFirmicutes , they displayed very different initial microbiota structures. Each had at least one dominant OTU from phyla other than Firmicutes; OTU00001  and OTU00006  dominated the donor 1 community, OTU00003 and OTU00004 (both Bacteroides spp.) constituted ~26% of the reads from the donor 2 community, and OTU00002  and OTU00006  were dominant in the donor 3 community. These initial community structures governed relative abundances in fermentations  being much more influential than glucan structure .Bifidobacterium spp.) across donors, although the population sizes were linked with α-glucan complexity (increasing in abundance with decreasing complexity) only with donor 2 microbiota. Additionally, members of order Bacteroidales  and family Lachnospiraceae  were very abundant and diverse in fermentations of polydextroses and/or resistant maltodextrins; however, the most abundant OTUs within these group varied by donor, largely based upon initial population sizes. Members of family Ruminococcaceae generally decreased in abundance from the inoculum irrespective of substrate. In general, however, community compositional responses to glucans appeared context-dependent and largely idiosyncratic to donors.Although changes in overall community structure were observed, these changes were relatively minor over the 48 h fermentation. Relative abundances of OTUs in post-fermentation communities strongly resembled the initial community structures, structuring significantly more strongly by donor than by glucan . HoweverBacteriodaceae were linear discriminants of resistant maltodextrins, whereas members of families Porphyromonadaceae, Erysipelotrichaceae, Veillonellaceae, and Lachnospiraceae were discriminants of polydextroses (LDA ≥ 3.5). However, within families Bacteroidaceae and Lachnospiraceae, many genera and species exhibited preferences for the other classes of glucans; for example, Bacteroides ovatus and Bacteroides uniformis were discriminants of polydextroses, whereas Fusicatenibacter saccharivorans and Roseburia faecis were discriminants of resistant maltodextrins (Bacteroides (including B. ovatus and B. uniformis) were most abundant in fermentations of resistant maltodextrin G, B. faecichinchillae was overrepresented in fermentations of resistant maltodextrin M , responses were glucan-specific and similar across all donors  than H, but similar preferences for the different resistant maltodextrins). However, for other species, responses to distinct glucans were strongly influenced by donor of origin. For example, although OTUs within genus Bacteroides responded with generally above-average growth on polydextroses and resistant maltodextrins across donors, they responded to mixed linkage α-glucans with above average growth rates uniformly in the context of donor 3’s microbiota and solely to glucan A in donor 1 and donor 2 communities. Further, different OTUs within Bacteroides displayed very different growth patterns on the resistant maltodextrins and polydextroses; for example, in donor 1 and 3 microbiota, OTU00003 responded more strongly to polydextroses than resistant maltodextrins, but vice versa in donor 2 microbiota. OTU00008 showed a similar pattern to OTU00003 for donors 1 and 2, but in donor 3 it was similarly responsive to all polydextroses and resistant maltodextrins. Similar OTU-level differences in glucan response based upon community context were observed within genus Blautia for all donors. Additionally, analogous dynamics occurred where a single dominant OTU was identified in a genus; OTU00005 (Fusicatenibacter sp.) responded strongly to polydextrose H but not to polydextrose E or resistant maltodextrins in donor 1 communities, but grew at an above-average rate across all glucans in context of donors 2 and 3. OTU00010 (Anaerostipes sp.) responded strongly to polydextrose H and resistant maltodextrins J-M in donor 1 communities, only to polydextroses  in donor 2 communities, and did not record above-average growth on any glucan in donor 3 microbiota. OTU00027, a Clostridium XIII sp., showed above-average growth on mixed linkage α-glucans in a donor 2 community and below-average growth in the community of donor 1; however, this organism grew at above-average rates on polydextroses and resistant maltodextrins . Taken together, these data suggest that organismal preferences for distinct glucan structures exist, but that actual species’ responses depend upon (1) strain-level differences among donors of origin that cannot be resolved in OTUs or (2) interaction with other community members (both positive and negative).To determine whether OTU-level specificities for distinct glucans were present but obscured by idiosyncratic donor responses, we compared OTUs by their change in abundance over fermentation in all three donor contexts. In this analysis, organisms growing faster than average display increases in relative abundance, while those growing slower than average will be seen to decrease in abundance. This revealed both similarity and context-dependence in OTU responses to different glucan structures and, further, differences in growth patterns in different OTUs classified within the same genus . For somBacteroides  were generally strongly correlated with one another across glucan classes, with the exception of moderate inverse correlation in growth between OTU00003 and both OTU00017 and OTU00004 on polydextrose H and resistant maltodextrins J and M , the level of coherence in growth response on glucans among the various OTUs depended upon the community of origin (Blautia) on essentially all polydextrose and resistant maltodextrin substrates. This OTU also showed above-average increases in relative abundance in donor 2 communities. However, OTU00007 showed above-average growth on resistant maltodextrins K, L, and M and OTU00062 responded strongly to poly-dextroses E and H and resistant maltodextrins D, G, and J . On the other glucans, the growth responses of these two OTUs were average or below. In donor 3 communities, only OTU00007 and OTU00026 show slightly faster growth than their other cousins in Blautia, though this effect was much smaller across glucans than in donor 1 and 2 communities. Taken together, these data suggest that interactions among OTUs and complex glucans depended upon (1) the fine structure of the glucan; and (2) the initial abundances of other community members.Cross-donor abundance fold change correlations revealed glucan-specific patterns. In many cases OTUs  behaved similarly within (or even across) glucan classes . For exa J and M . Howeverf origin . In donoIn nature, variation in polysaccharide structure is overwhelmingly associated with differences in the ratios of glycosyl residues that compose the polymer (starch and