Elaeis guineensis) fraction to protect osteoblast MC3T3-E1 and osteocyte MLO-Y4 cells against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage and the mechanisms involved in its protective action in MC3T3-E1.Natural antioxidants are considered as promising compounds in the prevention/treatment of osteoporosis. We studied the ability of purified δ-tocotrienol (δ-TT) isolated from a commercial palm oil  contents.MC3T3-E1 and MLO-Y4 cells were treated with δ-TT (1.25–20 µg/ml for 2 h) followed by t-BHP on cell viability and apoptosis reaching a maximum protective activity at 10 and 5 µg/ml in MC3T3-E1 and MLO-Y4 cells, respectively. This protective effect was due to a reduction of intracellular ROS levels and an increase in the defense systems shown by the increase in the GSH/GSSG. GSH loss induced by an inhibitor of GSH synthesis significantly reduced the δ-TT-positive effect on ROS levels. δ-TT prevention of oxidative damage was completely removed by combined treatment with the specific inhibitors of PI3K/AKT (LY294002) and Nrf2 (ML385).δ-TT significantly prevented the effects of The δ-TT protective effect against oxidative damage in MC3T3-E1 cells is due to a reduction of intracellular ROS levels and an increase of the GSH/GSSG ratio, and involves an interaction between the PI3K/Akt–Nrf2 signaling pathways. Several studies have shown that oxidative stress due to increased production of reactive oxygen species (ROS) or reduction of antioxidant defense systems exerts a crucial role in the development of age-related diseases including bone diseases such as osteoporosis . Bone stIt is also well known that ROS can significantly affect the generation and the survival of osteoblasts and osteocytes, former osteoblasts encased in the mineralized matrix which plays a key role in sensing and bone adaptation to mechanical loading . High ROFurthermore, clinical reports showed higher oxidative stress parameters in patients with fractures than in healthy subjects  and a maAlthough the notion that free radicals could act as signals is well established, only recently it has been underlined a difference between oxidative stress and oxidative damage. Oxidative stress is a reversible alteration of redox status of cell compartments that precede oxidative damage. When the redox status of the cell becomes more reduced, oxidation can be reversed by the activation of specific antioxidant enzymes, whereas oxidative damage is irreversible and occurs when reducing systems cannot cope with the rate of the oxidation of cell components. Thus, the preventive use of antioxidant compounds that could reverse oxidative stress and prevent oxidative damage could be useful to delay the onset of disability and promote health in an elderly population.In the last decade, a strong association between a lower incidence of age-associated degenerative diseases and a diet particularly rich in fruit and vegetables which represent a good source of antioxidant compounds has been shown .Among several natural antioxidant compounds with a potential therapeutic utility for the prevention and/or treatment of osteoporosis, there is an increasing interest in the effects of vitamin E supplement. Vitamin E can be classified into tocopherols and tocotrienols (TTs). There are four distinct isomers  in each group depending on the position of methyl group on the chromanol ring.For many years, α tocopherol was the main focus of vitamin E research, while only in the last decade the biological actions of TTs have been investigated. Although tocopherols and TTs are structurally very similar, TTs have been found to exhibit superior anti-inflammatory and antioxidant activities , suggestConversely, the bone protective action of TTs mixture , purifieFurthermore, osteoporosis has been correlated with low intake and serum levels of vitamin E and, specifically, of TTs .The protective action of TTs on bone seems to be due to positive regulation of bone turnover since TTs stimulate bone formation and inhibit osteoclastogenesis by inhibiting the mevalonate pathway . Among tHowever, the exact role of individual TT isomers in the regulation of bone cell activity and in the prevention or reduction of oxidative stress at cellular level remains to be clarified.Elaeis guineensis) fraction, to counteract the negative effects of oxidative stress on MC3T3-E1 osteoblast-like cell or MLO-Y4 osteocyte-like cells viability. Oxidative stress was induced by treating cells with tert-butyl hydroperoxide (t-BHP), an organic hydroperoxide widely used to induce oxidative stress in different cells [The aim of the present study was to examine the ability of purified δ-tocotrienol (δ-TT), extracted from a commercial palm oil , LY294002, EX-527, ML385, farnesylpyrophosphate (FPP), geranylgeranylpyrophosphate (GGPP), l-buthionine-sulfoximine (BSO) were purchased from Sigma-Aldrich Chemical, Italy.δ-TT and γ-TT were kindly provided by Prof. Giangiacomo Beretta . δ-TT and γ-TT were extracted by liquid chromatography using LC-940 Liquid Cromatography instrument  from the fraction rich in tocotrienols/tocopherols contained in the commercial oil of Elaeis guineensis , as previously described  with minl-glutamine, 100 µg/ml streptomycin and 100 U/ml penicillin at 37 °C in 5% CO2 atmosphere. Cell culture medium was replaced twice a week and MC3T3-E1 was trypsinized weekly.Murine osteoblastic cell line from ATCC (cat. Num. CRL-2593), MC3T3-E1, was seeded in High Glucose DMEM , in the presence of 10% FBS , 2% 2 atmosphere. The medium was supplemented with 5% newborn calf serum, 5% FBS and 1% penicillin–streptomycin. The cells were seeded in 10 mm dishes and were trypsinized twice a week.Murine osteocyte-like cells, MLO-Y4, were a gift from Dr. Milena Romanello . Plates of 10 mm were coated with type I collagen. MLOY-4 cells were cultured in α-MEM at 37 °C in 5% CO3 cells/well for MC3T3-E1 in 48 multiwells or at the density of 104 cells/well in a 24-multiwell plate for MLO-Y4. Briefly, after treatment, cells were incubated at 37 °C with 0.5 mg/ml 3--2,5-diphenyltetrazoliumbromide  for 3 h. After the supernatant removal, formazan crystals were suspended in dimethyl sulfoxide. The 550 nm absorbance was read at a microplate spectrophotometer .To test cells viability, MC3T3-E1 and MLO-Y4 were cultured in multiwell plates. Cells were seeded at the density of 15 × 103 cells/well) were seeded on 22-mm glass coverslips. Briefly, after treatment, the cells were fixed in 4% formaldehyde in 0.2 M sucrose, permeabilized with 0.1% TritonX100 in PBS for 5 min, and marked with 10 μg/ml Hoechst-33258 for 5 min. The fixed cells were observed at a fluorescence microscope (Axioplan) using 10 × and 20 × objectives. At least, 200 cells were counted for each sample.Apoptotic cells were evaluated by Hoechst-33258  staining, analyzing the chromatin condensation. MC3T3-E1 and MLO-Y4 cells (5 × 10t-BHP (250 µM) was added. DCF fluorescence was measured with a microplate spectrophotometer  at the 485 nm excitation and 530 nm emission wavelengths at 30, 60, 120 and 180 min after t-BHP treatment.ROS production was evaluated using 5(6)-carboxy-2′,7-dichlorofluorescein diacetate  as previously described . The celAs previously described , the intp value less than 0.05 was considered significant.Statistical analysis was performed by GraphPad Prism5 . The results are expressed as the mean ± SEM of six independent experiments (six replicates for each experiment) and evaluated by one-way ANOVA followed by post hoc Bonferroni’s test when data were parametric. A Kruskal–Wallis test followed by Dunnett’s test was used for non-parametric data. A We first established the effects of δ-TT on MC3T3-E1 cell viability in basal conditions using MTT test  exerted bone protective effects in postmenopausal women [t-BHP-induced cytotoxicity. We found that γ-TT protects MC3T3-E1 cells against oxidative damage even if at a lesser extent than δ-TT  was significantly reduced by δ-TT pre-treatment.We further examined the ability of δ-TT to prevent t-BHP-induced oxidative stress by reducing intracellular ROS levels. As shown in the Fig. t-BHP stimulates the intracellular ROS levels in a statistical significant manner (267–300%) as compared to those detected in control-treated cells. The addition of δ-TT significantly reduced ROS to a level similar to the one detected in the control cells starting from 30 min until 180 min of incubation. To highlight intracellular antioxidant factors potentially involved in the protective effects of δ-TT against t-BHP-induced oxidative damage, we focused our attention on glutathione (GSH). The GSH oxidation to glutathione disulfide (GSSG) and the consequent decrease in the GSH/GSSG ratio is related to oxidative stress and is considered a suitable indicator of cellular redox state [t-BHP (30 and 180 min) induced a significant reduction in GSH/GSSG ratio that was significantly prevented by δ-TT treatment , an inhibitor of GSH synthesis [δ-TT exerts its beneficial effects against ox state . As expeent Fig. a, b. Theynthesis  used at ynthesis , 36  and PI3K/Akt which are known to play a key role in cell survival against oxidative stress.As shown in Fig. t-BHP. Neither FPP nor GGPP (5 µM 2 h before δ-TT 10 µg/ml) could counteract the δ-TT protective effect against oxidative damage on cell viability . We used t-BHP 125 μM, for 3 h, since MLO-Y4 cells were more sensitive to damage than MC3T3-E1 cells (data not shown). As shown in Fig. t-BHP-treated cells. Treatment with δ-TT (5 µg/ml) significantly reduced t-BHP-induced apoptosis  treatment had no effect on MLO-Y4 cell viability in basal conditions Fig. a but wassis Fig. a, b.Fig.t-BHP-induced oxidative stress.In the present study, we have shown a stimulatory effect of δ-TT on MC3T3-E1 cell viability and provided evidence that δ-TT protects both MC3T3-E1 and MLO-Y4 against The δ-TT protective effect involves a reduction of intracellular ROS levels and is reflected by the increase of GSH/GSSG ratio, an indicator of cellular redox state. Finally, our data indicate that the interaction between the PI3K/Akt–Nrf2 pathways is essential in regulating δ-TT beneficial effects against oxidative stress in MC3T3-E1 cells.Our results, showing that purified δ-TT increased cell viability in basal conditions, are in line with previous reports obtained with long-term incubation of MC3T3-E1 cells with annatto-derived TT . Furthert-BHP-induced cell death and apoptosis as evidenced by MTT assay and Hoechst 33258 staining. The evidence that γ-TT is less effective than δ-TT in protecting MC3T3-E1 cells against oxidative damage indicates that δ-TT might be considered the major antioxidant compound in annatto seeds.We provided the first evidence that δ-TT protects cells against t-BHP by reducing intracellular ROS and increasing intracellular antioxidant activity. As an intracellular antioxidant factor potentially involved in the protective effects of δ-TT against t-BHP-induced oxidative damage, we specifically analyzed GSH. The GSH oxidation to GSSG and consequent decrease in the GSH/GSSG ratio are related to oxidative stress and are considered a suitable indicator of cellular redox state [δ-TT exerts its protective effect against oxidative damage induced by ox state , 36.t-BHP-treated cells was significantly prevented by δ-TT pre-treatment since the redox status of GHS was found to be similar to that detected in control MC3T3-E1 cells. It is possible that the protective effect of δ-TT against ROS levels induced by t-BHP could be due to its ability to modulate intracellular GSH levels. In fact, GSH depletion by BSO, an irreversible inhibitor of the rate-limiting enzyme of GSH synthesis, glutamate cysteine ligase , signift-BHP-induced oxidative damage in differentiated MC3T3-E1 cells by measuring the expression of several molecules related to bone resorption (RANKL and OPG) or bone formation . δ-TT did not modify the effects of t-BHP on the expression of RANKL, OPG, ALP, OC and Col1a (data not shown). The discrepancy between our data and previous reports could be due to the different experimental procedure .