Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA. In mammals, after completing development in the testis, sperm continue to mature as they transit a long convoluted tube known as the epididymis. This process entails extensive molecular remodeling, with changes to the lipid composition of the sperm membrane, gain of key proteins, and a dramatic overhaul of sperm RNA contents. Here, to explore whether cytosine methylation patterns might also be edited during this process, we characterized cytosine methylation genome-wide at seven stages of sperm development in the testis and epididymis. Overall, we find remarkably stable methylation patterns throughout, supporting the classical view of mammalian cytosine methylation dynamics. In addition, while our data initially suggested a major methylation remodeling event in the caput epididymis and vas deferens, followup studies revealed that the aberrant methylation signature resulted from contamination by cell-free DNA. Curiously, this DNA was associated with citrullinated histones, suggesting that it might be generated by a cell death process akin to NETosis. While it will be interesting to determine whether epididymal cell-free DNA serves any biological function, the more immediate implication of our results is technical, as cell-free DNA could contaminate studies of the sperm epigenome, and so should be eliminated to improve the robustness of sperm methylation measurements. In addition to contributing a haploid genome to the next generation, germ cells also deliver epigenetic information to progeny that can impact early development and later phenotypes. In mammals, this is best characterized in the context of genomic imprinting, a situation where genes exhibit monoallelic expression from either the maternal or paternal allele. In the classic examples of imprinted gene regulation, the heritable marking of imprinting control regions relies on the covalently-modified cytosine derivative 5-methylcytosine –4. In maCytosine methylation patterns in mammals undergo two major reprogramming events; the first during primordial germ cell development, and the second occurring upon fertilization. In the zygote, sperm methylation is rapidly erased shortly after fertilization, apparently via active demethylation, while oocyte methylation patterns are lost more slowly via passive demethylation (replication without maintenance methylation). However, a small number of genomic loci escape this demethylation process, including imprinting control regions and a subset of evolutionarily-young repeat elements ,6. The mAlthough the majority of the sperm methylation landscape is erased upon fertilization, the existence of escaper loci suggests the possibility that environmentally-regulated changes to the sperm methylome could play a role in modulating phenotypes in the next generation. Indeed, several studies have documented changes to sperm cytosine methylation in response to various diets or toxin exposures –13, raisIntriguingly, an early study on sperm methylation suggested the surprising possibility of a third cycle of methylation reprogramming, occurring during post-testicular sperm maturation in the epididymis . Brieflyet al study, we therefore revisited cytosine methylation dynamics during post-testicular maturation, using the gold standard whole genome bisulfite sequencing (WGBS) to characterize the methylation landscape genome-wide, at single-nucleotide resolution, in seven germ cell populations from primary spermatocytes to vas deferens spermatozoa . We found remarkably consistent methylation profiles in five of the seven germ cell populations, with strong correlations between the various testicular germ cell populations and corpus and cauda epididymal sperm. This general persistence of methylation patterns was interrupted by caput epididymal sperm, which exhibited modest global hypomethylation accompanied by hypermethylation of germline-associated CpG islands. We ultimately identified cell-free DNA, presumably derived from somatic cells of the epididymis, as the cause of the unusual “caput sperm” methylome–most definitively, we show that treating caput or vas deferens sperm with DNase I prior to sperm lysis restored the aberrant sperm methylation landscape to the same pattern seen in all other sperm populations from testicular through cauda epididymal sperm. Cell-free DNA was associated with the citrullinated histone H3 (citH3) that is characteristic of arginine deimination by peptidylarginine deiminase (PAD) enzymes, and, curiously, was detected only in virgin males. Taken together, our data support a static view of methylation patterns stably persisting throughout post-testicular sperm development, and reveal an intriguing but transient stage of programmed cell-free DNA production in the male reproductive tract.Motivated by the Ariel Fig 1A). For each population we collected samples from seven different males, isolated and pooled genomic DNA, and prepared libraries for whole genome bisulfite sequencing (WGBS), obtaining an average of ~300 million reads per pool (S1 Table). The resulting data recapitulate well-described features of the sperm methylome [Figs 1C and S1), supporting the quality of our dataset.We set out to build on prior low-throughput studies documenting differences in cytosine methylation between testicular sperm and sperm obtained from various regions of the epididymis . To thisethylome –22, suchFig 1B and 1C). Intriguingly, we found somewhat lower global methylation in caput sperm and, to a lesser extent, in sperm from the vas deferens. Examination of metagenes revealed that these two sperm populations deviated from the characteristic methylation profile observed in the other five samples, with lower methylation levels across coding regions, along with subtly increased methylation at promoters .We next turned to the question of how the sperm methylome changes over the course of epididymal transit. Comparing all seven populations, we noted similar overall methylation profiles, with global methylation interrupted by hypomethylated CpG islands in all seven samples . Overall, we found that five germ cell populations exhibited nearly-identical methylation landscapes, with strong correlations between the methylation datasets for the three testicular samples, and for corpus and cauda sperm. Importantly, this means that the differences in methylation between round spermatids and caput sperm are reversed in corpus sperm, rather than these methylation changes being part of an ongoing process of progressive methylation maturation in the epididymis. In other words, the nearly-identical methylation profiles for testicular spermatocytes/spermatids and cauda sperm  indicates that the sperm methylome is essentially unchanged by the process of epididymal maturation, and strongly argues against a major role for the epididymis in modulating sperm methylation.To more systematically search for methylation changes between germ cell populations, we averaged methylation levels over 200 bp regions tiled across the genome and compared methylation levels in these tiles between all pairs of samples in our dataset . Overall, as noted above, caput sperm were slightly less methylated than these other sperm samples–examination of hypomethylated loci in caput sperm revealed diffuse hypomethylation over a wide range of both coding and intergenic genomic loci (S2 Table). This signature was also detectable in metagene averages in the highly-methylated regions distant from CpG islands, with caput sperm exhibiting a small global deficit at these loci .In contrast to the nearly-identical methylation profiles obtained from testicular and corpus/cauda sperm, comparisons between caput sperm and any of these samples revealed large-scale changes in methylation across hundreds of 200 bp tiles . Together, these analyses reveal extensive methylation changes at regulatory