glycogen being notable exceptions) . For exaOur results demonstrate that microbiome responses to structurally distinct resistant glucans depend upon both fine glucan structure and community context, and community metabolic phenotypes emerge from the interaction of the two. With respect to metabolic outcomes, predominant SCFA outputs were largely determined by resistant glucan class , although some donor-dependent differences were observed among individual glucans within a class. Importantly, whether propiogenesis or butyrogenesis dominated in fermentation of a resistant glucan class was idiosyncratic to donors and inversely correlated, suggesting that (1) microbiomes have potential to produce either SCFA, but that (2) microbiomes are poised to produce one of the two from any given glucan class. It is important to account for differences in digestion of these glucans in the upper gastrointestinal tract, which may differentially alter the glucan structures finally encountered by gut microbiota . HoweverBacteroides, when considered at the level of glucan class, were overrepresented in cultures on resistant maltodextrins, with some species instead preferring polydextroses. When individual glucans were considered, however, this shifted to alternate preferences for resistant maltodextrins G and M, indicating that preferences of these OTUs for substrates within the resistant maltodextrin class was not equivalently high. Similarly, family Lachnospiraceae was a linear discriminant of polydextroses with respect to glucan classes, but of resistant maltodextrin D when all glucans were considered individually. Members of the genera Bacteroides and Parabacteroides as well as Anaerostipes (within Lachnospiraceae) have been observed to increase in abundance in feeding trials using some types of resistant starches [With respect to community composition, however, individual glucans within a class markedly diverged from one another in donor-dependent ways. Although we observed some consistency in responses across glucan classes at higher taxonomic levels, these relationships often shifted when individual glucans were considered independently. For example, members of genus starches ,36,37,38starches ,40,41,42starches , which sBacteroides OTUs varied as a function of both glucan and initial microbiota composition; although members of Bacteroides were responsive across individuals, the specific OTU-glucan associations varied. Interestingly, however, in many cases the same OTUs responded strongly to glucans across multiple donors  irrespective of initial abundances, suggesting that fundamental associations exist for some OTU-glucan pairs. Together, these data suggest that species may have preferences (possibly based on hydrolytic enzyme or transporter gene content) for particular glucan structures; however, it further suggests that an organism’s fitness on these substrates depends on competitive  interactions with other species in the community. It should be noted that organisms can compete (and cooperate) in ways that are independent of carbohydrate consumption , andIf our observations of strain specificity and context dependence in competition among fecal microbiota for variant resistant glucan structures in vitro are recapitulated in gut microbiomes in vivo, it suggests two related corollaries: (1) that outcomes of resistant glucan feeding trials are likely to initially be highly individual, based upon initial microbiome structure; but (2) over time, extended resistant glucan feeding should cause gut microbiomes to converge as the most efficient organisms increase in abundance across individuals. Further, it would suggest that this level of individual variability is likely to obscure outcomes of specific interactions between gut microbiota and variant fiber structures. Furthermore, as previous studies have asserted, it suggests that responses to well-defined fiber structures may be predictable given known microbiome community structures , and pot


Results: In the survey, 1026 participants took part, including 370  health professionals and 656  participants from a non-medical field. A total of 330 (89.18%) from group 1 and 528 (80.49%) respondents from group 2 expressed willingness to donate their corneas. The main reason for refusal of donation in both groups was a lack of knowledge concerning eye donation (7.8%). A social campaign (64.6%) was the most frequently chosen solution for increasing the number of potential donors by health professionals and medical students. In the group of doctors, not knowing how to report a potential donor was chosen as the greatest source of difficulty in donor reporting (40%). Conclusions: In the present study, the willingness to donate one’s eyes was substantial in both groups. Social campaigns and improving knowledge concerning the donor reporting process among health professionals might be beneficial in expanding the donor pool.Background: Limited access to corneal tissue for transplantation remains a challenge in many parts of the world. To date, little attention has been paid to the problem of the cornea donor shortage in Poland, where the number of waiting patients exceeds the number of transplants performed three-fold. The aim of this study was to assess the knowledge and willingness towards participating in corneal donation among different social groups in Poland. Methods: This prospective, cross-sectional study was conducted among health professionals, medical students, clerics, teachers, journalists, employees and patients of the District Railway Hospital in Katowice. Online and paper questionnaires were used to collect socio-demographic data and information regarding awareness of, knowledge about and attitudes toward corneal donation. For health professionals and medical students, the questionnaires contained additional questions concerning knowledge and solutions for expanding the donor pool. Descriptive analysis and associations were evaluated using the chi In contrast, Poland, also being a developed country, is distinguished by an exceptionally low keratoplasty rate of 32 × 10−6. Limited access to graft tissue remains a challenge in many parts of the world, with 32 countries not performing corneal transplants and 31 with a corneal transplant rate below 5 × 10−6, mainly in Asia and Africa [Corneal transplantation is a highly effective procedure in treating multiple corneal disorders, with great potential to improve visual acuity. However, its performance is limited by the availability of corneal tissue from deceased donors. The most important problem to be solved in transplantology is the shortage of donors, and thus the shortage of organs and tissues in relation to the number of potential recipients. The drastic disproportion between