+-dependent deacetylase which can modulate a variety of histones and non-histone proteins resulting in the modification of the expression of several genes involved in oxidative stress resistance [Various cellular signaling pathways participate in the maintenance of redox homeostasis. δ-TT treatment was found to increase cellular GHS content and attenuate mitochondrial ROS levels through activation of Sirtuin-1 which is an NADsistance . Howevert-BHP. At variance with that observed for the inhibitory action of annatto TT on bone resorption and osteoclast differentiation [Considering that δ-TT was found to down-regulate HMG CoA reductase at the transcriptional level  and thatntiation , we obset-BHP-induced oxidative stress, we investigated the PI3K/Akt signaling pathway. This pathway plays a key role in the control of osteoblast differentiation and homeostasis [To dissect other possible mechanisms which could mediate the δ-TT protection against eostasis  as well eostasis .t-BHP on MC3T3-E1 cell viability but partially reversed the protective effect of δ-TT.Pre-treatment with LY294002, a PI3K-specific inhibitor, exacerbated the cytotoxic effect of t-BHP [cis-regulatory module antioxidant response elements (AREs) NRF2 targets include ARE-bearing effector genes such ROS scavenging enzymes , phase-2 defense enzymes H quinone oxidoreductase (NQO1), [Another signaling pathway which has emerged as a master regulator of intracellular antioxidant response through transcriptional activation of antioxidant and detoxifying enzymes is the Nrf2 pathway , 47. Intt-BHP , 49. To t-BHP . The NRF (NQO1), ).In the presence of ML385, we found results similar to those obtained with LY294002 suggesting the involvement of both cytoprotective pathways in the δ-TT antioxidant effect. Indeed, when administered together, LY294002 and ML385 completely prevented the beneficial effects of δ-TT on cell viability. This is not surprising since cytoprotective systems are organized as interactive communication networks that are finely turned by regulators and are critical features of physiological signaling designed to protect cells against endogenous or exogenous stressors. We are aware of some limitations of our study since the molecular pathways involved in the functional interaction between PI3/Akt and Nrf2 pathways in modulating the protective action of δ-TT against oxidative stress remain to be clarified. However, considering the complex interaction between these signaling pathways, a clear understanding of the molecular events that regulate the cross-talk between PI3/Akt and the antioxidant Nrf2 pathway, in the presence of δ-TT, should be the object of further studies. It could be interesting to study whether the activation of PI3/Akt is a prerequisite for the action of δ-TT on Nfr2 activity and the downstream proteins of PI3/Akt involved in controlling Nfr2. Among protein kinase downstream of Akt, GSK-3β was reported to directly phosphorylate Nfr2 . Moreovet-BHP involves a modulation of intracellular GSH levels by promoting GSH synthesis since treatment with BSO, a specific inhibitor of GSH synthesis, significantly prevented the protective action of δ-TT on ROS levels. The possibility that this protective action of δ-TT could be due to Nrf2 activation is in line with previous reports showing that Nrf2 controls both basal and inducible expression of genes that encode GLC [t-BHP  in parallel .Accumulating evidence supports a central role for osteocytes in bone remodeling, mineralization and in the repair of microdamages and microfractures , 59. Ostt-BHP-induced apoptosis of the MLO-Y4 osteocyte-like cell line, which has similar phenotype and shares many characteristics of mature osteocytes and represent a model to study osteocyte viability and apoptosis [Excessive oxidative stress induces apoptosis of osteocytes  and recepoptosis . FurtherOne major point to bear in mind to translate in vitro results obtained with functional food products into potential health benefits in living organisms is the pharmacokinetic profile of the natural compound used. Alfa-tocopherol is the most bioavailable form of vitamin E and is considered the predominant isoform to be accumulated in tissues when orally administered . Despitet-BHP-induced oxidative stress both in MC3T3-E1 osteoblastic and in MLO-Y4 osteocyte-like cells. The ability of δ-TT to reduce bone loss in experimental and clinical conditions of osteoporosis could be due to a complex and coordinated action on bone cell activities. In particular, δ-TT could have an important role in maintaining bone health in condition of excessive ROS production such as aging by increasing osteoblast activity and by reducing an excessive apoptosis of osteocytes resulting in an unbalance of the remodeling process.In conclusion, the present study provides evidence that δ-TT exerts a strong inhibitory action on δ-TT protective effect in MC3T3-E1 cells is due to a reduction of intracellular ROS levels, is reflected by the increase of the GSH/GSSG ratio, and involves an interaction between the PI3K/Akt–Nrf2 signaling pathways.


Explanted corneae are highly needed for the surgical management of patients with severe corneal diseases. The aim of this study was to determine whether the body donors from the Institute of Anatomy are a suitable source of donor corneae.At the Institute of Anatomy at Saarland University Medical Center in Homburg, corneae are prelevated from body donors who had consented to the removal of tissues for transplantation purposes during their lifetime. Following the report of death, the LIONS Eye Bank is informed and the contraindications of corneal explantation are clarified. Obtaining a blood sample within 24 h postmortem is mandatory.2 (range: 511–2944 cells/mm2)). Contraindications were present in 19 (12.6%) cases. The corneae were extracted from 49 (32.7%) body donors. Out of these 98 corneae, 46 (46.9%) were successfully transplanted. Of all non-transplanted corneae, 6 (6.1%) were microbiologically contaminated, 10 (10.2%) had a positive serology, 22 (22.5%) had an endothelial cell count < 2000 cells/mm2 and 6 (6.1%) are at time of this analysis still in culture medium. The non-transplanted tissues were used for research.The Institute of Anatomy had 150 body donors in the time period from January 2018 to June 2019. Out of these, 68 (45.3%) were reported to the Eye Bank. The age of the donors (median 82 years (range: 57–96)) is not critical since the quality of the corneae depends on the number of endothelial cells (mean: 2109 ± 67 cells/mmExplanted corneae from the Institute of Anatomy are a valuable option in obtaining grafts for corneal transplantation, which is why we are working toward on expanding cooperation with this department. Blindness due to corneal diseases represents a major public health burden that is estimated, according to the World Health Organization, to affect 1.5 million people worldwide , 3.This success translated immediately in an ongoing increase in corneal transplantation worldwide. In the United States of America alone, techniques like lamellar keratoplasty showed an astonishing increase from 5 to 58% of all corneal transplantation in the past decade . In GermDespite all the remarkable progress that characterises corneal transplantation, there continues to be a tremendous number of patients worldwide who are in need of a donor cornea. The Global Survey of Corneal Transplantation and Eye Banking showed that worldwide about 53% of the population has no access to corneal transplantation, with only 35.7% having satisfactory access to donor corneae . GeneralThere are many identified factors that may contribute to the present corneal graft scarcity including: lack of efficient notification system of potential donors , reducedThe local demand on donor corneae in our Department of Ophthalmology at SUMC in Homburg is very high. The waiting list of 350 patients remains stable for years despite an increased number of corneal excisions performed by the LIONS Eye Bank Fig. . In ordeTherefore, the aim of this study focuses on determining the role of the Institute of Anatomy as an alternative source of donor corneae as well as the evaluation of the suitability of obtained corneal grafts for transplantation.Data for this study was collected retrospectively from January 2018 to June 2019 from body donors from the Institute of Anatomy who consented to the prelevation of tissues for transplantation purposes during their lifetime. Contraindications for prelevation included among others: positive serology for HIV, syphilis, hepatitis, multiresistent bacterial infections, hematological neoplasia etc. An extended list of contraindications to cornea prelevation is provided in Table The collected data included numerical and categorical variables. Numerical variables are reported as mean and standard error of the mean. Categorical data is presented as absolute number and percentage. Comparison of the mean was performed using t-test for numerical data and chi-square test for categorical data. Correlation analysis was performed using Pearson correlation. All the hypotheses were tested at a level of significance of 0.05. The Bonferroni method was applied to adjust for multiple comparison. The statistical analyses were performed using SPSS .From January 2018 to June 2019, the Institute of Anatomy in Homburg/Saar accepted 150 (100%) body donors. During this period, 68 (45.3%) donors, of which 43 (28.7%) in 2018 and 25 (16.6%) in the first 6 months of 2019, were reported to our Eye Bank. The remaining 82 (54.7%) cases were excluded from this study as they were announced to the Eye bank after 24 h postmortem or they did not meet the donor inclusion criteria  body donors in 2018 and 19 (27.9%) in 2019. The remaining 19 (27.9%) body donors either presented contraindications (Table Median age of the 49 included body donors was 82 years (range 57–96 years). Figure Candida albicans in the culture and in 4 (4.1%) corneae, contraindications to excision were found retrospectively. 4 (4.1%) corneae could not be used because of insufficient blood samples needed for serology testing. At the time of this analysis, 6 (6.1%) corneae were still in culture medium  could be used for transplantation, 22 (22.4%) had endothelial cell damage, 10 (10.2%) showed a positive serology for hepatitis B virus, 6 (6.1%) were microbiologically contaminated with ium Fig. .p < 0.01) and positive serology for hepatitis B virus . Of the two variables, only endothelial cell damage rates showed a statistically significant Correlation with the donor age . The rate of Contamination with Candida spp. in the culture was significantly lower in the corneae from the Institute of Anatomy .Compared to cornea from other sources, corneae from the Institute of Anatomy had statistically significant higher rates of endothelial cell damage . Corneae from the Institute of Anatomy had also a higher contraindication rate compared to other sources .All 46 (46.9%) corneae with contraindications to transplantation were used as educational or scientific material. Regarding corneae from other sources than the Institute of Anatomy, the transplantation rate at our Department of Ophthalmology reached 84.5% in 2018. The difference between the transplantation rate of corneae from the Institute of Anatomy and all other sources was statistically significant in our study  corneae had an ECN < 2000 cells/mm2. In this group mean value of ECN was 1431 ± 67 cells/mm2, with a range from 511 cells/mm2 to 1995 cells/mm2. ECN could be obtained in 44 (95.6%) of the 46 transplanted corneae. Data on ECN in 2 (4.4%) corneae was missing. Corneae used for transplantation had a mean ECN of 2350 ± 57 cells/mm2. ECN in transplanted cornea ranged from 1314 cells/mm2 to 2944 cells/mm2 .Following corneal excision, the endothelial cells number (ECN) was analysed. Data on ECN was present in 71 (72.4%) corneae. ECN was not analysed in 27 (27.6%) corneae with contraindications. Mean ECN was 2109 ± 67 cells/mmThe operative techniques, in which excised corneae were used, included: 24 penetrating keratoplasty (PKP 52.3%)), 13 Descemet Membrane Endothelial Keratoplasty (DMEK (28.2%)), 7 penetrating keratoplasty à chaud (15.2%) and 2 corneoscleral grafts (4.3%) , 13 DeCurrent data suggest a permanent increase in the number of keratoplasties performed worldwide . The supThe number of performed keratoplasties as well as the number of patients