elements in caput sperm, primarily reflecting hypermethylation of CpG islands in this sample (dots above and to the left of x = y) with a relatively small number of hypomethylated islands in caput sperm. This is further illustrated in Fig 2B, which documents methylation dynamics for differentially-methylated CpG islands across all seven germ cell populations, highlighting the dramatic changes in CpG island methylation in caput sperm . Again, we note that regulatory elements that exhibit aberrant methylation in caput sperm universally return to the spermatocyte/spermatid methylation patterns in the corpus and cauda samples. In other words, whatever methylation changes apparently occur in caput sperm are soon reversed in later sections of the epididymis .In addition to the widespread hypomethylation of the caput sperm genome, a large group of hypermethylated tiles was readily apparent in these scatterplots. Examination of these tiles revealed that they are largely associated with CpG islands. To visualize this, we calculated the average methylation across all annotated CpG islands; S3 Fig). Hypomethylated islands in caput sperm are significantly enriched for a handful of processes related to neuronal function . However, these enrichments appear to be primarily driven by the fact that caput-hypomethylated CpG islands are found within transcribed regions–the hypomethylation at these loci presumably reflects the more general hypomethylation characteristic of caput sperm . More intriguingly, hypermethylated islands are enriched for a variety of annotations associated with meiosis- and sperm-specific functions, including piRNA biogenesis, ion signaling, and reproductive process.What biological pathways are affected by epididymal methylation dynamics? To address this question, we sought gene ontology categories enriched in genes associated with caput sperm hypo- and hyper-methylated CpG islands  by pyrosequencing of bisulfite-converted DNA. As shown in Fig 3A, methylation differences between caput and cauda were robustly reproducible in many additional, independent pairs of sperm samples. Moreover, the caput methylome proved to be quite stable over an animal’s lifespan–methylation at two target loci assayed remained abnormal in caput sperm obtained from ten month old animals .To validate our genome-wide dataset, and to develop a set of cost-effective targets for follow-up mechanistic studies, we analyzed methylation levels at a number of target loci (Methods). For all samples we routinely determined, based on the characteristic hook-shaped morphology of the murine sperm head, that our caput sperm preps were >99% free of somatic cell contamination.These data validate our overall dataset, and we focus on these target loci in the mechanistic follow-up studies described below. Importantly, although we considered the possibility that a signature of hypermethylation of germline regulatory elements might reflect contamination by somatic cells, we ensured that the methylation differences between caput and cauda sperm were robust to several sperm purification protocols including one based on detergent washing of caput epididymis luminal contents, and an alternative Percoll-based isolation of caput sperm in the absence of detergent treatment . However, none of the buffer conditions tested were able to substantially influence the caput sperm methylome.We therefore set out to test the hypothesis that sperm methylation dynamics result from “pre-loaded” genome-associated DNA modification enzymes  whose ongoing activity could be inhibited or activated by changes in either substrate levels  or buffer conditions (DNMT3a activity is highly pH-dependent ). To tesFig 3B). This was due to the caput methylation profile shifting to match that of cauda sperm; cauda sperm methylation was totally unaffected by the male’s mating status.During the course of these studies, we found one animal in which the methylation level at our target genes was nearly identical for caput and cauda sperm. Further investigation revealed that a female had accidentally been weaned into an otherwise all-male cage, suggesting that mating might influence the methylation dynamics described above. Indeed, direct testing of this hypothesis confirmed that methylation of our target genes was nearly identical in caput and cauda sperm obtained from animals who had successfully sired offspring . We next considered the possibility that caput sperm obtained from virgin animals represent an unusual first wave of spermatogenesis [Fig 3C), refuting the hypothesis that first wave sperm carry an unusual methylome.We considered and rejected two hypotheses regarding the change in methylation status of caput sperm in mated animals: 1) that conversion of the testicular sperm methylome to the unusual caput state might require extended incubation of newly-arrived sperm in the caput luminal environment, and 2) that the virgin caput methylation profile reflects sperm originating from the unusual first meiotic wave of spermatogenesis and somehow being captured in the caput epididymis. The first hypothesis was rejected based on the finding that the cauda-like methylation program in caput sperm from mated animals was stable for at least nine weeks following mating . These webs were not detected in cauda epididymal sperm preparations . To visualize this extracellular DNA in situ, we stained histological sections of several epididymal regions with DAPI. Intriguingly, in both caput sections and in the vas deferens we find a DAPI-staining rim associated with the apical region of the epithelium . DAPI-positive rims were not observed in the cauda epididymis, and were diminished or lost from the caput epididymis and vas deferens following mating . We cannot be certain that these DAPI rims are the source of the free DNA that contaminates caput sperm preps–the DAPI rims appear to coincide with the cell bodies of the epididymal epithelium –but it is notable that these rims were only observed in the same samples  that suffer from cell-free DNA contamination.What then could account for the unique methylation program observed in caput sperm from virginal males? Careful inspection of our genomic DNA preparations revealed that although testicular and cauda sperm samples were characterized by uniformly high molecular weight genomic DNA, there was a dim additional “cloud” of lower molecular weight DNA in the caput sperm preps. This suggested the possibility that caput sperm might be contaminated by cell-free DNA. Indeed, DAPI staining of caput sperm preparations revealed not only the expected hook-shaped sperm heads, but at higher exposure times we noted the presence of web-like DNA structures, often ensnaring multiple sperm , demonstrating that contaminating cell-free DNA present in the caput epididymis is responsible for the unusual caput methylome observed in virgins. To ensure that this finding was not idiosyncratic to the FVB/NJ strain background used throughout this study, we also carried out pyrosequencing in epididymal and vas deferens sperm samples obtained from C57Bl6/J males , again confirming 1) that caput and vas deferens sperm exhibited aberrant methylation at our target genes in this strain background, and 2) that this aberrant methylation profile was eliminated by DNase treatment of sperm preparations prior to sperm lysis and genomic DNA extraction.To definitively test whether extracellular DNA is responsible for the methylation profile of virginal caput sperm, we treated various sperm samples with DNase I to eliminate any extracellular DNA prior to extraction of genomic DNA for pyrosequencing analysis. Remarkably, DNase I treatment completely restored the caput sperm methylome to the methylation levels observed in testicular or cauda epididymal sperm (Discussion). We note that the cell-free DNA observed in our sperm preparations is reminiscent of extracellular