the supply and demand of donor corneas worldwide, in which there are 70 patients per one acquired cornea, has been reported in a recent global survey of eye banking and corneal transplantation . In affld Africa . Differed Africa ,5. NeverThis descriptive cross-sectional survey followed the principles of the Declaration of Helsinki. In order to assess the awareness and attitude of various social groups toward eye donation, two questionnaires were drawn up—one for medics and a second for other groups . Therefore, two major groups were included—medical and non-medical. In the healthcare professionals group, doctors, nurses and paramedics from three local hospitals were enrolled. Only doctors from the anesthesia, neurology, internal medicine and surgical departments were enrolled in the study, as they represent potential professionals reporting donors. To extend the group, other health professionals were also asked to fill out the questionnaire via e-mail. The student group consisted of those having lectures in the Ophthalmology Department, District Railway Hospital in Katowice. The non-medical group included patients admitted to the hospital for COVID-19 vaccination, teachers from local schools, journalists and priests asked to fill out the questionnaire personally or through e-mail. The questionnaire for healthcare professionals consisted of questions regarding demographic data, including sex, age and profession, one about the source of knowledge about corneal transplants, nine questions concerning the process of corneal procurement, all single-choice and closed, four questions connected with willingness to donate corneal tissue, and one regarding solutions for improving the situation, which was a multiple-choice question with pre-set answers. The second questionnaire contained, apart from demographic data, such as sex, age, educational level and profession, three questions concerning knowledge about eye donation and five connected with willingness to donate one’s corneas. In both questionnaires, questions regarding reasons for donation refusal included the option other, with the opportunity to write their own answer.Respondents under 18 years old and who did not consent to participate were excluded from the study. Informed consent was obtained from all participants. The study was conducted between January 2020 and May 2021. Throughout this period, questionnaires were successively collected. Healthcare professionals were the first group studied, and questionnaires from this group were collected until August 2020. In the second half of 2020, teachers, journalists and priests were enrolled in the study. With the beginning of COVID-19 vaccinations in our hospital, patients scheduled for vaccination were asked to fill out the paper questionnaire. Participants taking part in the online survey required approximately two or three recalls to fill the questionnaire, while paper forms were mostly filled out after one recall. 2 test was used to compare qualitative variables between groups, while quantitative variables were compared using Student’s t-test in the case of two groups or the analysis of variance (ANOVA) with Tukey’s post hoc test for more than two groups. p values < 0.05 were considered significant. The statistics were prepared using MS Excel and R language in the Rstudio environment (RStudio Team (2020). RStudio: IntegratedDevelopment for R. RStudio, PBC, Boston, MA URL http://www.rstudio.com/, accessed on 1 September 2021).Data from paper questionnaires as well as online surveys were collected and compiled and analyzed in Excel spreadsheets  separately for the medical and non-medical groups. Data are presented as the percentage of people indicating a given response for qualitative variables and the mean and standard deviation for quantitative variables. The chi2 test with the numbers above 300 was 0.99. The test power calculated using the G power software for the chip < 0.000).This study consisted of 1026 participants—370 health professionals and 656 participants from a non-medical field. A total of 446 healthcare professionals were contacted, which resulted in an 83% response rate. Approximately 90 doctors from local hospitals were excluded, due to them belonging to departments other than anesthesiology, surgery, and internal medicine, including cardiology and neurology. All respondents from the other groups were enrolled in the study. In the non-medical group, 1986 participants were contacted, providing a 51.66% response rate. In the first group, the most populous age group was in range of 18–30 years (59.73%), in which 22.16% of respondents were males and 77.84% were females. In this group, 34.32% of participants were nurses, 30.27% were doctors, 28.11% were students and 7.3% were paramedics . The larMedia was the main source of knowledge (34.87%), followed by university (24.32%) and the workplace (17.84%). Only 5.14% of medics had ever reported a potential donor, and only 26.22% were aware of the low reporting rate of donors in Poland .p = 0.137). Paramedics and nurses declared their willingness to donate cornea less frequently than other groups (p = 0.026). The level of knowledge showed a positive correlation with willingness to be a donor (p < 0.000), and the other professions did not differ significantly.Not knowing how to report a potential donor was the most frequent obstacle chosen by respondents (13.51%), with a lack of time occupying second place (4.05%). Most of the health professionals (78.65%) had knowledge regarding the current form of consent in Poland; however, as many as 21.35% were not aware. In this group, willingness to donate was very strong—90.54% of participants would consent to being a donor after death, and 91.08% would not have an objection to a corneal retrieval from their close relatives. The main reason for donation refusal, including from a family member, was the lack of knowledge regarding eye donation (3.24% for themselves and 2.70% for relatives). The greatest number of people opposed to consenting to a transplant was found in the 30–50 and 50–70 age groups. There seems to be a tendency towards a reluctance to donate one’s cornea that increases with age ( a donor . Doctorsp = 0.104).As for the most effective solutions for improving the situation in Poland regarding the shortage of potential donors, participants chose a social campaign (68.92%) and a computer application facilitating donor reporting (46.22%) . In the A substantial number of participants in the second group had never heard about corneal donation, and the questionnaire was the first source regarding this issue (42.44%) .The vast majority of respondents did not have knowledge about the form of consent in force in Poland (70.23%) .Additionally, in this group, 