on a waiting list for a corneal transplantation has increased continuously over the past decade in Germany . In ordep < 0.01). Increased donor age did not correlate with the ECN in transplanted cornea . These results confirm that increased donor age was independently of ECN associated with lower transplantation rates in the corneae obtained from the Institute of Anatomy. An increase in contraindication rates along with donor age could explain the observed differences in transplantation rates. Our study showed that increased donor age is statistically significant associated with endothelial cell damage . Endothelial cell damage rate could therefore be a cofounder in the donor age and transplantation rate correlation.The transplantation rate of corneae from the Institute of Anatomy was statistically significant lower compared to corneae supplied from all other sources. Furthermore, 46.9% of corneae from the Institute of Anatomy were declared as unsuitable for transplantation purposes in our study. This represents a 3-fold increase in unsuitable corneae compared to all other sources. Compared to these results, the most recently published data by the European Eye Bank Association (EEBA) categorized 31% of the excised corneae unsuitable for transplantation purposes . There ap < 0.01). The positive serology rate in our study is consistent with the results included in the EEBA, which showed a positive serology in 9% of cases [Donor related factors include positive donor serology. Corneae obtained from the Institute of Anatomy were more often explanted from patients infected with hepatitis B virus compared to other cornea donor sources . This difference in culture contamination could be attributed to the fact that the highest contamination rate is usually found in corneae procured in clinical departments [An important donor related factor with an impact on the transplantation rate is the corneal culture contamination. The culture infections with artments . A similartments , 14.2 vs. 2424 ± 17 cells/mm2, p < 0.05). Furthermore, our data showed similar results regarding the mean ECN in transplanted corneae  . The low rate of PKP à chaud could reflect a good quality of the corneae obtained from the Institute of Anatomy  but important number to our donor pool. As such, the main way to cover the need of corneae at our department of ophthalmology remains through further implementing and improving the existing hospital cornea retrieval programmes (HCRP).These results, as reflected by the current study, confirm throughout the above-mentioned limitations that the Institute of Anatomy represents a valuable alternative for obtaining donor corneae in the context of an acute global need for cornea donors. This is especially true in the context of a robust anatomy department, such as the one we cooperate with.Our study supports the supplementary role of the Institute of Anatomy in increasing the corneal donor pool. Advanced age and presence of contraindications to extraction and transplantation represent major restrictions. Further improvements in the collaboration with the Institute of Anatomy including a better donor notification system will help overcome the initially relatively small number of possible cornea donors.


GetUsPPE.org has built a centralized platform to facilitate matches for PPE donations, with an active role in matching donors with the appropriate recipients. A manual match process was limited by volunteer hours, thus we developed an open-access matching algorithm using a linear programming-based transportation model. From April 14, 2020 to April 27, 2020, the algorithm was used to match 83,136 items of PPE to 135 healthcare facilities in need across the United States with a median of 214.3 miles traveled, 100% of available donations matched, met the full quantity of requested PPE for 67% of recipients matched, and with 46% matches under 30 miles traveled. Compared with the period April 1, 2020 to April 13, 2020, when PPE matching was manual, the algorithm resulted in a 280% increase in matches/day. This publicly available automated algorithm could be deployed in future situations when the healthcare supply chain is insufficient. The risk to healthcare professionals (HCPs) of infection with COVID-19 appears significant7, with HCPs reflecting over 11%  of all cases confirmed as of April 16, 2020 in the state of California8; similar rates have been reported in other states that document whether confirmed positive cases were among HCPs9. The PPE shortage was met with a multifaceted response. The US Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) expanding the quality and approval standards for filtering facepiece respirators  allowed for use in medical efforts during the pandemic10. A variety of private-sector manufacturers expanded or redirected their efforts to produce medical supplies, in some cases under mandate of the Defense Production Act11. In addition, numerous grassroots efforts arose to respond to the need. Organizations with 3D-printing and related fabrication capabilities began producing open-source models of PPE with guidance from the FDA15. Other groups formed at both local and national scales to help facilitate and match PPE donations from individuals, small business, and other private donors to medical facilities in need18. Originally formed as a coalition through the viral Twitter hashtags #GetUsPPE and #GetMePPE, GetUsPPE.org quickly became a nationwide organization focused on a mission to “build a national, centralized platform to enable communities to get PPE to healthcare providers on the frontlines of the COVID-19 pandemic”19. The original platform specification empowered donors with smaller quantities of PPE  to facilitate their own donations by selecting an organization in need from a map that populated potential recipient organizations from the database. These requirements quickly changed due to the need for recipient organizations’ information to be protected, given large amounts of PPE-related scams and reports of retribution by healthcare organizations toward employees who raised concerns about PPE shortages22. Additionally, the wide variation among donations in terms of quantity, item type, and geography demonstrated a need for the organization to assume an active role in matching donors with the appropriate recipient.The arrival of Coronavirus Disease 2019  in the United States and subsequent reports of community transmission in states including Washington and California greatly increased awareness of potential medical supply shortages for responding to a widespread respiratory disease outbreak. Early concerns about inadequate ventilators and hospital/ICU beds necessary to handle a surge of patientsGetUsPPE assembled a volunteer team to facilitate connections between donors and local recipients. This required a volunteer to segment lists of both donors and recipient sites by geographic region , confirm that suppliers still had inventory available for donation via phone/email, search for a recipient organization in the database that both requested the items available and is in reasonable (subject to the volunteer’s judgment) vicinity of the donor, possible by volunteer transport. The volunteer confirmed recipient need and acceptance of supplier quality and then linked the donor and a possible recipient via email to confirm delivery location and time. The match process often necessitated a third-party transportation volunteer if the donor was unable to get the supplies to the recipient independently. This challenge was made more complex by trying to maximize the amount of need that could be met while minimizing or eliminating the financial and time costs of shipping or independent transportation for either donors or GetUsPPE.org volunteers. Efforts to manually match donors to recipients was not a scalable solution, with donations and requests substantially outpacing volunteer capacity to match and subsequently process them.GetUsPPE and our collaborators aimed to identify ways in which the donor-to-recipient matching process could be automated, allowing for faster matching of larger sets of supply and demand data. In particular, this manuscript documents the development of an open-access matching algorithm to optimize PPE donation and request matches. The algorithm is based primarily on PPE quantities offered and requested and geographic proximity as initial variables to maximize the amount of need met while minimizing delivery miles for GetUsPPE and its donors and partners.From April 14, 2020 to April 27, 2020, we used the PPE-matching algorithm to match 83,136 items of PPE to 135 healthcare facilities in need across the United States (Table PPE donor–recipient matches had a median distanced traveled of 214.3 miles. All available donations were used (100% of supply). The algorithm distribution met the full quantity of requested PPE for 67% of recipients matched  . A straight average of pre-algorithm matches yields 2.62 matches per day on average. Supply on the GetUsPPE Platform is growing but is volatile. The strict linear relationship between the number of matches and time during the manual matching phase despite volatile supply indicates a system with a matching capacity (measured in volunteer hours) bottleneck23. Although one could add more volunteers to speed up the manual matching process, automated matching speeds up the process substantially and the the number of matches begins to track supply much more than earlier, revealing that matching capacity is no longer a bottleneck. The extent of growth in matching due to the algorithm is visually captured in the graph as the gap between the extrapolated linear trendline of manual matches and the actual the number matches from April 14. Specifically, after the algorithm was employed, there was a 280% increase in the number of matches made per day  if the donor’s supply cannot be split across recipients.The PPE-matching algorithm described above efficiently allocates PPE donations to healthcare facilities and has allowed GetUsPPE to scale up PPE donation coordination efforts significantly. Specifically, the advantages of the algorithm are two-fold:The algorithm relies on PPE demand that is self-reported by healthcare facility representatives and PPE supply that is voluntarily reported by individuals or organizations for donation through GetUsPPE.org. Using these inputs, the algorithm maximizes the demand met while minimizing the shipment-miles-traveled, which results in lower shipping costs, shorter volunteer drives for drop-offs, and faster distribution.We were unable to analyze for the significance of the difference in pre-algorithm and post-algorithm matches/day due to confounding factors, including continued onboarding of additional volunteers and improved workflows. While the algorithm takes distance, PPE type, and quantity into consideration, it does not currently account for geographic and facility-level variations in COVID-19 prevalence, which would likely influence ongoing PPE needs. Additionally, this initial matching algorithm does not inherently prioritize any organization over the other, preventing us from factoring in equity and bioethical concerns such as high urgency of need  or organizations that serve particularly vulnerable populations . Further, the algorithm is unable to account for organizations that have not self-reported demand to the public GetUsPPE website; the geographic distribution of demand input data inevitably affects the allocation of PPE supply. To address these limitations, moving forward, we are performing targeted outreach to increase the GetUsPPE supply and demand data, pursuing partnerships with public health and bioethics experts to address equity concerns in distribution, and collaborating with epidemiology and predictive modeling experts to better account for COVID-19 patterns of disease spread.27. One limitation of the current software is that usage is restricted to the continental United States. Further research and development is needed to expand the geographic reach of this open-source algorithm.The ability to use a PPE-matching algorithm to connect PPE donors and recipients en masse has significant ramifications for controlling the spread of