DNA released by neutrophils, known as Neutrophil Extracellular Traps, or NETs [Padi2 in this tissue, largely confined to principal cells of the caput epididymis and vas deferens . Moreover, consistent with the caput cell-free DNA being produced downstream of PAD-dependent deimination, we found robust citH3 staining of our caput sperm preparations, with negligible signal in cauda sperm preps .Based on its impact on caput sperm methylation, it seems likely that the cell-free DNA originates from somatic cells rather than germ cells . Based on the methylation levels at imprinted regulatory elements, as well as changes in DNA yield following DNase I treatment of caput sperm prior to gDNA isolation, we roughly estimate that in our hands caput sperm genomic DNA is contaminated by ~20% somatic cells. Of course, this level of contamination by intact cells would readily be detectable by inspection of our caput sperm preps, emphasizing the fact that this contamination arises from cell-free DNA rather than intact somatic cells. That said, it remains plausible that the contaminating DNA comes from cells that lyse during dissection and sperm preparation, although it would be unusual for such DNA to be associated with citrullinated histones.Our study also revealed a surprising feature of the male reproductive tract, in which cell-free DNA is present in the caput epididymis and the vas deferens of virgin males. This DNA clearly derives from somatic cells rather than dying sperm, since the methylation profile of virgin caput sperm departs from the characteristic germline methylome to incorporate aspects more typical of somatic cells. This is apparent in the hypermethylation of key germline regulatory elements, which are unmethylated and active during spermatogenesis but methylated and repressed in most somatic tissues. This is also clear at imprinting control regions, which are either completely methylated or unmethylated in the sperm genome, but are 50% methylated in somatic cells–the contaminated caput methylome exhibits a shift towards 50% methylation at many imprinting control regions ; 3) our inability to detect substantial staining of the neutrophil marker myeloperoxidase in the caput cell-free DNA (not shown); and 4) the expression of Padi2 specifically in principal cells in our single-cell atlas of the epididymis [It is unclear which cells are responsible for production of cell-free DNA. Extracellular DNA traps are most famously produced by neutrophils during a process known as NETosis, but they have been reported in multiple inflammatory cell types ,34. In tididymis . AlthougPadi2 specifically in the two regions exhibiting cell-free DNA, and by the confirmed presence of abundant citH3 staining in caput sperm preps . This raises the question of what purpose extracellular DNA in the epididymal lumen might potentially serve. Extracellular DNA plays a key role in defense against pathogens in several contexts–NETs in the mammalian bloodstream entangle pathogens and aid in their engulfment by antigen-presenting cells [P. sativum) roots was shown to protect against fungal infections [Fig 3B). As infections would presumably be more likely in sexually-active animals than in virgins, we therefore disfavor this hypothesis, although it is plausible that the system serves to ensure the reproductive machinery is protected until a first mating is achieved. Alternatively, we speculate that the DNA webs observed here could ensnare sperm rather than pathogens, potentially playing a role in removal of early rounds of defective sperm, or masking sperm from inducing an immune response. Of course, the simplest hypothesis would be that the extracellular DNA documented here is a vestige of some earlier developmental process–death of epithelial cells during epididymal morphogenesis, for instance–and serves no biological function. That said, the use of PAD-dependent cell death would be unusual for typical programmed cell death in development.Whatever the cell of origin of the extracellular DNA here, it seems likely that it is produced via a PAD-dependent genome decondensation event analogous to the one that drives the production of Neutrophil Extracellular DNA Traps, or NETs . This hyng cells , while efections . While aS10 Fig), demonstrating that at least this observation is not an artifact of some transient early process in virgins. That said, other aspects of the sperm maturation process may differ between virgins and mated males, emphasizing the importance for reproduction studies to explicitly state the mating status of males being used.Although the function, if any, served by extracellular DNA in the epididymis is unclear, the more pressing implication of our findings is that they raise two important technical considerations for reproductive epigenetics studies. First, one important finding here is that at least some aspects of sperm populations are different between virgins and mated males, raising the question of whether other features of the epididymal sperm epigenome–often assayed in virgin males–are stable throughout reproductive life. For example, we and others have previously documented substantial differences between the small RNAs carried by caput and cauda epididymal sperm, with a variety of genomically-clustered microRNAs, and a subset of tRNA fragments such as tRF-Val-CAC, being far more abundant in cauda sperm than in caput –17. We tSecondly, our work has important technical implications for studies of the sperm methylome, as future efforts focused on cytosine methylation in sperm must contend with the possibility of contamination by cell-free DNA. Although most such studies do not utilize caput epididymal sperm, it is well known that different labs differ in whether “mature sperm” used for molecular studies or IVF are isolated only from the cauda epididymis, or from a mixture of cauda and vas deferens. Given our findings here with vas deferens sperm, it is clear that dissections that capture variable lengths of the vas deferens will result in variability in sperm methylation due to the contamination of these samples by DNA with a somatic cell methylation program. In addition, although we found contaminating DNA specifically in the caput and vas of virgins, it is plausible that cell-free DNA could be more persistent or produced in different parts of the epididymis in other strain backgrounds or in males subject to different diets or stressors. A simple solution to this issue in future studies would be to treat all sperm samples with DNase I prior to genomic DNA isolation for methylation analyses.Animal husbandry and experimentation was reviewed, approved, and monitored under the University of Massachusetts Medical School Institutional Animal Care and Use Committee (Protocol ID: A-1833-18).Unless otherwise specified, tissues were obtained from 10–12 week old male FVB/NJ or C57Bl6/J mice.FVB mice, euthanized at 10–12 weeks of age (unless otherwise noted) according to IACUC protocol, were dissected into four segments that roughly corresponded to caput, corpus, cauda, and vas deferens. Caput, corpus, and cauda epididymis as well as vas deferens were placed into Donners complete media  and tissSperm collection from the caput epididymis is performed as described above, but without somatic cell lysis. Instead, caput sperm are purified using a Percoll gradient as described  to separ2HPO4, 0.6 mM KH2PO4, 0.23% Sodium DL-Lactate, 0.1% Glucose, 0.1% BSA, 0.011% Sodium Pyruvate, 1.2 mM MgSO4 and 1.2 mM CaCl2) containing 25 μg/ml liberase (Roche Diagnostics GmbH) for 30 min at 37°C with gentle agitation every 5 min. The cell suspension was mixed by pipetting 20 times with a 10-ml plastic pipette. After homogenization by pipetting 10 times through a P1000 pipette, the single cell suspension was filtered twice through a 40-μm cell strainer (Fisher Scientific) on ice and centrifuged at 1500 rpm at 4°C for 10 