58.32% of participants were unaware about the cornea donor age limit, and 50.84% did not know that cornea donation is only possible after death. Regardless of the poor awareness, willingness to donate corneal tissue in the second group was also strong (80.61%); however, only 59.39% of respondents would have signed up for the central donor register, if there was one. At the same time, slightly fewer respondents were not against donation from their relatives (73.59%). Similar to the first group, a lack of knowledge was the main reason behind refusing donation (10.23% and 9.31% for relatives) .p = 0.000), as well as consenting to obtain corneas from relatives (p = 0.008) and registering as a cornea donor (p = 0.005)  . p = 0.000 and p = 0.000, respectively). The greatest reluctance to become a potential donor (p = 0.000), to consent to a cornea donation from a family member (p = 0.000) and to show willingness to sign up to a donor registry (p = 0.000) was observed in the group of people with basic education. The majority of incorrect answers regarding the form of consent was observed among people who gave their friends (p = 0.000) or the survey (p = 0.000) as a source of knowledge. People pointing to the media responded slightly better, as well as people who knew someone after a transplant, people who obtained this knowledge during their studies and who drew from other sources of knowledge. In the question about donor age limit, most of the correct answers were indicated by people who knew someone after a corneal transplant (p = 0.000). Slightly fewer correct answers were given by people declaring their university education and the media as sources of knowledge. The worst results were those indicating other sources of knowledge. Belonging to a certain professional group had no association with willingness to donate one’s corneas.The highest number of correct answers regarding the form of consent in Poland and the possibility of retrieving corneas from a living donor was observed among respondents with higher education ; therefore, it seems that religious issues are not a barrier to donation in Poland [Since Poland is an 88% Christian country and donation issues used to arouse ethical and religious controversy , in our n Poland . Addition Poland ,12. Accon Poland . The conThere is also a general dispute regarding whether the type of consent has an impact on the effectiveness of organ and tissue procurement. In a global survey conducted by Gain P et al., it was described that an opt-out system promotes donation . The conLimited knowledge and limited awareness were revealed to be contributing factors for donation refusal in our study, and are cited as the only modifiable factors that may change the attitude toward this issue ,15 The mOther reasons for organ and tissue scarcity have been discussed, such as non-recognition of a potential donor, which depends on a system between different hospitals and the eye banks ,18. It iThe time after death to cornea procurement in Poland is limited to 24 h; however, for example, in Germany, this period is extended to 72 h. There are also differences regarding cornea procurement from oncology patients even between hospitals in Poland, where some hospitals collect tissue from such patients and others do not. This also narrows the donor pool, while the only definitive contraindications according to EEBA include retinoblastoma, hematological neoplasm, and malignant tumors of the anterior segment of the eye. Finally, only 5.14% of surveyed health professionals have ever reported a cornea donor, and as the main problem in this process, respondents indicated a lack of knowledge regarding this procedure. From the solutions proposed in the responses to the questionnaire for increasing the donor pool, respondents most frequently chose social campaigns and a computer application facilitating donor reporting, while a salary increase for those reporting donors was preferred by almost one-third of respondents.This study has several limitations. First of all, despite the large number of overall participants, the studied group was heterogeneous in terms of profession and educational level, which may influence the generalization of these results and presents a sample of willingness to donate corneas in Poland. Secondly, the two studied groups differed in terms of demographic data, however the aim of the study was not to compare both groups but to assess the attitude towards cornea donation separately for medical and non-medical respondents, assuming that healthcare professionals have more knowledge on this topic. Furthermore, most of the participants were from the Silesia region, and may not exactly reflect the situation in different parts of our country. Finally, the response rate in the second group was relatively low, and therefore the opinion of those who were not willing to take part in the study is not known, and it is possible that they have a more negative attitude toward cornea donation than the participants.To conclude, the results of the study indicate that the willingness to donate corneas in Poland is significant; however, raising awareness in the Polish population through social campaigns could have a positive effect on increasing the donation consent rate. Work should be undertaken to improve and facilitate donor reporting, including an incentive system and innovative tools such as computer applications to make this process more effective.


Immune markers are altered in Parkinson's disease (PD), but relationships between cerebrospinal fluid (CSF) and plasma cytokines and associations with neurodegeneration‐associated proteins remain unclear.CSF and plasma samples and demographic/clinical measures were obtained from 35 PD patients. CSF samples were analyzed for cytokines (together with plasma) and for α‐synuclein, amyloid β(1‐42) peptide, total tau, and phospho(Thr231)‐tau.There were no CSF–plasma cytokine correlations. Interleukin (IL)‐8 was higher and interferon‐γ, IL‐10, and tumor necrosis factor–α were lower in CSF versus plasma. In CSF, total tau correlated positively with IL‐8 and IL‐1β, whereas α‐synuclein correlated positively with amyloid β(1‐42) and negatively with semantic fluency (a known marker of PD dementia risk).Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.CSF and peripheral cytokine profiles in PD are not closely related. Associations between CSF IL‐8 and IL‐1β and tau suggest that CSF inflammatory changes may relate to tau pathology within PD. CSF α‐synuclein/amyloid β may reflect the risk of developing PD dementia. © 2020 The Authors.  