COVID-19 and preserving the healthcare workforce. Given that the global stockpile of PPE was inadequate prior to the emergence of COVID-19, GetUsPPE.org’s role in developing an automated algorithm to efficiently match the existing donated supply of PPE to nearby recipients serves as a critical stopgap to break the chain of transmission, while new global PPE production pipelines and supply chains are established. This publicly available matching algorithm has implications for emergency supply chains beyond the world of PPE. In any humanitarian context, that is characterized by fragmentation of supply and demand quantities, significant geographic dispersion in those supply / demand points and fluctuations in these points as a function of time, is a potential candidate for large scale application of this algorithm at a regular cadence. Such scenarios are widespread across delivery of medical supplies and food to vulnerable populationsInput data Fig.  was collMinimizes the shipment-miles-traveled in the network (miles traveled weighted by shipments)Balances supply and demand with a few specific conditions:All supply is exhausted if demand exceeds supply.If supply exceeds demand (a rare event in this scenario), all demand is met by minimizing shipment miles and excess supply is held back for future requests.28.Recipients may get less than what they ask for if demand exceeds supply . This is measured by the metric called fill rate Eq. 28.3.Minimize logistics complexity for small donors by precluding multiple shipments from the donor. “Small” donor is defined by a parameter on donor capacity. For example, if this parameter is 50 for masks, then a donor with 50 or fewer masks to donate will never be asked to ship to multiple recipients. This parameter is defined by the match volunteers, based on product type and fill rate considerations. While the “small” donor threshold for gowns might be 50, for gloves that threshold might be better set at 200 due to packaging volume.The data collected by GetUsPPE.org was input into an open-source matching algorithm. The key objectives that this matching allocation satisfies are:30. In our experience, the choice of distance traveled per unit of PPE as a key element of the objective function mattered for several reasons:Donors often prefer donating locally.Donors are more likely to deliver their PPE supply on their own if the recipient is closer.If the donor is unwilling to deliver, finding a volunteer for the same activity is easier if the recipient is closer.If neither donor nor volunteer is available, GetUsPPE ships through its logistics partners. The logistics is funded through $ donations by financial donors, which is limited. Therefore, minimizing distance while maximizing impact is a core goal of the organization in order to stay within allocated budgets. Every $ saved on logistics can be used to buy scarce PPE and other resources required for the organization’s primary mission.The construction of the objective function and the constraints followed best practice in the design of matching marketsThe code is written in Python and follows several steps as described below:29, which is also referred to in layman’s terms as “as the crow flies”. However, unlike Euclidean distance, Haversine distance incorporates the shape of the Earth in the computation of distance.Step 1: We use the distance function “Haversine” to compute the distance between every donor zip code and every recipient zip code. For example, if we have 3 donors and 2 recipients, there will be 3*2 = 6 possible distances. This distance function converts every zip code into a latitude and longitude specification. Distance between any two zip codes (measured by latitude / longitude) can be computed using basic coordinate geometry. We use the notion of Haversine distance, a generalized form of Euclidean distanceStep 2: We create a unique identifier for each donor and recipient and assign the above distances to a distance matrix marked by these identifiers. We also ingest supply capacity and donor request into supply and demand arrays marked by these identifiers.Step 3: We calculate total supply by adding all donor capacity and total demand by adding all donor requests. If total supply is less than total demand, we leave supply and demand arrays as it is. If total supply exceeds total demand, we create a dummy recipient with a large demand value . We assign a large distance  between this recipient and every donor in our donor pool.30, an open-source Linear Programming Solver to solve the optimization problem as defined below:Step 4: We use Google Linear OptimizationThe mode is specified for an individual PPE type but is repeatable across each type:Notation:i = 1,2,3….Let the donors be indexed by j = 1,2,3…Let the recipients be indexed by X be the quantity shipped by donor i to recipient j (this is the variable)Let d be the distance from donor i to recipient j (this is a parameter)Let S(i) be the available supply at donor i (parameter)Let A(j) be the “ask” at recipient j (parameter).Let The optimization problem is as described in Eq. subject to the following constraints as described in Eq. X values >0. These are the baseline shipment allocations and a positive match between Donor i and Recipient j. If total supply exceeds total demand (measured in Step 3), then any  match where recipient j is the dummy recipient is excluded from the matching set. Donors i who remain unmatched are flagged and potentially carried forward to a future allocation.Step 5: We filter out all i for whom more than 1 match  is >0 and donor supply A(i)≤ parameter specified by user (default = 50). For each donor i identified by this process, we sort X values in descending order; define Xmax as the highest of these values. In case of a tie, we pick the highest X with a lower distance d. In case of a tie on distance, we pick one of the candidates at random. We recalibrate the shipment quantity to reduce number of shipments to 1 for donor i using the following formulas  values and matches  and render them back to the user in the form of a matching table (Fig. Step 7: We extract final ble Fig. .Fig. 5AlVolunteers are given matching tables and coordinate with donors and recipients over email and/or phone to facilitate and confirm drop-off and delivery. After a volunteer verified that the donor still had the PPE donation to give, a donor was considered “matched” to the recipient facility and that “match” was documented. This project was undertaken as a Quality Improvement Initiative and as such was not formally supervised by the Institutional Review Board per author institutional policies.Further information on research design is available in the Reporting Summary


Moreover, the ratio of the intATFL-CFL location based on the anterior fibular border for all cadavers was 0.386. The present study suggests a reference ratio that can help surgeons locate the fibular tunnel for a more anatomically accurate reconstruction of the lateral ankle ligament. Also, it may be necessary to make a difference in the location of the fibular tunnel according to the number of ATFL bundles during surgery.We aimed to describe the location of fibular footprint of each anterior talofibular ligament (ATFL) and calcaneofibular ligament (CFL), as well as their common origin in relation to bony landmarks of the fibula in order to determine the location of the fibular tunnel. In 105 ankle specimens, the center of the footprints of the ATFL and CFL  and the intersection point of their origin (intATFL-CFL) were investigated, and the distances from selected bony landmarks (the articular tip (AT) and the inferior tip (IT) of the fibula) were measured. Forty-two (40%) specimens had single-bundle ATFL, and 63 (60%) had double-bundle patterns. The distance between intATFL-CFL and IT was 12.0 ± 2.5 mm, and a significant difference was observed between the two groups ( Ankle sprain is the most common sports-related injury that usually involves the lateral ligament complex (LLC) of the ankle , 2. AmonTo achieve a good clinical result for CAI treatment, many surgical techniques with anatomical repair or reconstruction of the ATFL and/or CFL have been proposed –14. EarlThe fibular origins of ATFL and CFL are concentrated in the lower part of the lateral malleolus and connected with connective fiber . Also, lTherefore, we aimed to describe the location of the fibular footprint of each ATFL and CFL, as well as the location of their common origin in relation to the easily identifiable bony landmarks of the fibula. Furthermore, we intend to suggest a reference ratio that can easily detect the location of the fibular tunnel as the common origin site of the ATFL and CFL by considering the anatomical variation of the ligaments.This study was approved by the Institutional Ethics Committee.105 specimens were included for this study. Of the 105 ankle specimens dissected from adult formalin-fixed cadavers, 40 (38.1%) were from females and 65 (61.9%) from males. The mean age of the donors at death was 76.4  years. Subjects with traces or scars from trauma or surgery on the skin and those with bony morphologic deformation due to bone union after fracture in the lateral aspect of the ankle were excluded. In addition, in order to discriminate the history of ligament injury, subjects in whom the shape of the ligament itself was damaged by previous injury after exposing the ligament through dissection were excluded. Even if the shape of the ligament itself was preserved, subjects in whom the tension of the ligament was not maintained and the fiber of ligament was abnormal when the ankle joint was placed in a neutral position were also excluded.The ankle specimens were stabilized in a lateral position with the ankle in neutral position. The lateral side of the ankle was completely exposed using detailed dissection to remove skin and soft tissue overlying the lateral hind foot. Care was taken to avoid injury or disruption to the native anatomy.The articular tip of the lateral malleolus (AT) was defined as the anterior fibular tubercle located most superior to the anterior border of the lateral malleolusThe inferior tip of the lateral malleolus (IT) was defined as the tip located most inferior to the anterior border of the lateral malleolusThe center of the fibular footprint of the ATFL (cATFL) was defined as the midcentral point bisecting the superior and inferior margins of the ATFL band at the fibular attachmentThe center of the fibular footprint of the CFL (cCFL) was defined as the midcentral point bisecting the superior and inferior margins of the CFL band at the fibular attachmentThe intersection point of the fibular origin of the ATFL and CFL (intATFL-CFL) was defined as the point where the most inferior and posterior fiber of the ATFL band and the most superior and anterior fiber of the CFL band intersect each otherAfter careful dissection, the number of ATFL bundles was noted to detect anatomical variability. Next, the anterior border of distal fibula  was confirmed using combined visual inspection and direct palpation of bone with all soft tissues removed. The following two reference points were marked: the articular tip of the lateral malleolus (AT) and the inferior tip of the lateral malleolus (IT). Further dissection to identify the center of the fibular footprint of the ATFL and CFL was performed . Finally, the intersection point of the fibular origin of the ATFL and CFL (intATFL-CFL) was identified by minimal dissection of the most inferior and posterior fibers of the ATFL and the most anterior fiber of the CFL . The poiIf the ATFL had multiple bundles, the center of the footprint including all bundles was used for measurements. The distances from the center of the fibular footprint of the ATFL and CFL to the inferior tip of the lateral malleolus were measured using a flexible surgical ruler. Also, the distance from the intersection point of the fibular origin of the ATFL and CFL to the inferior tip of the lateral malleolus and the distance from the articular and inferior tips of the lateral malleolus were measured. The identification of landmarks and measurements of the distances were performed independently by two researchers. Each independent researcher repeatedly measured