min, and then the pellet was resuspended with 20 ml elutriation buffer. Separation of testis cell populations was performed by centrifugal elutriation using a JE-5.0 elutriation system and a 4-ml standard elutriation chamber (Beckman Coulter). The assembly of the system followed the manufacturer’s instruction. The precise elutriation conditions are as follows: Fractions 1–3 were run at 3000 rpm and fractions 4–5 were run at 2000 rpm. The flow rate was 14, 18, 31, 23, and 40 ml/min for fractions 1–5, respectively. During elutriation, the elutriation chamber was maintained at 4°C and the cells were collected into 50-ml conical polypropylene tubes that were packed on ice. Cells in tubes of fractions 3 to 5 were pelleted by centrifugation at 1500 rpm at 4°C for 10 min. All pellets from the same fraction were combined and resuspended in 200 μl of elutriation buffer. Percoll (Sigma-Aldrich) gradient (23–35%) was prepared by using a Gradient Master 108 (Biocomp) following standard program: S1/1, 2:26 (time), 82.0 (angle), 13 (rpm). After loading the cell suspension, centrifugation was carried out with SW40Ti Rotor (Beckman Coulter) at 11,000 rpm at 4°C for 15 min. The cells were then collected in 15-ml conical polypropylene tubes and pelleted by centrifugation at 1500 rpm at 4°C for 15 min. The cell pellets were stored at -80°C and ready for DNA extraction. Based on cell morphology and small RNA data, Fraction 5 was determined to correspond to primary spermatocytes, with fractions 4 and 3 being relatively early and late round spermatids, respectively.For each isolation, two testes were acquired from one FVB/NJ mouse at 10–12 weeks of age. Cell suspension was prepared by incubating the testes without their tunica albuginea in 5 ml elutriation buffer  with 5 μL RNase A (Qiagen 19101) for 2 hours after which samples were treated with 5 μL Proteinase K (Qiagen 19131) overnight. Following phenol chloroform extraction (UltraPure Phenol:Chloroform:Isoamyl Alcohol ThermoFisher 15593031) using phase lock gel tubes (Quantabio 10847–802), aqueous phase was transferred to a fresh tube containing 1.5 μL glycogen (20 mg/mL), 1.3 μL 5 M NaCl, and 100% EtOH. Genomic DNA integrity was determined using NanoDrop and quantified for downstream applications using Qubit (DNA BR)750 μL Extraction Buffer  was added to sperm pellets and incubated for 2 hours at 56 For each population we collected sperm samples from seven different males, isolated genomic DNA, and prepared libraries for whole genome bisulfite sequencing (WGBS); Bisulfite converted DNA was prepared according using the EZ DNA Methylation-Lightning Kit (Zymo D5030). Library construction was performed using Accel-NGS Methyl-Seq DNA Library Kit (Swift) according to the manufacturer’s instructions. 30ng bisulfite converted DNA was used and barcoded following the (Swift) manufacturer’s instructions including 6 rounds of PCR. All final libraries were analyzed by Fragment Analyzer and quantitated using the Qubit as well as the KAPA Library Quantitation qPCR kit.www.bioinformatics.babraham.ac.uk/projects/seqmonk/). See S1 Table for summary statistics of read counts, mapping and duplication rates, and bisulfite conversion efficiency. Analyses in Figs 1, S2 were carried out using evenly-spaced 200 bp tiles, using only tiles with at least 1 CpG and at least 10 methylation-informative reads in every one of the seven datasets. For analyses in Figs 2B and S3, each CpG island was assigned to the nearest gene based on proximity to the transcription start site (within 50 kb).Sequences were trimmed with Trim Galore —Swift libraries were trimmed by 10 bp from their 5’ ends for both R1 and R2. The non-CG methylation levels were consistently very low (~0.2%) indicating good bisulfite conversion rates. The resulting trimmed sequences were mapped to the mouse GRCm38 genome using Bismark  (v0.17.0S4 Table). Primers (IDT) were designed using the PyroMark Assay Design software (Qiagen).Pyrosequencing was performed using the PyroMark Q24 (Qiagen: 9001514) according to the manufacturer’s instructions. Bisulfite converted DNA was prepared using the EZ DNA Methylation-Lightning Kit (Zymo D5030). PCR was performed with 10–20 ng of bisulfite-converted material for each locus of interest (DL-Lactate (1% vol/vol), NaHCO3 (200mM) as well as Donners complete buffered to pH 6.5, 7, and 7.4. Additional conditions include: Donners basic (stock solution) [3 , Donners basic supplemented with BSA , Donners basic supplemented with Sodium DL-Lactate , and finally Donners basic buffered to pH 6.5, 7, and 7.4.Prior to genomic DNA extraction, sperm were incubated for 4 hours at 37°C in the following conditions: Donners complete media [Genomic DNA isolation).DNase treatment of caput sperm was performed prior to gDNA isolation as per manufacturer’s instructions (Qiagen79254). After DNase treatment, sperm were spun for 30 sec to (14000 rpm), supernatant aspirated, and sperm samples processed for genomic DNA extraction , fixed for 10 minutes in 4% paraformaldehyde solution, washed 3x with PBS, and permeabilized for 10 min with 0.1% triton in PBS. Blocking was performed with 10% BSA in PBS for 1 hour at room temperature in a humidified chamber. Staining with primary antibody, Anti-Histone H3 (citrulline R2 + R8 + R17 (Abcam ab5103), was performed for 2 hours at room temperature in 1% BSA PBSt at a concentration of 1:250 in a humidified chamber. Primary antibody was decanted and slides washed three times with PBS for 5 minutes each. Goat anti-Rabbit 488 secondary antibody (Invitrogen A11008) was performed for 1 hour at room temperature at a concentration of 1:500 in a humidified chamber. Secondary antibody was decanted and slides washed three times with PBS for 5 minutes each followed by VECTASHIELD Antifade Mounting Medium with DAPI (Vector Labs H-1200), sealing coverslip, and imaging on the Zeiss Axioskop 2 Plus. All studies were repeated with at least two distinct sperm samples. Exposures for different sperm preps were captured using identical light intensity and exposure times, set to enable visualization of cell-free DNA in DAPI images, and set just below saturation for cit-H3 images. Note that secondary alone controls for cit-H3 were clean (not shown).Virgin or retired breeders of approximately the same age were anesthetized and perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA)/PBS. Epididymides were explanted and further incubated in 4%PFA/PBS at 4°C overnight. After washing the excess of PFA with PBS, the sample was incubated at 4°C in 30% sucrose, 0.002% sodium azide in PBS until organ sank to the bottom of the tube. The sucrose solution was replaced and once organ remained at the bottom of the tube the same volume of optimal cutting temperature compound (OCT) was added to the vial and kept ON at 4C under agitation. Samples were then mounted in OCT and frozen at −80°C until sectioning. Sectioning was done at a thickness of 5 μm by the UMASS morphology core. Slides were stored frozen at -20°C.S8 Fig shows characteristic images for a range of exposure times to illustrate the presence of a DAPI rim in virgin caput samples, but not mated caput or cauda epididymis and show that this rim is not simply an artifact of different exposure times used for different samples.Slides were placed at a 37°C warm plate for 10 minutes to ensure proper attachment of the section to the slide, then washed three times for 5 minutes in PBS 0.02% tween 20 (PBS-T) to remove OCT, followed by DAPI stain  for 5 min. After washing off the excess of DAPI with PBS-T, slides were mounted with ProLong gold Antifade (Thermofisher P36930) and imaged the following day. Males from the same litter were split into the following groups: not mated, mated and recovered for three weeks, and mated and recovered for six weeks. Mating was confirmed by the formation of blastocysts in culture. All males were dissected at the same age (14 weeks). Caput and cauda sperm were isolated as described and small RNA sequencing was performed. Isolation of 18–40 nts small RNAs was carried out as previously described . Size seS1 TableDeep sequencing statistics for WGBS datasets used in this manuscript.