Parkinson's disease (PD) is associated with central and peripheral immune changes.The key neurodegeneration‐associated protein involved in PD is α‐synuclein, with multiple factors leading to abnormal aggregation and pathology. CSF total α‐synuclein concentration is generally decreased in PD when compared with controls,Increased levels of inflammatory cytokines  have been detected in PD CSF when compared with controls,The relationships between CSF cytokine changes and neurodegeneration‐associated protein levels are largely unknown. Currently, limited studies have investigated this in PD,This study aimed to examine the relationships between central and peripheral cytokine levels as well as CSF cytokine and neurodegeneration‐associated protein associations in a well‐characterized, moderate‐stage PD cohort, to provide further insight into the drivers of central immune activation in PD.Ethical approval was obtained from the Cambridgeshire‐2 Research Ethics Committee (08/H0308/331). Patients were recruited from the PD Research Clinic at the John van Geest Centre for Brain Repair in Cambridge. Following screening for contraindications to lumbar puncture, written informed consent was obtained. Clinical data gathered included demographic, medical/drug history, Movement Disorder Society–Unified Parkinson's Disease Rating Scale (MDS‐UPDRS), Addenbrooke's Cognitive Examination–Revised (ACE‐R), semantic fluency (predictive of dementia in PD),Lumbar punctures were performed in the left lateral position at the L3/4 or 4/5 space using aseptic technique, 1% lignocaine as local anaesthetic and a 22G spinal needle; ~2 to 5 mL of CSF was collected. A subset of patients (n = 22) had concurrent ethylenediamine tetraacetic acid (EDTA) venous blood sampling.g for 15 minutes. Supernatant was stored in ~500 uL aliquots at −80°C. Plasma was extracted from blood by centrifugation at 2000 rpm (~670g) for 15 minutes and stored at −80°C.CSF samples were centrifuged at 3000https://www.mesoscale.com/): V‐PLEX proinflammatory panel‐1 cytokines  in CSF and plasma, and α‐synuclein, phospho(Thr231)‐tau, total tau, and amyloid β(1‐42) in CSF. Plates were read using the Meso Scale Diagnostics SECTOR Imager. Data were processed using Meso Scale Diagnostics Discovery Workbench software.Samples were analyzed using the Meso Scale Diagnostics  electrochemiluminescence platform. Assays were performed in duplicate, at 1:2 dilution, and according to the manufacturer's instructions  Statistical Package for the Social Sciences (SPSS) version 25 and GraphPad  Prism 7. Cytokine and protein variables within the CSF and plasma with assay‐detected values in >75% of participants were included in the analysis. Logt tests, with Bonferroni correction for multiple testing as appropriate. Bivariate correlations were assessed between CSF cytokines and neurodegeneration‐associated proteins and between CSF markers and clinical measures. Variables with uncorrected significant correlations (P < 0.05) were included in linear regression analyses with adjustments for relevant confounders.Plasma and CSF cytokine profiles were compared using a repeated‐measures analysis of variance and paired on treatment) 31.0 (12.1), ACE‐R score 90.3 (9.4), Beck Depression Inventory score 9.1 (7.7), and semantic fluency score 23.9 (8.4).Demographic and clinical measures in the PD cohort (n = 35) were expressed as mean (standard deviation) or percentage: age 65.4 (7.6) years, sex 48.6% male, years of education 18.7 (3.9), disease duration 5.4 (5.6) years, Movement Disorder Society–Unified Parkinson's Disease Rating Scale part III . Paired comparisons between CSF and plasma values indicated that IL‐8 was significantly higher, whereas IFN‐γ, IL‐10, and TNF‐α were significantly lower in CSF versus plasma following Bonferroni correction for multiple testing  indicated no correlations between CSF and plasma levels in this cohort. Repeated‐measures analysis of variance indicated a significant overall difference between CSF and plasma profiles , total tau and IL‐1β , total tau and IL‐8 , and phospho‐tau and IL‐2  were the most abundant measured neurodegeneration‐associated proteins in CSF, whereas total tau and phospho‐tau were present at low concentrations , tau and IL‐1β , and α‐synuclein and amyloid β(1‐42) , with age included as covariate  as well as nominally significant associations (P < 0.05) between semantic fluency and IL‐6, ACE‐R and IL‐6, and age and both IFN‐γ and IL‐1β , but not the associations between ACE‐R and CSF variables  and CSF total tau in PD. In addition, we have confirmed a positive correlation between CSF α‐synuclein and amyloid β(1‐42) and found a negative correlation between CSF α‐synuclein and semantic fluency—a key clinical predictor of dementia in PD.Previous studies investigating relationships between CSF and serum/plasma cytokines in PD have reported elevated TNF‐α in CSF compared to serum and correlations between CSF and serum levels of IL‐6, IL‐1β, and IL‐10 in PD and controls.Within PD CSF, the proinflammatory cytokines IL‐1β and IL‐8 correlated positively with total tau, whereas the anti‐inflammatory IL‐2 correlated negatively with phospho‐tau. Potential causal links between tau species and immune changes are unclear, but inflammation has been shown to influence tau production/pathology.The positive correlation between CSF α‐synuclein and amyloid β is consistent with previous studies.The limitations of this study include the small sample size, lack of plasma samples in all subjects, and the absence of matched healthy control samples. Furthermore, not all cytokines could be adequately measured in the CSF and plasma using this assay, and higher sensitivity assays may be needed for improved resolution of the low‐level cytokines. However, the assessment of multiple neurodegeneration‐associated proteins and cytokines in simultaneously obtained CSF and plasma samples from a clinically well‐characterized cohort has uniquely allowed interrelationships between these factors to be explored further in PD. Longitudinal assessments of these relationships in larger PD cohorts and matched healthy controls would now be of interest.Data are available upon reasonable request.(1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the first draft, B. Review and Critique.R.S.W.: 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3BS.F.M.: 1A, 1B, 1C, 2C, 3BK.F.: 1B, 1C, 2C, 3BD.P.B.: 1B, 1C, 2C, 3BR.A.B.: 1A, 1B, 2C, 3BC.H.W.G.: 1A, 1B, 1C, 2A, 2C, 3BTable S1‐ Summary of significant correlations between neurodegeneration‐associated proteins and cytokines. * = Remained significant post Bonferroni correction.Table S2 – Significant correlations between clinical variables and CSF markers on bivariate analysis. * = Remained significant post Bonferroni correction.Table S3 ‐ Linear Regression with ACE‐R score and semantic fluency as the dependent variables. *p < 0.05.Click here for additional data file.