the distance twice after identifying the landmarks. The averages of the two researchers' measurements were adopted as data for each specimen.t-test was used. All statistical analyses were performed using SPSS 25.0 software , and a p value less than 0.05 was considered statistically significant.Inter- and intraobserver reliabilities for all measurements were calculated by the intraclass correlation coefficient (ICC). According to the definition of Landis and Koch , ICCs ofIntraclass correlation coefficients were generated for all measurements. All measurements were higher than 0.8 (indicating acceptable reliability) and were employed in the study.The distance (mean ± standard deviation) between the center of the fibular footprint of the ATFL and inferior tip of the lateral malleolus was 15.9 ± 3.2 mm. The distance between the center of the fibular footprint of the CFL and inferior tip of the lateral malleolus was 8.6 ± 2.9 mm. The distance between the intersection point of the fibular origin of the ATFL and CFL and inferior tip of the lateral malleolus was 12.0 ± 2.5 mm . The disp < 0.001). The average distance from cATFL to IT was 17.5 ± 3.2 mm in type 1 and 14.8 ± 2.7 mm in type 2. However, there was no significant difference between the two groups in the distance from cCFL to IT . In terms of the distance from the intersection point of the fibular origin of the ATFL and CFL to IT, significant differences were observed between the two groups .Of the 105 specimens, 42 (40%) specimens had single-bundle ATFL (type 1) and 63 (60%) specimens had a double-bundle pattern (type 2). There were no triple-bundle pattern specimens . With reIn addition, the three distances from the inferior tip of the lateral malleolus to anatomic footprint sites of the lateral ankle ligaments were calculated as a ratio of the length between the articular tip and inferior tip of the lateral malleolus. All ratio values are listed in A clear understanding of the anatomical location of the ligaments in relation to the bony landmark is important for surgeons performing anatomic reconstruction of the lateral ankle ligaments. The first contribution of the present study is to propose that surgeons can use a reference ratio to locate the fibular tunnel for anatomic reconstruction of the lateral ankle ligament, particularly in a patient who is much smaller or larger than average. The second is that there is a difference in the location of the fibular tunnel for anatomic reconstruction of the lateral ankle ligament between the single and double fascicular ATFL.Most ankle stabilization surgeries involve repairing or reconstructing the ATFL and/or CFL. Numerous surgical procedures for chronic ankle instability have reported good clinical results ; but in The previous cadaveric study by Matsui et al.  suggesteThe intATFL-CFL was previously identified at the fibular obscure tubercle (FOT) by Buzzi et al. . Based oA systematic review showed that the origins of the ATFL and CFL were located around 10-14 and 5-8 mm from the IT, respectively . On the Although ligament injury combined with bony avulsion fracture may interfere with the reference using the bony landmarks, the importance of the applicability of this ratio using surgically relevant bony landmarks for reference during the anatomical reconstruction procedure cannot be underestimated. The indication for performing an anatomical reconstruction procedure is instability from chronic rupture rather than acute rupture of the lateral ligament. A sufficient incision may be made to expose the ligament attachment, or an arthroscopy may be used to navigate the ligament attachment. However, in chronic ankle lateral instability (CALI), the surgeon is more likely to face a complex situation in which the native ligament centers are not visible due to severe ligament damage. Use of this ratio using bony landmarks facilitates consistent and anatomical placement. Additionally, it may clinically enable minimally invasive anatomical reconstruction of the lateral ligament. A previous study has mentioned that sufficient incision is the key to anatomic reconstruction of the ligaments, but it is emphasized that attention to wound complications and nerve damage is essential . PositioThe ultimate purpose of ligament reconstruction is to recreate the course of the injured ligaments . Thus, lPrevious studies reported anatomical variations of the ATFL with regard to the number of bundles , 23, 25.Although no conclusions were reached regarding frequencies of ATFL bundle patterns, there may be a possibility that the location of the fibular tunnel may differ depending on the type of ATFL bundle patterns. To date, there is no description of a comparative analysis of intATFL-CFL according to the number of ATFL bundles in the literature. Although the measured value was the difference in small units such as mm, our findings have shown that both the absolute value and the ratio of the location of intATFL-CFL differ significantly according to the number of ATFL bundles. In the double fascicular ATFL, the fibular tunnel for more anatomically correct ligament reconstruction would have to be located more distal than that in single fascicular ATFL, although achieving this small difference may not be surgically feasible.This study was limited by the use of fixed cadavers to evaluate the morphological characteristics of the lateral ankle ligament. With regard to postmortem changes, there may be differences in the measurements taken for a live person and those from a cadaver. Further, the cadavers were limited to those of elderly individuals  and the trauma history of the ankle may not be fully certain because the corpse specimens donated for research do not provide the past medical history.The present study suggests a reference ratio that can help surgeons to locate the fibular tunnel for more anatomically correct reconstruction of the lateral ankle ligament. Also, it may be necessary to make a difference in the location of the fibular tunnel according to the number of ATFL bundles during surgery. Further clinical trials on this will be needed in the future.


RBP4 gene polymorphisms essential from a cardiovascular perspective.Although many preventive and treatment approaches have been proposed, cardiovascular disease (CVD) remains one of the leading causes of deaths worldwide. Current epidemiological data require the specification of new causative factors, as well as the development of improved diagnostic tools to provide better cardiovascular management. Excessive accumulation of adipose tissue among patients suffering from obesity not only constitutes one of the main risk factors of CVD development but also alters adipokines. Increased attention is devoted to bioactive adipokines, which are also produced by the adipose tissue. The retinol-binding protein 4 (RBP4) has been associated with numerous CVDs and is presumably associated with an increased cardiovascular risk. With this in mind, exploring the role of RBP4, particularly among patients with obesity, could be a promising direction and could lead to better CVD prevention and management in this patient group. In our review, we summarized the current knowledge about RBP4 and its association with essential aspects of cardiovascular disease—lipid profile, intima-media thickness, atherosclerotic process, and diet. We also discussed the  Current2 . Excessi2 ,16,17. A2 . Most of2 ,21,22,232 . In thisRetinol-binding protein 4 (RBP4), presented in RBP4 (MIM 180250) located on chromosome 10 (10q23.33) between coordinates 93,591,694 and 93,601,744 bp according to human genome reference assembly GRCh38.p13  occurrence in the world population based on 1000 Genomes Project data . It is worth noting that all of the listed variants are located outside the coding gene region and that there can be a potential relationship between the regulation and the change in gene expression levels.Since RBP4 may be correlated with conditions related to Type 2 diabetes mellitus (T2DM), obesity, or CVD, the ications . Additioe levels ,56,57. Thickness . These hAs mentioned before, the secretion of adipokines is frequently abnormal among patients with obesity. Adipocyte hypertrophy, ectopic fat accumulation, and adipose tissue inflammation may cause adverse adipokine secretion, which, in turn, can be associated with a number of health consequences, including metabolic, inflammatory, or cardiovascular diseases . HoweverPossible explanations regarding different concentrations or changes in RBP4 may include both ethnic and age differences  . DespiteRBP4 and retinoids are involved in the lipid status since they influence the metabolism of triglycerides . RPB4 leAlthough RBP4 may be involved in different periods of the atherosclerotic process, its role in lipid metabolism remains unclear and should constitute the focus of further research. -/macrophages, which suggests that alternative pathways may be considered [Chronic vascular inflammation plays a critical role in the development of atherosclerosis . It beginsidered . In the nsidered . Moreovensidered . On the nsidered . Anothernsidered . RPB4 leAs the research studies have demonstrated, RBP4 is associated with insulin resistance (IR), which is further associated with chronic subclinical inflammation and can promote vascular inflammation ,85,86,87As has already been mentioned, the prevalence of CVD, of which atherosclerosis is the major component, is still increasing. There is a strong need for undertaking both standard and novel diagnostic methods among men and women for an early prevention plan to be implemented. Identification of atherosclerosis at the subclinical stages would primarily promote earlier diet prevention and a selection of more effective treatment methods, which may lead to a better prognosis . UltrasoAlthough the intima-media thickness measurement is a well-established method, its correlation with serum RBP4 levels is not quite well understood and remains undefined. To evaluate whether there is any interaction between these two parameters, further research is still necessary. In the Mansouri et al. study, there was no correlation between cIMT and RBP4 levels in Type 2 diabetes patients (T2DM) . SimilarNutritional and lifestyle care are some of the essential approaches to CVD management and prevention. Standard dietary patterns, such as the Dietary Approach to Stop Hypertension (DASH) or Mediterranean diet (MeD), as well as the new plant-based diets, are recommended in CVD risk reduction . The queCompliance with the DASH diet was independently associated with lower RBP4 serum levels among middle-aged and elderly adults . FurtherCurrent studies are focused on finding new biomarkers which would provide an added value to the well-known CVD risk factors and could serve as a diagnostic tool. Retinol-binding protein 4 should be investigated from the biomarker perspective since RBP4 levels were observed to be higher among individuals with CVD and could be involved in the atherosclerotic process. According to Alkharfy et al., serum RBP4 levels correlated significantly well with the existing risk factors of cardiovascular disease in women, regardless of body weight . This reRBP4 gene polymorphisms, should also be included in the extensive studies.Retinol-binding protein 4 is one of the adipokines potentially associated with an increased risk of developing cardiovascular disease, particularly among patients with obesity. The role of RBP4 in the atherosclerotic process is mainly associated with an increased expression of pro-inflammatory cell surface adhesion molecules and soluble pro-inflammatory factors. RBP4 has also been connected with an unfavorable lipid profile and an increased intima-media thickness; however, these associations should constitute the focus of further extensive research. To investigate the role of RBP4 in CVD more precisely, other factors, such as diet or It is difficult to determine at this point whether RBP4 could constitute a novel biomarker useful in CVD and which pathological process it could represent . Additionally, further research should be conducted to confirm whether RBP4 is yet another risk factor of CVD, as RBP4 can represent a promising indicator in the treatment and diagnosis of CVD.