(XLSX)Click here for additional data file.S2 TableRows show genomic coordinates, nearest genes (within 50 kb), and methylation levels in all seven sperm populations for all 200 bp tiles that are at least 20% hypo- or hyper-methylated in caput sperm compared to cauda sperm.(XLSX)Click here for additional data file.S3 TableRows show methylation levels and location of nearest gene for annotated CpG islands.(XLSX)Click here for additional data file.S4 TableFigs 4.Sequences for pyrosequencing primers used in (XLSX)Click here for additional data file.S1 Figet al [Scatterplot shows methylation levels for 200 bp windows tiled across chromosome 1, comparing data for cauda sperm from Wang (TIF)Click here for additional data file.S2 FigFig 1D and 1E, for all pairwise comparisons in this dataset.Scatterplots are shown as in (TIF)Click here for additional data file.S3 Fig10) for selected categories enriched among hyper (red) and hypo (green) methylated CpG islands.For all CpG islands exhibiting >20% methylation differences between caput and cauda sperm, nearest genes were identified and enriched gene ontology categories were identified using Funcassociate . Bar plo(TIF)Click here for additional data file.S4 FigFig 3A). For all five loci, pyrosequencing qualitatively confirmed the methylation trends observed in WGBS; while there was some quantitative disagreement (eg 83% vs 42% for methylation at Tdrd12 in caput sperm), we note that values inferred for small numbers (~4–5) of CpGs in the WGBS dataset are expected to be somewhat noisy given the relatively low sequencing depth. B) As in panel (A), but for selected imprinted genes. Notably, while testicular germ cells and corpus and cauda sperm exhibit the expected 0% or 100% methylation, our caput sperm (and to a lesser extent vas deferens sperm) data are closer to the 50% methylation expected of somatic cells. C) Scatterplot comparing methylation levels at imprinting control regions , while Ariel et al reported minimal methylation at this site in caput sperm along with higher methylation in cauda sperm. There are countless potential technical differences that might account for this, including strain differences (CD1 vs FVB and C57), mating status and age , sperm preparation , or genomic DNA extraction , and of course the methylation readout .A) WGBS data for genomic loci selected for targeted follow-up. At each locus, red and blue dashes represent individual reads for a given CpG, with red and blue showing methylated or unmethylated reads respectively. Top panels show a wider view of the genomic context, with boxes indicating regions shown in the zoom-in bottom panels. For these five regions, methylation levels obtained from the WGBS dataset are shown for caput and cauda sperm samples, along with methylation levels obtained in followup pyrosequencing validation Click here for additional data file.S5 FigPyrosequencing data for the two indicated target loci in four unmated males at ten months of age, confirming that the caput methylome is stable throughout a typical male’s lifespan.(TIF)Click here for additional data file.S6 Fig3, sodium pyruvate, sodium DL-Lactate, or adjusted to pH 6.5, 7.0, or 7.4 (red triangles). Although not indicated in the figure, cauda sperm samples (right) included samples subject to most of the buffer incubations used for caput sperm samples, none of which affected methylation in these samples.Pyrosequencing data for caput and cauda sperm samples. Caput sperm samples were either processed for genomic DNA shortly after isolation, or were incubated for four hours at 37°C in various buffer conditions, as indicated. Buffer conditions were based on either Donners Basic (DB) or Donners Complete (DC), and were supplemented with various levels of NaHCO(TIF)Click here for additional data file.S7 FigFig 4A) but not cauda sperm. In the case of cauda sperm, DAPI staining was completely confined to sperm heads, and no webs of DNA were observed even at the highest exposures. Also notable here is the high purity of our sperm samples, with no round DAPI-stained nuclei contaminating the field of sperm heads in any of the four samples. These representative samples illustrate the >99% purity of our sperm preparations that we routinely ensure by microscopic examination prior to processing for molecular studies.For all samples, left panels show DIC images and right panel shows DAPI staining. Panels show mock-treated and DNase-treated caput sperm (A) and cauda sperm (B), revealing extensive DNase-sensitive background “fog” and fibrous tangles in the mock-treated caput sperm Click here for additional data file.S8 FigA) Images of DAPI-stained epididymis sections from the indicated samples: virgin caput epididymis (3 samples shown), mated caput, virgin cauda, and mated cauda. Top five rows are images taken from C57Bl6/J animals (at 25 weeks of age), where the “rim” of DAPI in the caput epididymis is more prominent, while bottom row shows a typical FVB caput epididymis (10 weeks of age) with a noticeable but dimmer DAPI rim. In each row, five exposures are shown from lowest (left) to highest (right) exposure to show that the virgin caput DAPI rim is not an artifact of mismatched exposures. B) Higher-resolution images are reproduced from the highest exposures from panel (A), as indicated. These images emphasize the presence of a DAPI-stained rim (examples highlighted with yellow brackets) in virgin but not mated caput epididymis. Although the presence of the DAPI rim corresponds to anatomical regions where sperm preparations are contaminated by cell-free DNA–virgin caput epididymis and vas deferens, but not cauda or mated caput–the physical nature of this rim is unclear. We note the presence of occasional nuclei within this rim in locations presumably corresponding to apically-located clear cell nuclei, indicating that the DAPI rim largely colocalizes with the cell bodies of the caput epididymal epithelium. Yet the absence of this DAPI rim in the caput epididymis of mated animals shows that this staining is not nonspecific background staining or autofluorescence. Further defining the nature and subcellular localization of this material will likely require super-resolution imaging and additional DNA staining approaches.(TIF)Click here for additional data file.S9 FigFig 4C. B) Data for the indicated samples obtained from 10–14 week old C57 males. Data here are shown for samples either mock-treated (left samples) or DNase-treated  prior to genomic DNA extraction. See also Fig 4D.A) Pyrosequencing data show methylation at the three indicated target loci in testicular spermatozoa, caput, corpus, and cauda epididymal sperm, and vas deferens sperm, as indicated. All samples were obtained from 10–14 week old FVB males, and sperm were DNase-treated prior to genomic DNA isolation. In all cases DNase treatment completely eliminated methylation differences between the various samples. See also (TIF)Click here for additional data file.S10 Fig2(Cauda+1)/(Caput+1)) for virgin males (x axis) compared to males 6 weeks after mating (y axis). Overall enrichments for small RNAs in cauda or caput sperm remain highly correlated after mating. D) Example of cauda-enriched microRNAs from the X-linked miR-465 and miR-880 clusters. Enrichment in cauda sperm, and absence from caput sperm, was unaffected by mating status.Several previous studies have documented significant differences between the RNA payload of caput and cauda sperm, including dramatically lower levels of genomically-clustered microRNAs in caput sperm relative to both testicular and cauda sperm –17. To d(TIF)Click here for additional data file.