To analyze the incidence of epididymal anomalies (EAs) associated to spermatic obstruction in patients with undescended testis (UT) according to testicular position and age.We studied 87 patients (110 testis) with cryptorchidism and analyzed the presence of EAs correlated with the testicular position, age and patency of the processus vaginalis (PV). To analyze the relations between the testis and epididymis we considered three situations: (a) Normal pattern: the epididymis was attached to the testis at the head and tail and epididymis totally attached to the testis; (b) EAs: when the epididymis was attached to the testis only at the head  and (c) The mean age of the patients was 5.18 years (SD=2.867). Of 110 testes analyzed, 14 were abdominal (12.72%); 83 inguinal (75.45%) and 13 suprascrotal (11.81%). Normal relationships between testis and epididymis were observed in 54 patients (62.1%) with no significant differences in relation to the patient's age (p=0.666). Epididymal tail disjunction was observed in 23 patients (26.44%), with no significant differences in relation to age (p=0.59). EAs associated to spermatic obstruction were observed in 16 patients (18.4%), also with no significant differences in relation to age (p=0.684). We did not observe significant correlation between the testis position and the incidence of EAs (p=0.119). We did not observe significant correlations between patency of the PV (64.7%) and incidence of EAs (p=0.742).Epididymal anomalies associated with spermatic obstruction are present in almost 20% of undescended testes, without significant correlation with age, testicular position and patency of the PV. This information needs to be correlated to the infertility risk of this congenital anomaly. Undescended testis can be associated with several anatomical anomalies. Epididymal anomalies (EAs) and patency of the processus vaginalis (PV) are among the most frequent, with a highly variable incidence reported in the literature: from 36 to 79% -4. UndesSeveral studies have examined the relationship of EAs and patency of the PV -11. On tWe hypothesized that disjunction of epididymal head and total disjunction of epididymis are usual in undescended testes and more frequent in testes situated in high position and with patency of the PV. The aim of this article is to evaluate the incidence of epididymal anomalies associated with spermatic obstruction in patients with undescended testis (UT) according to testicular position, patient age and patency of the PV.This retrospective single-center study was approved and was carried out in accordance with the ethical standards of the hospital's institutional committee on human experimentation - IRB number 30706720.8.0000.5259.From January 2011 to January 2020, we studied 87 patients with cryptorchidism (110 testes). All patients underwent orchidopexy and the testes were divided into three groups according to their position: A) abdominal: testes located above the inner inguinal ring; B) inguinal: testes located between the inner and outer inguinal rings; and C) suprascrotal: testes located below the outer inguinal ring. During surgery, three parameters were analyzed: A) testicular position; B) relationship between testis and epididymis; and C) persistence of processus vaginalis (PV).To analyze the relations between the testis and epididymis, we used a previous classification , 13: TypAll parameters were statistically processed and graphically described. The Wilcoxon-Mann-Whitney test was used for comparison of quantitative data (p <0.05). The Chi-square test was used to analyze the population contingency under study (p<0.05), calculated by Graph Pad.All the 87 patients were aged between 1 and 10 years (mean=5.18/SD=2.867). Of these patients, 29 (33.33%) had cryptorchidism on the right side, 35 (40.22%) on the left side and 23 (26.43%) were bilateral. Of 110 testes, 14 were abdominal (12.72%), 83 inguinal (75.45%) and 13 suprascrotal (11.81%). The correlation between EAs and patient age can be seen in We did not observe epididymal atresia  in our sample. The normal relationships between testis and epididymis (Type I and II) were observed in 54 patients (62.1%), with no significant differences in relation to patient age (p=0.666). We observed normal relationships between testis and epididymis in 12 of the suprascrotal testes (92.3%); 50 of inguinal testes (60.2%) and 7 abdominal testes (50%).The disjunction of epididymal tail (Type III) was observed in 23 patients (25.3%), with no significant differences in relation to patient age (p=0.59). Disjunction of epididymal tail was observed in 5 (35.7%) of abdominal testes and 18 (21.7%) of inguinal testes. This anomaly was not observed in suprascrotal testes.Disjunctions of epididymal head and total disjunction of epididymis  were observed in 16 patients (18.4%), also with no significant differences in relation to patient age (p=0.684). The anomalies associated with spermatic obstruction were observed in 15 (18.1%) of the inguinal testes, 2 (14.3%) of the abdominal testes and only 1 (7.7%) of the suprascrotal testes.We did not observe significant correlation between the testis position and the incidence of epididymal anomalies in our sample (p=0.119). We observed 13 patients (11.31%) with bilateral cryptorchidism. In these 13 patients, 8 of them (61.53%) had epididymal anomalies and in 3 of them (23.07%) the epididymal anomalies were bilateral. In bilateral cases of undescended testis the anomalies associated with spermatic obstruction (Types IV and V) were observed in 6 cases (46.15%) and in 2 of them (15.38%) the patients had total disjunction between the testis and epididymis (Type 5) in both sides .Open PV was found in 74 cases (64.7%) of cryptorchidism. We did not observe significant correlations between patency of the PV and incidence of EAs (p=0.742). The normal relationships (Type I and Type II) occurred in 24 (66.7%) of the cases of PV obliterated and in 45 (60.8%) of the cases of PV patency. Epididymal tail disjunction occurred in 6 cases (16.7%) in obliterated PV and 17 cases (23%) in patency of the PV, and the anomalies associated with infertility (Types IV and V) were observed in 6 cases (16.7%) of obliterated PV and 12 cases (16.2%) of patency of the PV.Epididymal anomalies can occur due to abnormal involution of the mesonephric duct adjacent to the testis . The epiKnowledge of EA associated with cryptorchidism is relevant in clinical practice to prevent accidents during orchidopexy and to counsel patients and predict infertility in the future, such as in cases of epididymis atresia, disjunction of epididymal head and total disjunction between the testis and epididymis , 17.Turek et al.  found hiEAs can be classified as disjunction anomalies, epididymal atresia and elongated epididymis , 13, 18.The epididymis is anatomically connected to the gubernaculum, which in turn is attached to the testis and scrotum. An interesting and controversial theory suggests that the epididymis is one of the organs responsible for testicular migration, through its peristaltic and secretory activity in the second gestational trimester . This thMollaeian et al.  studied Some studies have found an association of patency of the PV with EAs and cryptorchidism, suggesting there is a common stimulus, probably androgenic, participating in the epididymal development, testicular migration and closure of the PV , 23. TheThe main limitations of our study are: a) small sample size; b) uneven age distribution; c) impossibility of histological analysis to observe patency of epididymal ducts ; and d) we do not have long-term follow-up of patients with types 4 and 5 epididymal abnormalities to ascertain if they have developed infertility. A perfect study would be to, prospectively, follow patients diagnosed with undescended testis until they reach adulthood.We found epididymal anomalies associated with spermatic obstruction in almost 20% of undescended testes and their incidence had no correlation with age, testicular position and patency of the PV. This information needs to be correlated to the infertility risk in this congenital anomaly.