Historical, educational, and technical barriers have been reported to limit the use of surface electromyography (sEMG) in clinical neurorehabilitation settings. In an attempt to identify, review, rank, and interpret potential factors that may play a role in this scenario, we gathered information on  current  Surface electromyography (sEMG) is a technique for non-invasive measurement of the electrical activity of a muscle through adequately positioned surface electrodes on the skin . sEMG haad-hoc European actions and to the efforts made by national scientific societies  and geographical origins was conducted.Through a literature scan of three biomedical databases  using common keywords (surface electromyography AND neurorehabilitation) and limiting to medical subject headings (MeSH) and MeSH Major Topic, we retrieved an initial set of 821 articles  to be then manually screened. Based on the title, abstract and keywords, pertinent articles were selected by two of the authors  . After removing duplicates across the databases, manual screening of the full-texts led to retain 32 articles specifically dealing with the topic under study. From these, a set of unique authors' names was extracted, leaving 82 authors who had published at least two articles which major topic was the application of sEMG to neurorehabilitation from a methodological/technical or clinical perspective, or both. Of these 82 individuals, 52 had authored at least two articles in a prominent role (first or second or last or corresponding author). After extracting contact information, electronic invitations were sent to these 52 authors. The participants were requested to respond anonymously to a questionnaire, which had been developed iteratively by a primary research team  and then reviewed and pilot-tested by an external board of eight “core experts” . Feedback received during review and piloting was incorporated into the survey.The survey  comprisehttp://survey-monkey.com] was used to deliver the questionnaire electronically. Identified experts were invited to participate via an e-mail that included key information about the study, its purpose, how it would inform consensus on sEMG employment, potential utility, professional figures involved in its usage, education, training and teaching, and potential barriers.An online software [SurveyMonkey Participants were asked to respond to each question on a 5-point Likert scale , and in two questions (11 and 21), by ranking items. They were also instructed to leave unanswered those questions that were perceived as outside their expertise/knowledge. We asked them for any additional comments/insights they wished to provide using free-text boxes. These comments were recorded and, based on an eventual trend , taken into consideration and commented upon when interpreting the survey results. The survey was available for 6 weeks. Four reminders were sent by e-mail to participants on days 14, 28, 35, and 42.p < 0.05.A cut-off of 75% agreement was chosen as the consensus threshold based on the findings of a systematic review of surveys and consensus studies . AccordiOf the 52 invitation e-mails sent , 35 invitees (67%) completed the 30-question survey. Responses were received from a minimum of 26 to a maximum of 35 participants (74–100%). Data were analyzed from the 35 respondents and the consensus threshold (75%) was calculated for each question relative to the number of respondents.The specific clinical subfields of the interviewed cohort were also checked (question 30). The majority of the respondents declared to engage in “Activities to improve mobility (movement), muscle control, gait, and balance”  and in “Exercise programs to improve movement, prevent, or decrease weakness caused by lack of use, manage spasticity and pain, and maintain range of motion” . Twelve (34%) specifically engaged in “Help with obtaining assistive devices that promote independence,” 9 (26%) in “Help with activities of daily living (ADLs),” and 6 (17%) in “Patient's education and counseling.” Two respondents declared in the comment areas to specifically engage in “Measuring human motion in clinics.” Eight out of 35 (23%) declared not to work in a clinical setting, but rather in clinical research.1. Current sEMG employment in clinical settings and potential utility (questions 1–10) – Consensus was reached on that sEMG is more frequently employed in technical/methodological research than clinical research , and that it should be used in neurorehabilitation to obtain information on neuromuscular function that is not provided by other assessment techniques/tools . With regard to its clinical utility, the respondents agreed by consensus that sEMG can enhance the assessment and characterization of neuromuscular impairments in patients , positively influences the intervention plan design , allows better tracking of changes in muscle activity from baseline when neurorehabilitation interventions are administered , allows evaluating the effects of non-invasive interventions designed to impact muscle activity  , and allows evaluating the effects of invasive interventions designed to impact muscle activity (such as surgery and neuromuscular blocks) . Moreover, its employment for biofeedback training in case of abnormal patterns of muscle activity that may be modified through motor learning was agreed upon by 30/35 (86%) of the respondents.When enquired specifically on the role of sEMG in patient's assessment, sEMG was deemed by consensus very likely to be useful to outline the sequential timing of muscular actions during given movements  , evaluate the appropriateness of the muscle activity during a specific movement  , and characterize the stretch reflex .Regarding the utility of sEMG in the definition of an intervention plan, sEMG was indicated as potentially useful when there is need to investigate or quantify abnormalities in the sequential timing of muscular actions during given movements , muscle imbalance/dyssynergia , and involuntary muscle activity  . No consensus was reached for 6 of the 8 items questioning whether sEMG information may prove useful to track changes induced by a therapeutic intervention. The cohort agreed that sEMG can, instead, track rehabilitation-induced changes in the sequential timing of muscular actions during given movements   and for involuntary muscle activation  .Regarding the employment of sEMG as a stand-alone technique or in combination with other methods used by neurorehabilitation professionals to assess muscle and physical function, only 18 out of 32 respondents (56%) suggested the stand-alone use of sEMG, whereas they agreed by consensus on the combination of sEMG with gait/motion analysis (with or without motion capture) , muscular hyperactivity/muscle tone assessment , accelerometry , and stretch-reflex assessment .When questioning about the employment of sEMG for biofeedback training in case of abnormal patterns of muscle activity, consensus was reached for its utility in allowing the patient to learn how to “change the coordination pattern of an agonist with respect to antagonists and synergists (muscle selectivity)” , “decrease the activity of overly tense and/or involuntarily hyperactive muscles” , and “increase the activity of weak and/or hypoactive muscles” .sEMG was expected to have practical utility in clinical neurorehabilitation for five neurological disorders: “Neuromuscular disorders” , “Stroke/cerebrovascular diseases” , “Spinal cord disorders” , “Peripheral nerve disorders” , and “Multiple sclerosis/demyelinating diseases” .2. Professional figures using sEMG and potential advantages of better qualified professionals (questions 11–15) – Interviewees were asked to rank the professional figures who are most frequently involved in sEMG recordings in neurorehabilitation settings. Respondents listed “Biomedical engineer” in first place  followed by “Physiotherapist”  and “Kinesiologist/Human movement scientist” .When judging the level of involvement of each of the professionals for sEMG signal acquisition, processing and quality control, consensus was reached only for “Biomedical engineer”  and “Kinesiologist/Human movement scientist”  but not for other professions. When asked about the involvement for sEMG interpretation, respondents agreed by consensus on five professional figures: “Kinesiologist/Human movement scientist” , “Clinical neurophysiologist” , “PM&R” , “Biomedical engineer” , and “Physiotherapist” . Both PM&R doctors and physiotherapists were professionals with the highest number of “very high” agreement .Participants were also asked whether greater qualification of neurorehabilitation professionals on sEMG would contribute to improve the quality of neurorehabilitation care and reduce the cost of its delivery. Consensus was reached for quality improvement  but not for cost reduction .3. Education, training, and teaching (questions 16–20) – Regarding the adequacy of the professional figures in the education, training, and teaching of neurorehabilitation professionals on sEMG, consensus  was reached for four figures: “Biomedical engineer” , “Kinesiologist/Human movement scientist” , “Physiotherapist” , and “Clinical neurophysiologist” . A combination of professional figures was also indicated as adequate . Interestingly, this option received the largest number of “very adequate” preferences , followed by “Biomedical engineer” , and “Physiotherapist” .Participants were asked to indicate how many years of practice/experience with sEMG techniques are necessary to qualify for providing education and training on the use of sEMG for clinical neurorehabilitation. They agreed by consensus that practice/experience <1 year is inadequate to very inadequate . At least 5 years were indicated as an adequate to very adequate period .When analyzing the technical skills for sEMG recording and interpretation that neurorehabilitation professionals should own , respondents agreed they should be able to recognize and filter out artifacts at the skin-electrode interface  , and choose the processing technique that is most appropriate for a given application .With regard to the importance of sEMG-derived variables for clinical applications, consensus was reached for the “Timing of muscle activation and their variability” , amplitude estimators  , “Signal quality/reliability indicators ” , and envelope time course .When asked to indicate further competencies to complement basic knowledge on physiological and non-physiological factors that influence sEMG, respondents agreed by consensus that neurorehabilitation professionals should own knowledge about sEMG patterns of recruitment in the main central and peripheral neuromuscular disorders , about the use of sEMG to assess muscular hyperactivity , about sEMG patterns of recruitment of healthy individuals , and about the pathologies affecting muscle fiber conduction velocity .4. Potential barriers to sEMG usage (questions 21 and 22) – Respondents agreed by consensus on 5 of the 12 suggested potential barriers limiting the widespread use of sEMG in clinical neurorehabilitation: “sEMG data analysis/interpretation is difficult to perform without specific education/training” , “Inadequate education for professionals in neurorehabilitation” , “Lack of widely accepted evidence that the use of sEMG improves treatment effectiveness” , “Inadequate education and training on sEMG in graduation courses” , and “Time-consuming” .We asked participants to rank which clinical environment is most likely to favor the usage of sEMG. Respondents listed “Privately operated clinic (with public or insurance-based reimbursement)” in first place  followed by “Publicly operated clinic (with either public or insurance-based reimbursement)”  and “Privately operated clinic (out-of-pocket)” .p = 0.01), and in a potential barrier perceived to limit its clinical translation (“Purchase and maintenance costs of sEMG equipment”), which was deemed relevant by 71% of the clinicians and by 19% of the non-clinicians (p < 0.0001).We conducted survey research to elucidate key aspects on four main themes including trends in the current employment of sEMG in clinical settings and its potential utility, professional figures