SARS-CoV-2is a health threat with dire socioeconomical consequences.As the crucial mediator of infection, the viral glycosylated spikeprotein (S) has attracted the most attention and is at the centerof efforts to develop therapeutics and diagnostics. Herein, we usean original decomposition approach to identify energetically uncoupledsubstructures as antibody binding sites on the fully glycosylatedS. Crucially, all that is required are unbiased MD simulations; noprior knowledge of binding properties or ad hoc parameter combinationsis needed. Our results are validated by experimentally confirmed structuresof S in complex with anti- or nanobodies. We identify poorly coupledsubdomains that are poised to host  epitopes and potentiallyinvolved in large functional conformational transitions. Moreover,we detect two distinct behaviors for glycans: those with strongerenergetic coupling are structurally relevant and protect underlyingpeptidic epitopes, and those with weaker coupling could themselvesbe prone to antibody recognition. This is a characteristicthat it shares with other members of the Coronaviridae family, allof which are characterized by a highly selective tropism that determinesthe onset of a variety of diseases in domestic and wild animals aswell as in humans, including central nervous system affections, hepatitis,and respiratory syndromes.tein (S) 1 is the 11 and the structural basis for the recognition of thespike protein’s receptor binding domain (RBD)  analyses allowedprecise determination of the structure of the full-length spike proteinin its trimeric formin (RBD) 1 by the 23 This would be a crucial first step in the selection and optimizationof candidate vaccines and therapeutic Abs  as well as in the development of additional serologicaldiagnostic tools.This detailed dynamic and structural knowledgecan set the stagefor understanding the molecular bases of S protein recognition bythe host’s immune system, providing information on which physicochemicaldeterminants are required to elicit functional antibodies (Abs). Suchunderstanding can then be exploited to design and engineer improvedantigens based on S, for instance by identifying antigenic domainsthat can be expressed in isolation or short sequences (epitopes) thatcan be mimicked by synthetic peptides.Even more significantly, knowledge acquiredtoday about such recognitionmechanisms could well mean that we are better prepared to tackle similarpandemics in the future by contrasting them more efficiently throughthe application of the same efficient and well-tested methods to newprotein variants. More specifically, upon emergence of a new pathogen,generally portable computational methods could be advantageously exploitedto rapidly identify and synthesize recombinant antigen- or peptide-basedvaccines.24 in order to predict immunogenic regions. To this end, a simple abinitio epitope prediction method that we previously developed forunmodified proteins29 is optimized and extended to cover glycoproteins. The method isbased on the idea that Ab recognition sites (epitopes) may correspondto localized regions that exhibit only low-intensity energetic couplingwith the rest of the structure. Otherwise put, putative interactingpatches are hypothesized to be characterized by nonoptimized intramolecularinteractions with the remainder of the protein. Actual binding toan external partner such as an Ab is expected to occur if favorableintermolecular interactions determine a lower free energy for thebound state than for the unbound state.30 Furthermore, minimal energetic coupling with therest of the protein provides these subregions with greater conformationalfreedom to adapt to and be recognized by a binding partner29 as well as improved tolerance to mutations at minimal energeticexpense without affecting the protein’s native organizationand stability in a way that could be detrimental for the pathogen.All of these properties are indeed hallmarks of Ab-binding epitopes.Here we analyze representative 3D conformations ofthe full-lengthtrimeric S protein in its fully glycosylated form 1, extracWe show that our approach is indeed able to identify regions—alsocomprising carbohydrates—that recent structural immunologystudies have shown to be effectively targeted by Abs. On the samebasis, our method predicts several additional potential immunogenicregions (currently still unexplored) that can then be used to generateoptimized antigens, either in the form of recombinant isolated domainsor as synthetic peptide epitopes. Finally, our results help shed lighton the mechanistic bases of the large conformational changes underpinningbiologically relevant functions of the protein.To the bestof our knowledge, this approach is one of the firstthat permits the discovery of epitopes in the presence of glycosylation(an aspect that is often overlooked) starting only from an analysisof the physicochemical properties of the isolated antigen in solution.Importantly, the approach does not require any prior knowledge ofAb binding sites of related antigenic homologues and does not needto be trained/tuned with data sets or ad hoc combinations of informationon sequences, structures, solvent-accessible surface area (SASA),or geometric descriptors. The procedure is thus immediately and fullyportable to other antigens.39 and discuss in full in To reveal the regions of the S proteinthat could be involved inAb binding, we employ a combination of the energy decomposition (ED)and matrix of low coupling energies (MLCE) methods, which we previouslyintroduced and validated40 (built from PDB entry 6VSB(41 which for a protomer composed of N residues (including monosaccharide residues on glycans) gives asymmetric N × N interactionmatrix M. Eigenvalue decomposition of M highlightsthe regions of strongest and weakest coupling. The map of pairwiseenergy couplings can then be filtered with topological information to identify localizednetworks of low-intensity coupling .Startingfrom six combined 400 ns replicas of atomistic MD simulationsof the fully glycosylated S protein in solutionntry 6VSB), we isoIn our model, when these fragments are located on or near the surface,contiguous in space and weakly coupled to the protein’s “stabilitycore”, they represent potential interaction regions .i, j) are identified by cross-comparison with theresidue–residuecontact map  regions for Ab binding through their isolatedparent domains may prove more advantageous in developing new immunogens.32The less restrictive 15% cutoff subdivides the full-length, fullyfolded S protein into potentially immunoreactive domains see .26,30,32The more stringent epitope definition (5% cutoff) narrowsthe focuson those  intradomain regions that could be directly involvedin forming the interface with Abs, which can then be used to guidethe engineering of optimized antigens in the form of synthetic epitopepeptidomimetics. In this context, to be defined as epitopes, the energeticallyuncoupled regions must be at least six residues long.42 (see PDB entry 6W41), known to targeta cryptic epitope that is exposed only upon significant structuralrearrangement of the protein12  (Fab(s)) of COV57, a novel Ab whose neutralizing activityshowed no correlation with that of RBD-targeting Abs44 1B,D. We 46 an Ab that was recently shown to cross-react with S proteinsof human, civet, and bat coronaviruses. This analysis also recognizesa potential antigenic region in a carbohydrate cluster located inthe S2 domain of the protein. Intriguingly, recent findings indicatethat an oligosaccharide-containing epitope centered around this predictedregion is targeted by the glycan-dependent antibody HIV-1 bnAb 2G1247 N-terminal region (42). Indeed, usingcocktails of antibodies to target different regions of S has recentlybeen proposed as a viable therapeutic option,43 and Ab cocktails have been successfully used in the treatment ofother epidemics such as Ebola. In this context, the company Regeneronis pursuing a cocktail-type approach for SARS-CoV-2 that is alreadyin clinical trials.49Overall, these findings support the validityof our approach inidentifying protein domains that can be aptly used as highly reactiveimmunogens, as they are most likely to be targeted by a humoral immuneresponse. Our analysis predicts that regions other than the S proteinRBD may represent alternative targets for neutralization or functionalperturbation of SARS-CoV-2. On the one hand, this may be importantin view of the fact that the RBD can also be the target of non-neutralizingantibodies ,we next focus on those regions of the S antigen that can be involvedin forming contacts with antibodies.44 S309 (PDB entries 6WPT and 6WPS),50 and AB23 (PDB entry 7BYR);51 with thenanobody H11-D4 (PDB entry 6Z43); and with a recently reported synthetic nanobody(PDB entry 7C8V) A-(352)A-(375)S-(434)IAWNS(438)-(442)DSKVGG(447)-(449)YNYL(452)-(459)S-(465)E-(491)PLQS(494)-(496)Q-(507)PYR(509)encompasses regions of the S protein in contact with the