Immune effector cell associated neurotoxicity syndrome (ICANS) remains a devastating, frequent complication of chimeric antigen receptor (CAR) T cell therapy for advanced-stage hematologic malignancies. Symptoms range from encephalopathy and headaches to aphasia, strokes, and diffuse cerebral edema. Persistent mild cognitive symptoms have also been reported. Unfortunately, the underlying pathophysiology driving ICANS is poorly understood. Current proposed models center on systemic inflammatory changes leading to endothelial dysfunction, blood-brain barrier (BBB) breakdown, and systemic cytokine and/or monocytes infiltration into the central nervous system (CNS). However, these models do not integrate predisposing risk factors for the development of ICANS. We previously demonstrated that pre-infusion plasma neurofilament light chain (NfL), a marker of neurodegeneration, may predict development of ICANS. Early elevations in NfL suggest development of ICANS is also related to pre-existing neuroaxonal injury. The longitudinal relationship between latent neuroaxonal injury, blood brain barrier (BBB) integrity, neuroinflammation, and cognition remains unknown.This prospective, observational trial examines the relationship between multi-modal , neuroimaging) biomarkers and cognition in a cohort of twenty patients undergoing standard-of-care CAR T cellular therapy. Biomarkers for neural injury include blood and CSF NfL and volumetric measures derived from structural magnetic resonance imaging (MRI). Biomarkers for neuroinflammation include blood and CSF glial fibrillary acidic protein (GFAP) and qualification of white matter hyper-intensity burden on MRI. BBB integrity will be quantified using the serum/CSF albumin ratio. Finally, neuropsychological performance testing will assay cognitive performance across multiple cortical domains including attention, memory, and executive function. Participants will undergo a baseline (pre-infusion) examination, followed by evaluation  on post-infusion day 3 (D3), D30, D90, and D180. The primary outcome is percent change in a given biomarker level.This ongoing trial has 2 of 20 planned participants enrolled.


Moreover, several lung donor biopsies before implantation  were evaluated, focusing on smoking-related lesions. Correlations were analyzed between all pathological data and smoking exposure, along with other clinical parameters. Results: Among the three groups, donor and recipient characteristics were comparable, except for higher Oto scores and age in group III. Group III showed a longer intensive care unit (ICU) and hospital stay compared with the other two groups. This finding was confirmed when SD history was considered as a continuous variable. However, survival and other mid- and long-term major outcomes were not affected by smoking history. Finally, morphological lesions did not differ between the three groups. Conclusions: In our study, SDs were associated with a longer post-operative course, without affecting graft aspects or mid- and long-term outcomes. A definition of pack-years cut-off for organ refusal should be balanced with the other extended criteria donor factors.Background: The use of smoking donors (SD) is one strategy to increase the organ pool for lung transplantation (LT), but the benefit-to-risk ratio has not been demonstrated. This study aimed to evaluate the impact of SD history on recipient outcomes and graft alterations. Methods: LTs in 293 patients were retrospectively reviewed and divided into non-SD ( Lung transplantation (LT) is a well-established procedure for several end-stage lung diseases. However, the extensiveness of this procedure is limited, mainly because of a globally diffuse organ shortage. Strategies proven to expand the lung donor pool include optimal intensive-care donor management, donation after cardiac death, and the use of extended criteria donors ,2,3,4. TAmong the extended donor criteria, a positive donor smoking history, generally considered as exposure of more than 20 pack-years, remains controversial, with some evidence of detrimental effects on early recipient outcomes but without a significant impact on long-term survival ,14,15,16However, to the best of our knowledge, no studies have investigated the effect of donor smoking history using precise pack-years exposure but have rather compared patients in wide clusters with distant pack-year cut-offs. The main goal of this study was to evaluate the impact of donor smoking exposure on lung transplantation outcomes. We also attempted to find a relationship between donor smoking history, microscopic graft damage, and post-transplant outcomes.In this single-center retrospective study, we reviewed a cohort of patients who underwent LT in our institution between May 1995 and June 2015. During this period, we performed 355 LTs . The main clinical information about the donor, the recipient, and the surgical procedure was collected. Donor data comprised the main clinical and functional characteristics, including Oto score . Donor sDue to a lack of precise smoking history information, we excluded 62 patients from the study, thus leaving 293 patients for further analysis. The institutional review board gave approval for this study (4539/AO/18).Preoperative recipient data included age, sex, weight, height, body mass index (BMI), native disease, and, eventually, emergency status . Surgical procedure parameters included ischemic time , operative room (OR) time, extracorporeal support, and type of transplantation .Post-operative data included the main early outcomes of ventilation time; intensive care unit (ICU) and hospital stay; and primary graft dysfunction (PGD) graded at 0, 24, 48, and 72 h . Thirty-Donor lung pathological examinations were performed in 97 cases, equally distributed between the three groups, and were available in cases of downsizing lobar resection during transplantation or single LT after bilateral harvesting. Biopsy tissue samples performed in apparently normal lung parenchyma were formalin-fixed, paraffin-embedded, and stained with hematoxylin-eosin. Different smoking-related lesions  were evaluated by a dedicated thoracic pathologist using a scoring system from 0 to 3 . The 3-year acute cellular rejection (ACR) index was quantified as the number of transbronchial biopsies (TBBs) with ACR ≥ 1/total number of TBBs × 100, using TBBs performed in the first three years post-transplantation. Similarly, the ACR ≥ A2 index  was also considered. The presence of airway inflammation was determined based on evidence of lymphocytic bronchiolitis (LB) in all biopsy specimens.Descriptive statistics were reported as quartile I/median/quartile III for continuous variables and as percentages (absolute numbers) for categorical variables. Wilcoxon-Kruskal-Wallis and Pearson’s Chi-square tests were performed to compare the distribution of continuous and categorical variables, respectively.p-value. An ordinal regression approach was employed for nominal outcomes. Results were reported as OR, 95% CI, and