involved in sEMG assessment and interpretation, educational aspects, and potential barriers to the incorporation of this technique in daily neurorehabilitation practice. By building consensus, we intended to gather information from experts in the field who published prominently on the topic for establishing a common platform to streamline future clinically-applied research on the topic. Importantly, the majority of the interviewed cohort were clinicians (57%). Consensus was reached on 17 of the 22 proposed questions encompassing the four main themes of the survey focused.Contrary to our first assumption, no consensus was reached on the statement that sEMG is rarely used in clinical neurorehabilitation 56%). This finding is in disagreement with previous reports –5, which6%. This sEMG was deemed by consensus to provide unique information on neuromuscular function that is not offered by other assessment techniques/tools. Among the advantages for which consensus was established , its use was considered to substantially enhance the quality of patient's assessment. This result supports the use of sEMG as a tool for motion analysis as it can record and quantify clinically important muscle-related activity. Given that sEMG is deemed to cause minimal burden to the subject or the patient  on whether it could be used to differentiate the many types of tone-regulations impairments such as myoclonus, dystonia, and tremors. It may be that these latter applications require more clinical validation before being employed in neurorehabilitation settings.As strongly agreed by the respondents, the potential of sEMG for accurate analysis of movement disorders is maximized by its combination with other quantitative and qualitative methods, such as gait/motion analysis, muscular hyperactivity/reflex/tone assessments, and accelerometry. Interestingly, the item on muscular hyperactivity was widely commented on. Two invitees for example, remarked that sEMG could differentiate between spasticity and rigidity, as suggested by Levin et al. , MullickThe cohort was also very clear in highlighting by consensus that sEMG is more frequently employed in technical/methodological reports than in clinical research, possibly suggesting that a substantial distance still exists between researchers and clinicians, in agreement with previous reports , 19, 22.Specific education and training of professionals were considered to play a decisive role, particularly to improve the quality of neurorehabilitation care. Accordingly, inadequate education and training on sEMG in graduate courses, lack of continuing education for professionals in neurorehabilitation and time-consuming set up were listed as the main barriers to the clinical employment of this technique.Acquiring and consolidating knowledge on physiological and non-physiological factors that influence sEMG was also put forward as a key point for the interpretation of sEMG findings by neurorehabilitation professionals. In particular, strong consensus (>90%) was achieved for three elements to be part of their educational background:  knowledgInterestingly, even though the cohort was mainly composed of clinicians, the professional figures who were deemed best qualified for teaching sEMG to neurorehabilitation professionals were not medical doctors nor physiotherapists. Indeed, biomedical engineers and kinesiologists/movement scientists were ranked, respectively, in first and second place, provided that they hold superior expertise and skills which, regardless of the professional figure, was indicated as the key factor.The item on potential barriers was extensively commented upon, with invitees converging on four additional factors:  poor relTaking together the interviewees' responses and comments on factors potentially limiting the usage of sEMG in neurorehabilitation, it appears that the integration of sEMG into an agreed framework for diagnosis and treatment is still challenging for clinicians. Furthermore, there is currently poor translational evidence to make sEMG part of a coherent diagnostic or measurement context and to use it to track the right clinical outcomes. For all these reasons, it is likely that sEMG is not used extensively in neurorehabilitation.As for teaching, biomedical engineers were deemed the professional figure most frequently involved in sEMG signal acquisition, processing, and quality control. Kinesiologists/human movement scientists, clinicians  and biomedical engineers were, instead, indicated as those most likely in charge of data analysis, post-processing, features extraction etc. Among the comments accompanying this specific item, the relatively new professional figure of “human motion analyst” was put forward by some respondents as a possible reference to manage sEMG assessments in the clinical setting.Data indicate that neurorehabilitation professionals should hold basic know-how  but also additional abilities to recognize and filter out artifacts, distinguish between cross-talk and co-activation, and also choose the most appropriate processing technique. More advanced expertise, such as importing data into external environments and further computing were regarded as “not required for all clinical neurorehabilitation professionals.” Overall, the item about the ideal technical skills was also widely commented. What clearly emerged from additional reasoning and elaborations from the experts was that a basic knowledge about the identification of “bad” signals  may be sufficient for clinicians, whereas familiarity with signal acquisition issues management  along with relatively more advanced analyses  are skills that are necessary for professionals working in the laboratory and dealing with human motion analysis.too many data–no data” paradox  and non-clinicians (46%) for several issues. For instance, unlike clinicians, the majority of the non-clinicians considered sEMG more relevant for research than clinical purposes. As for those factors potentially limiting the usage of sEMG in clinical practice, different beliefs emerged between the two categories being “inadequate education” and “purchase and maintenance costs of sEMG equipment” perceived as more relevant barriers by clinicians. These findings reveal that for selected items the professional figure background influence the questionnaire responses.The findings of the present survey research may be limited by the characteristics and geographical location of the selected contributors who participated in the survey, possibly subjecting the survey findings to selection bias as the usage of sEMG may vary by country. In particular, 86% of the interviewees were from Europe and 54% from Italy, possibly reflecting a greater sensitivity and interest to the problem in this country. Although we collected responses from the majority (67%) of the sEMG experts invited, it cannot be excluded that responses from a larger number of individuals with different backgrounds may have led to different results, possibly leading to reduced likelihood of consensus. Another element that needs to be considered in the interpretation of the findings is the choice to interview only scientists, researchers, and clinicians who had authored indexed articles on the topic rather than clinicians who did not. Therefore, the results of the survey and their external validity and generalizability may be limited and biased by this choice and will need to be compared to a larger sample of clinicians who specifically engage in neurorehabilitation on a daily basis, but not necessarily on research. Moreover, while being iterative in the conception and planning of the survey, we acknowledge that the methodological robustness of this study could have been enhanced by adding one or more rounds to the present one-round survey.Finally, while we attempted to be comprehensive in the development of the survey questions and sub-items, other questions could have been asked to address specific issues that were here possibly overlooked.This survey research clarified several aspects of sEMG in neurorehabilitation ranging from current trends in its use, educational, technical, and methodological features as well as the translational outreach and potential utility of this technique for clinicians and patients.In particular, sEMG was indicated as practically useful in clinical neurorehabilitation for patient assessment, to define the intervention plan and complement/optimize other methods used to quantify muscle and physical function. Nevertheless, the aggregate opinion of the interviewed experts clearly revealed that sEMG is more frequently employed in technical/methodological than clinical research. Moreover, the slow dissemination of research findings, lack of education on sEMG and lack of incorporation of the patients' goals when applying the technology seem to prevent prompt translation into practice. Additionally, multidisciplinary competences are necessary to face the challenges of the complexity of the matter, beside the identification of more specific procedures, for increasing clinical use and benefits for the patients.Future translational studies aimed at testing whether the addition of sEMG brings clinically important benefits are required to fill the gap between research and clinical practice, which may itself limit the employment of sEMG in neurorehabilitation. With the present survey findings obtained by bringing together the expertise, guidance, and insights of leading experts in the field, we are now better positioned to open a debate on the appropriateness and value of sEMG for neurorehabilitation professionals and its potential translation into clinical practice.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Institutional Review Board Department of Biomedical Sciences University of Sassari, Italy. The patients/participants provided their written informed consent to participate in this study.AMa, AC, and FD conceived the idea and managed all aspects of the work. LB-O, AB, UD, MK, NM, DM, AMe, SR, and AT developed the survey questions, developed and piloted the survey. All authors provided critical feedback and helped shape the research, analysis, and manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DAZL) is an RNA binding protein coding gene in autosomal, playing important roles in testicular development and gametogenesis. In this paper, we found that DAZL is extremely highly expressed in testis compared with other organs and reaches to a peak at sex maturity (6-month old) in testis. Two single nucleotide polymorphisms (SNPs) within DAZL were found to have significant effect on the variation coefficient between left and right epididymis weight.The deleted in azoospermia-like (DAZL) gene encoding an RNA binding protein is pivotal in gametogenesis in lots of species and also acts as a pre-meiosis marker. The current study was conducted to detect expression profiles and single nucleotide polymorphisms (SNPs) of DAZL in sheep using qPCR, DNA-pooled sequencing, improved multiplex ligase detection reaction (iMLDR®) and restriction fragment length polymorphism (RFLP) methods. The results confirmed that ovine DAZL showed the highest expression level at six-months of age across five developmental stage. At six-month stage, DAZL expressed primarily in testis across seven tissues analyzed. The abundance of DAZL in the large-testis group is higher than that in the small-testis group although it is not significant. In addition, six SNPs (SNP1-SNP6) were identified in DAZL. Of those, SNP1 (p < 0.05) and SNP6 (p < 0.01) were significantly correlated with the variation coefficient between left and right epididymis weight (VCTW). The current study implies DAZL may play important roles in testicular development and its SNPs are associated with testicular parameters, which supply important indicators for ram selection at early stage.The deleted in azoospermia-like ( The males with high quality semen not only tend to have more offspring than females, but also help accelerating genetic improvement ,2. There = 0.67) , which iDAZ gene and two autosomal homolog genes: deleted in azoospermia-like (DAZL) and boule-like RNA-binding protein  [DAZL is conserved throughout the vertebrates with single