antibodiesC105 (PDB entry 6XCN),ry 7C8V) 3.50 with aminoacid sequence (332)ITNLC(336)-(361)C and with the (N334-linked) fucosylated N-glycan chitobiose core (Manβ1–4GlcNAcβ1–4[Fucα1–6]GlcNAcβ-Asn).52 This predicted region sits notably close tothe RBD interaction surface with ACE2.Interestingly, an additionalpredicted patch comprising a set ofdecorating carbohydrates is correctly predicted to be part of theinterface with antibody S309,42 (Antibody EY6A (PDB entry 6ZDH) binds the RBD inthe region of the cryptic epitopedescribed by Wilson and collaborators42 4. Import42 .43 GN(185)-(242)LAL(244)-(246)R-(248)Y-(258)WTAGA(262)contains residues R246 and W258, which were described as central determinantsfor contact between the N-terminal domain and antibody 4A843 5.46 Specifically, our identified reactive sequence is the following:(1076)TTAPAICH(1083)-(1087)A-(1092)REG(1094)-(1096)FVSNGHWFVTQRN(1108)-(1112)P-(1114)I-(1116)T-(1118)DN(1119)-(1126)C-(1129)V-(1132)IVNNTVYDPLQPELD(1146).Finally, the restrictive predictionidentifies the sequence spanningresidues 1076–1146, which includes amino acids 1111–1130,experimentally identified as the epitope for mAb 1A9.In general, our approach is able to identify potential immunoreactivedomains and epitopes of the spike protein on the basis of only structuraland energetic information: our approach correctly predicts 20–80%of the amino acids engaged by mAbs in reported X-ray structures. Asdifferent (combinations of) antigen residues may be shared among differentantibodies in a polyclonal response (such as the one taking placein the host organism), capturing even the minimal sequence endowedwith potential immunoreactivity can aptly represent a useful steptoward designing molecules that can elicit Abs capable of interferingwith viral entry or replication.In this framework, sequencespredicted to be reactive using therestrictive epitope definition (5% cutoff) can be used to generateoptimized antigens in the form of synthetic peptide epitopes. Engineeringsuch epitopes would entail the synthesis of conformationally preorganizedpeptidomimetics of the “natural” reactive regions—withintra- and extracellular stability enhanced through, e.g, a combinationof natural and non-natural amino acids—that could reproducethe main structural and energetic conditions required to elicit ahumoral immune response and thus constitute candidates for vaccinedevelopment. Furthermore, reactive peptides thus identified may besuitable for use as baits in serologic diagnostic applications  to capture and detect not only circulatingantibodies that are expressed in response to SARS-CoV-2 infectionsbut also those that are endowed with neutralization activity, thuspotentially predicting the infection outcome. As a further application,these peptide-based baits can represent a useful tool for isolatingnew mAbs and screening small molecules for drug development.One of the most significant aspects of our approach is that theS protein’s entire glycan shield is explicitly taken into accountin the prediction of the immunoreactive regions. Indeed, the variousoligosaccharide chains appear to behave differently shielding carbohydrates. In light of this, it is reasonable to suggestthat the protective effect of these particular carbohydrates may becircumvented and neutralized by exposing the underlying peptidic substructures.Furthermore, information on oligosaccharides identified as epitopeconstituents can be exploited to design glycomimics or glycosylatedpeptides as synthetic epitopes.The latter aspect is indeedparticularly relevant: small syntheticmolecules that mimic antigenic determinants  offer enticing opportunities to developimmunoreagents with superior characteristics in terms of ease of handling,reproducibility of batch-to-batch production, ease of purification,sustainable cost, and better stability under a variety of conditions.Furthermore, production of these molecules greatly reduces the riskof cross-reactivity with any copurified antigens, which is insteadrife when dealing with recombinant proteins. In contrast to smallerpeptides or sugar-decorated peptidomimetics, a full-length recombinantantigenic protein (or any protein-based detection device) would typicallyrequire more stringent conditions  for storage, transport, and management in order to preservethe protein in its properly folded active form. The same would betrue for other vaccinal solutions such as deactivated pathogens.Overall, our work confirms how simple and transparent structuraland physicochemical understanding of the molecule that is the keyplayer in SARS-CoV-2 viral infection can be harnessed to guide theprediction of  regions thatare involved in immune recognition and to understand its molecularbases. Agreement with experiment confirms that knowledge generatedin the process has the potential to be translated into new moleculesfor vaccine and diagnostic development. In this context, we have alsoidentified potentially reactive regions in the S protein stalk thatare currently under experimental synthesis and testing.Furthermore,potential functional implications offered by the approachare illustrated by the fact that domains/regions that are relevantfor the protein’s biological activation are naturally identified.This renders the approach well-suited to identify subtle functionalvariations in possible mutants of the S proteins that are expectedto emerge as a result of further viral diffusion and host adaptation.Finally, the possibility of accurately partitioning such a complexsystem in functional subunits could aptly be exploited in the parametrizationof coarse-grained models to simulate the system at longer time scales.This kind of structure-based computational approach can clearlyexpand the scope of simple structural analysis and molecular simulations.In applicative terms, generation of synthetic libraries based on predicted/identifiedepitopes (with possible addition of sugars) would definitely boostselection and screening of antigens for vaccine development.13 based on PDB entry 6VSB. Throughout this work, we retained exactly the same force fieldparameters used by Woods et al. in their MD simulations: all residuesexcept glycosylated asparagines were treated using the ff14SB forcefield,54 whereas glycans and glycosylatedasparagines were modeled using the GLYCAM_06j force field.52Coordinates of the fully glycosylated SARS-CoV-2 S protein’s“RBD up” protomer featured in this work originated fromMD simulations by Woods and co-workerscpptraj utility in AmberTools (version 17)55 after all sixindependent MD replicas13 were concatenatedand then aligned with the “autoimage” command. The chosenmethod was the hierarchical agglomerative algorithm56 with ε = 0.5. From each of the three most populatedclusters, we isolated one representative frame, from which we retainedthe “RBD up” protomer and its glycans and again used cpptraj to discard all solvent molecules and ions and thetwo “RBD down” protomers. All subsequent calculationson these three “RBD up” protomer models are listed chronologicallyin what follows.Clustering was based on the root-mean-squaredeviation of Cαatoms of the RBD domain in the “RBD up” protomer andperformed with the sander in the AMBER software package (version 18).55 The protomers were minimized using the generalizedBorn implicit solvent model as parametrized by Onufriev et al.,57 with a universal 12.0 Å cutoff appliedin the calculation of Lennard-Jones and Coulomb interactions . For this stage, the concentrationof (implicit) mobile counterions in the GB model was set to 0.1 M,and the SASA was computed according to the linear combinations ofpairwise overlaps (LCPO) method.58A 200-step minimization of each of the threeprotomer models wascarried out using the default procedure  implemented in the MD engine 41 were performedon each of the three minimized “RBD up” protomers usingthe dedicated mm_pbsa.pl utility in AmberTools (version17). The purpose of these calculations was to obtain a breakdown ofnonbonded energy interactions  between every possible pair of residues in the protomer; for a protomer composed of N residues, this leads to a symmetric N × N interaction matrix M.60MM/GBSA calculationsThe implicit GB solvationmodel used in these calculations wasidentical to the one used in the preceding minimization step (videsupra), except that the implicit ion concentration was set to 0.0M and the SASA was computed with the ICOSA method (based on icosahedraaround each atom that are progressively refined to spheres).Mij ofthe symmetric interaction matrix M obtained from separateMM/GBSA calculations (vide supra) on each of the three S protein protomermodels under study (with N residues) can be expressedin terms of the eigenvalues and eigenvectors of M asα isthe αtheigenvalue and viα is the ith component of the corresponding eigenvector.The elements v1 , which is associatedwith the lowest eigenvalue λ1, allows the identificationof most of the crucial amino acids necessaryfor the