p-value. A Gamma model was employed for continuous outcomes, given the non-normal distribution of all continuous outcomes considered. Results were reported as marginal effect, 95% CI, and p-value. The marginal effect was computed from the partial derivatives of the marginal expectation.A regression approach, both univariable and multivariable, was employed to assess the effect of recipients’ smoking history on clinical outcomes of interest. A logistic regression approach was adopted for binary outcomes. Results were reported as odds ratio (OR), 95% confidence interval (CI), and Analyses were performed using R software   with rmsAmong the 293 patients considered in the study, 225 recipients had no positive donor smoking history (group I), 45 had a donor smoking history between 1 and 20 pack-years (group II), and 23 had a donor smoking history of 20 pack-years or more (group III).p < 0.001). In addition, as expected, group III showed a higher Oto score compared with groups I and II .p = 0.04) and ICU stay  was observed with increasing smoking history. At the same time, group III also presented a longer duration of post-transplant hospitalization and reduced respiratory function compared with the other two groups, although these differences were not statistically significant. While the ACR index was similar among groups (p = 0.165), the probability of grade A2 or worse decreased significantly as donor smoking history increased .Finally, chronic rejection prevalence was not different between groups, nor were PGD rate and short-term mortality .p = 0.02) but not on ventilation time. Multivariate analysis  confirmep = 0.01).At the same time, this analysis found a correlation between group III and longer hospital stay  was observed.Finally, an association between recipient female sex and PGD rate at 72 h and in-hospital mortality , especially when considering only ≥A2 grade cases . Concerning respiratory performance, a worse 12-month FEV1 % was observed in female recipients  and in patients with restrictive diseases and specifically idiopathic pulmonary fibrosis patients .p = 0.44).Regarding long-term outcomes, overall 5-year survival  was simip = 0.04) as well as ICU and hospital stay  as donor smoking history increased. No effect was observed on the other outcomes evaluated, particularly on 12-month FEV1.Univariate analysis  showed ap = 0.04) and hospital stay  but not on ventilation time.Multivariate analysis  confirmep = 0.03) and anthracosis  was observed.Considering donor smoking history as a continuous variable for multivariate analysis , no evidThe use of extended criteria organs is one of the most employed and extensively investigated strategies to overcome the lung donor shortage and expand lung transplantation activity. Despite some minor evidence of worse outcomes , liberalWe do not have a precise explanation for the increase in ICU stay of patients transplanted with donor organs with a history of smoking, considering similar ventilation time and PGD among the three groups. Nevertheless, this may be related to the older age of both the recipients and the donors in group III, which could have led to careful and protective anesthesiologic management in these patients.Long-term results showed no differences among the three groups in survival rate, early and late complications , and pulmonary function. Multivariate analysis did not reveal a correlation between the donor’s smoking history and lung function; however, compared with groups II and I, recipients of group III showed a 21.5% and 15% lower FEV1 at 6 months post-LT, a 10% and 14% lower FEV1 at 12 months, and a 14% and 27% lower FEV1 at 24 months, respectively.This fact may be related to the presence of significantly older donors in the group of patients showing lower respiratory performance, a factor known to impact recipient respiratory function tests . A commoBearing in mind these considerations, we could define donor smoking history as a non-modifiable risk factor that needs to be considered at the time of lung offer. As a single criterion, donor smoking history does not appear sufficient for organ refusal, and it should be weighed with other extended criteria when present as well as other recipient risk factors .Obviously, to carefully evaluate a lung donor as suitable, the evaluation of a CT scan is mandatory before retrieval to exclude the presence of lung nodules, parenchymal thickening, anatomical abnormalities, atelectasis, and emphysema.In our center, the donor’s evaluation is always performed by a senior surgeon and, especially in the case of smoking history, the visual evaluation of the CT scan is important to quantify emphysema. Unfortunately, in our study, a CT scan was not always available before retrieval and, for this reason, the quantification of emphysema was done directly at the retrieval time through the visual inspection, palpation, and recruitment maneuvers of the lungs.In our opinion, there is no direct correlation between pack-years and the extension of emphysema. We retrieved lungs from 23 donors with a smoking history ≥ 20 pack-years, considering them suitable; this can be explained by the fact that emphysema is a multifactorial event not only caused by smoking.Thus, a correct donor-recipient match seems vital, as demonstrated by Pierre et al. , who desAnother peculiarity of our study is the investigation of the correlation between donor smoking history and allograft microscopic alteration at the time of transplantation. Different from other work  that anaOutside the field of lung transplantation, different studies have documented the growing prevalence of microscopic alterations with the increase of smoking history, but they do not completely fit our field of investigation since they are based on populations that are very different from the lung donor population; the former individuals are older, have a much higher smoking exposure, and often have associated pulmonary diseases, even neoplastic ones ,30,31,32Our study was limited by several factors, mostly related to the asymmetric distribution of donor smoking history, with a small population sample in the heavy-smoker donor group. This fact, in addition to post-transplant mortality, very likely affected long-term outcomes as few heavy-smoker donors could be evaluated.In this light, a multi-center study would be desirable. In addition, the study period covering approximately 20 years of activity certainly involved limitations for the evolution of both pharmacological and anesthetic treatments.In conclusion, in our experience, the use of donors with a positive smoking history is associated with a longer post-operative course proportional to pack-years, without any influence on other major early- and long-term results or on pulmonary tissue preservation. Thus, in our opinion, the decision to accept this type of graft should consider not only the number of pack-years but also other donor and/or recipient risk factors, along with the donor shortage situation in the country and waitlist overcrowding.