RNA recognition motif (RRM), while DAZ is conserved confined to Old World monkeys, apes and human [DAZL can be a germ cell marker [DAZL can act as an intrinsic inducer of meiotic initiation in post-migratory germ cells that its expression dramatically increases at the onset of meiosis [DAZL-deficient mice, germ cells eventually underwent apoptosis rather than initiating gametogenesis [DAZL have been found to have significant influence on male fertility of the Chinese population [The deleted in azoospermia (DAZ) family is a kind of RNA binding proteins (RBPs), which plays important roles in spermatogenesis, gametogenesis and postnatal male germ cell development ,7. This s BOULE) . Of thosnd human ,10. DAZLl marker  and showl marker . Additio meiosis ,14. In Dogenesis . In humapulation .DAZL has 11 exons with a length of 22 kb and is located on chromosome 1, encodes a polypeptide chain consisting of 258 amino acids. Li et. al (2020) detected the expression of DAZL in testis across several developing stage, finding that DAZL showed the highest expression level in the testis after sexual maturity [DAZL in Hu sheep at different testicular developmental stages and in different tissues was still unclear. Therefore, the current study detected the expression patterns of DAZL in Hu sheep’s heart, liver, spleen, lung, kidney, longissimus dorsi and testis and compared its expression in testis among neonatal, pubertal, sexual maturity, body mature and adult stages. In addition, the DNA-pooling sequence, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and improved multiple-temperature ligase detection reaction (iMLDR®) methods were applied to screen SNPs within DAZL that associated with testis size and related traits. This study aims to provide theoretical and practical significances for animal breeding and supply molecular markers for marker assisted selection (MAS) at early stage of Hu sheep.In sheep, maturity . HoweverAll experimental procedures were carried out following the experimental field management protocols (file No: 2010-1 and 2010-2) approved by Lanzhou University. All efforts were taken to minimize animal suffering.longissimus dorsi. All samples were immediately preserved in liquid nitrogen after collection until DNA and RNA extraction.Hu sheep, because of high fecundity and perennial estrus, is one of the most widely raised sheep breeds for intensive sheep production system (house feeding) in China. In the current study, a total of 352 healthy male Hu lambs with similar birth weight at similar birth age and without cryptorchidism, were selected from Zhongtian Industry Co. Ltd . This population was divided into 5 groups which were M0 , M3 , M6 , M12  and M24  slaughtered at 0 day, 3 months, 6 months, 12 months and 24 months, respectively. After slaughtered, the testis and epididymis weights and volumes were measured, and samples of testis and epididymis were collected immediately. In addition, 3 Hu sheep closed to the average testis weight at 6 months among all individuals (n = 340) were selected to collect the sample of heart, liver, spleen, lung, kidney and longissimus dorsi at M6 (n = 3 for each tissue) and testis at M0, M3, M12, M24  and M6  using RNAsimple Total RNA Kit  following the manufacturer’s protocols. Aliquots (200 ng) of total RNA were used for cDNA synthesis using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix  following the manufacturer’s protocols.The genomic DNA was extracted from 340 testis tissues at M6 using a standard phenol-chloroform method . The totDAZL in five developmental stages in Hu sheep’s testis, seven different tissues and different size of testis at 6 months was quantified by RT-qPCR. The RT-qPCR was performed on Bio-Rad CFX96 Real-Time PCR System  using 96-well plates. All the operations were followed the MIQE guidelines [DAZL expression detection were designed using Premier v. 6.0 software  based on ovine DAZL sequences (XM_027964130.1) obtained from the NCBI database . Comparison of gene abundance between large- and small-testis groups was implemented by Wilcoxon Rank Sum test in R 3.6.1.The expression of idelines . Primersdatabase . Each 25DAZL genomic sequence (NC_019458.2) to amplify 10 exons and their flanking regions , 2.5 μL NEBuffer (1×) and 17 μL ddH2O. The system was incubated at 37 ℃ overnight and then visualized in 1% agarose gels.A total of 11 pairs of primers were designed based on  regions . The gennnealing  for 30 sThe testis parameters including variation coefficient between left and right testis weight (VCTW), variation coefficient between left and right epididymis weight (VCEW), testicular index (TI), the ratio of right epididymis weight to right testis weight (REW/RTW), the ratio of left epididymis weight to left testis weight (LEW/LTW), the ratio of total epididymis weight to total testis weight (TEW/TTW) were calculated using Excel, the calculation formula are as follow:PIC), effective allele number (Ne) and expected heterozygosity (He), as well as Hardy–Weinberg equilibrium (HWE) testing p value—were directly calculated following previous description [https://www.r-project.org/) fitting a linear model:ijk is the trait measured on each of the ijkth animal; µ is the overall population mean; Gj is the fixed effect associated with jth genotype; and Eijk is the random error. Differences in mean phenotype among genotypes were tested using the LSD test in agricolae R package (https://cran.r-project.org/web/packages/agricolae/) and Dunn’s Kruskal–Wallis post hoc. p < 0.05 was to be considered significant. p < 0.01 was considered to be highly significant. Bonferroni correction was applied for multiple testing between genotype groups. The Least Absolute Shrinkage and Selection Operator (LASSO) regression in lasso2 R package (https://cran.r-project.org/web/packages/lasso2/) was used to find the representative SNP. Moreover, the linkage disequilibrium (LD) of identified SNPs was performed using HAPLOVIEW software [In addition, genotypic, allelic frequencies and genetic parameters—including the polymorphism information content  was detected by RT-qPCR. Comparison of gene abundance among five growth stages was implemented by Kruskal–Wallis rank sum test. The results revealed that DAZL had the highest expression in M6 (DAZL in M6 was significantly higher than that in M3 (p = 0.01). No statistically significant difference was detected between the expression abundance in M6 and M0, in M6 and M12 and in M6 and M24 (p > 0.05)  . These rDAZL was evaluated in different tissue and testes with different size at M6, because of DAZL showing the highest expression level at this stage. The samples, including heart, liver, spleen, lung, kidney, longissimus dorsi and testis, in addition, three of the biggest (L) and smallest (S) testis from 340 Hu sheep at M6, were selected to compare DAZL expression. The result of DAZL expression in different tissues showed that DAZL expressed extremely high in testis, significantly higher than that in kidney .The mRNA expression level of n kidney A. The exis group B. HoweveAluI (A^GCT) and was genotyped by PCR-RFLP. Three genotypes of GG, GA and AA were observed in SNP6. Within SNP6 locus, the GG genotype showed a 159 bp band in agarose gel electrophoresis. Heterozygote GA genotype showed three bands of 159 bp, 92 bp and 67 bp. AA genotype showed two bands of 92 bp and 67 bp  were genotyped by iMLDR. All 5 SNPs were successfully genotyped and the genotypic and allele frequency, He, Ne, PIC and Hardy–Weinberg Equilibrium testing p < 0.05); SNP6 showed a strong significant association with VCEW (p < 0.01). The remaining SNPs had no significant effect on testis parameters  phenotypes were removed for further analysis. The results from Shapiro test indicated that all phenotypes have deviated from normality . In orderameters . Considerameters .DAZL was calculated by HAPLOVIEW software, as the results showed that all the SNPs were in strong linkage disequilibrium, excepting SNP2 unlinked with SNP1  and SNP6  (2 = 0.373). In addition, 5 haplotypes were combined. TTCGGG was the domain haplotype with the highest frequency of 0.537, CTTAAA was on the second with the frequency of 0.224, TTTAAG, TGCAAG and CTTAAG were with frequencies of 0.189, 0.027 and 0.014, respectively.The linkage disequilibrium among the 6 SNPs identified in = 0.009) . MoreoveIn addition, we also analyzed the genotype haplotypes of 6 SNPs. In total, 4 haplotypes  with the number greater than 12 were used for further analysis in this population . The resDAZL was evaluated in testis across five developmental stages, including M0 (neonate), M3 (puberty), M6 , M12 (body mature) and M24 (adult). The results revealed that DAZL showed the highest expression level in testis at M6, which is significantly higher than M3. Our result was in line with previous studies, which found DAZL was highly expressed in pubertal (1–1.5 year) and then declined in post-pubertal (2–3 years) and adult (4–8 years) testes of stallion [DAZL showed the highest expression level in the testes of Tibetan sheep at 12 months of age, which was inconsistent with the current study [DAZL in testes between 5-month-old and 12-month-old, at which Small Tail Han Sheep had not completely reach sexual maturity. The highest expression of DAZL in testis at six months in this study maybe consistent with the development of seminiferous tubules and spermatogenesis in the pubertal stage testis according to the study that reported DAZL only expressed in the cytoplasm of partial spermatogonia localized within seminiferous tubules at the pre-pubertal stage while in spermatogonia and primary spermatocyte at pubertal stage, in the cytoplasm of spermatogonia at post-pubertal stage [DAZL may mainly function in testicular development and play a key role in regulating spermatogenesis.In this study, the mRNA expression level of stallion . In contnt study . This dial stage ,23. ThisDAZL showed the highest expression level in testis among seven tissues. Meanwhile, large testes had more abundant expression than small testes at this stage though it was not significant. The results also indicated that DAZL has important roles in testis development from another point of view. The same results were also found in 12-month-old Small Tailed Han sheep, which revealed that DAZL was highly expressed in testis, slightly expressed in heart, while no expression was observed in liver, spleen, lung, kidney and muscle [DAZL has functioned in spermatogenesis [DAZL expression. These results may explain the function of DAZL in spermatogenesis indirectly, while the possible mechanism that how DAZL functioned in spermatogenesis still need further study.At the six-month stage, d muscle . The pred muscle ,24. Hencogenesis  that theDAZL was investigated in this study, six SNPs were found by DNA-pooling sequencing and genotyped by iMLDR and PCR-RFLP. Association analysis was performed between these six SNPs and 13 testicular parameters, finding that SNP1 and SNP6 have a significant association with VCEW. Epididymis where not only the mature of sperm, but also sperm transport, concentration, protection and storage happens, is also an important organ in male reproduction for its function in form vibrant and fertilized sperm [In ram breeding, individuals with large size testis and epididymis are preferred because testis size is positively correlated with ejaculated volume, sperm motility and sperm density and negatively associated with sperm abnormality ,5. Thereed sperm . The sized sperm . The varDAZL expression profiles were investigated in Hu sheep, revealing that DAZL expressed primarily in testis when compared to other tissues, and large testes had more abundant expression than small testes at six months. Additionally, the expression level of DAZL reached to a peak at six-month testis during testicular development. A total of six SNPs were found in DAZL which SNP1 and SNP6 had a significant effect on the variation coefficient between left and right epididymis weight. These findings revealed that DAZL is crucial during testicular development, and its SNPs which associated with testicular parameters can supply important indicators for ram selection at early stage.In this study, the