stabilization of a protein fold and consequently those aminoacids that are minimally coupled to such a core. The latter were shownto correspond to potential interaction regions.It was previouslyshown in a number of cases that eigenvector M was thus decomposed instead viathe alternative approach developed by Genoni et al.30 In that scenario, the aim is to select the smallest setof Ne eigenvectors that cover the largestpart of residues  with the minimum redundancy underthe assumptions that (a) for each domain there should exist only oneassociated eigenvector recapitulating its most significant interactions;(b) each “domain eigenvector” has a block structure,whereby its significant components correspond to the residues belongingto the identified domain; and (c) combination of all of the significantblocks covers all of the residues in the protein. Matrix M can thus be reformulated as a simplified matrix M̃ with elements M̃ij given byNe essential eigenvectors insteadof N residues. As detailed by Genoni et al.,30 the essential folding matrix M̃ is subsequentlyfurther filtered through a symbolization process to emphasize thesignificant nonbonded interactions, yielding the matrix M̃S, which is finally subjected to a proper clustering procedureleading to domain identification. The final simplified matrix M̃S resulting from domain decomposition thusreports only those residue pairs in the protomer that exhibit thestrongest and weakest energetic interactions.In the caseof multidomain proteins such as S, the first eigenvectoris not sufficient, and additional eigenvectors are needed to capturethe essential interactions for folding/stability and binding. Theinteraction matrix 27 This approach allows the identification of nonoptimized contiguousregions on the protein surface that are deemed to have minimal couplingenergies with the rest of the structure and thus have a greater propensityfor recognition by Abs or other binding partners.Final epitopepredictions were made using the MLCE method, in whichanalysis of a given protein’s energetic properties is combinedwith that of its structural determinants.M̃S resulting from domain decomposition (vide supra) witha pairwise residue–residue contact matrix C. Thelatter matrix considers a pair of residues to be spatially contiguous if they are closer than an arbitrary6.0 Å threshold; contact distances are measured between Cβatoms in the case of non-glycine amino acid residues, H atoms in thecase of glycine residues, and C1 atoms in the case of glycan residues.The MLCE procedure entails cross-comparison of the simplifiedpairwiseresidue–residue energy interaction matrix M̃S and C yields the matrix of the local pairwisecoupling energies, MLCE, whose elements are given byThe Hadamard product of the two matrices


Simultaneously having two pathologically distinct neoplastic lesions causing critical spinal stenosis is exceedingly rare. When such lesions are near one another but occupy different spinal compartments, significant challenges arise.We present the case of a patient with metastatic non-small cell carcinoma to the thoracic spine and an intradural meningioma occurring two spinal segments from each other. A 66-year-old female presented with one month of progressive mechanical back pain and two days of lower extremity weakness and urinary retention. She was found to have a left upper lobe lung mass. An urgent biopsy demonstrated non-small cell lung carcinoma. MRI of her thoracic spine demonstrated a T9 intradural-extramedullary enhancing lesion simultaneously with a destructive lesion of the T11 vertebral body extending into the anterior epidural space with significant cord compression at T9 and T11. The patient was taken for an urgent posterior decompression from T9 to T11, T9 left-sided pediculectomy with resection of intradural tumor, T11 corpectomy with anterior cage reconstruction, and instrumented fixation from T7 to L2. The pathology from the T9 lesion demonstrated findings consistent with a meningioma while the T11 lesion confirmed metastatic non-small cell lung adenocarcinoma. The patient improved neurologically postoperatively and regained the ability to ambulate within one week of surgery.Pathologically distinct spinal lesions in close anatomic proximity, but in two separate compartments are exceptionally rare. We performed a simultaneous posterior approach for resection of the T9 meningioma and a T11 corpectomy for the metastatic lesion with rapid neurologic recovery. Having multiple concurrent spine tumors of differing etiologies is exceedingly rare, especially in patients without neurofibromatosis or von-Hippel Lindau disease. Most cases present within the literature which contain concurrent spinal canal lesions within close proximity to one another comprise a combination of meningiomas, neurofibromas, and schwannomas -2. To ouA 66-year-old female presented to us with a one-month history of progressive mechanical back pain and two days of subjective lower extremity weakness and urinary retention. She neurologically declined rapidly and was no longer able to ambulate upon evaluation by our service. She had no history of neurologic symptoms or deficits prior to this. MRI of her thoracic spine Figure  demonstrThe patient was taken for an urgent posterior decompressive laminectomy from T9 to T11 , T9 left-sided pediculectomy with resection of intradural tumor Figure , T11 rigPathologically distinct spinal lesions in close anatomic proximity, but in two separate compartments are exceptionally rare in the absence of neurofibromatosis or von-Hippel-Lindau disease. During our literature review, the majority of concurrent spine tumors at either the same or adjacent spine levels involved meningiomas, schwannomas, and one case of a neurofibroma -5. To ouThe MRI is widely considered to be the best preoperative imaging technique to diagnose spinal tumors. Deciphering spinal meningiomas from schwannomas on an MRI may be difficult when encountering an intradural, extramedullary mass. According to De Verdelhan et al.’s single institution review of 52 spinal meningiomas and schwannomas, meningiomas are more likely to occur within the thoracic spine, whereas schwannomas frequently arise within the cervical or lumbar spine. With a gadolinium contrast enhanced MRI, meningiomas tend to have homogenous, moderate enhancement with a dural tail . SchwannOur patient’s rapid neurologic decline likely stemmed from pre-existing severe cord compression from the presumably long-standing meningioma at T9 in addition to the metastatic lesion at T11. Concurrent tumors at nearby spinal levels present unique treatment challenges. Given the severe spinal stenosis present from both the intradural lesion at T9 and the anterior compressive lesion at T11 on preoperative MRI, we advocated for treatment of both disease foci with one operation. We performed a simultaneous posterior approach with a left pediculectomy for resection of the T9 meningioma and a right trans-pedicular T11 corpectomy for the metastatic lesion involving the anterior vertebral body. Pedicle screw fixation was performed from T7 to L2 given the potential instability caused by resection of the left pedicle at T9 and the right pedicle at T11 which were required for an adequate operative access window to simultaneously treat both pathologies.To our knowledge, this is the first documented case of a concurrent spinal meningioma and metastatic lesion. Different pathologies in nearby compartments present unique treatment challenges and require a thorough review of preoperative radiographic imaging. Given the presence of two separate lesions at nonconsecutive vertebral levels, a unique operative plan was formulated in order to treat both levels of critical spinal stenosis as well as the underlying pathology.


Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease that targets motor neurons (MNs) in the brain and spinal cord. It leads to gradual loss of motor signals to muscles leading to atrophy and weakness. Most patients do not survive for more than 3–5 years after disease onset. Current ALS treatments provide only a small delay of disease progression. Therefore, it is of utmost importance to explore new therapeutic approaches. One of the major hindrances in achieving this goal is poor understanding of causes of the disease. ALS has complex pathophysiological mechanisms in its genetic and sporadic forms. Protein aggregates are a common hallmark of ALS regardless of cause making protein pathways attractive therapeutic targets in ALS. Here, we provide an overview of compounds in different stages of pharmacological development and their protein pathway targets. Amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease is the most common motor neuron (MN) disease. It is a progressive and fatal neurodegenerative disease, which affects both upper MNs in the motor cortex and lower MNs in brain stem and the anterior spinal cord . ALS patProtein aggregation is an important feature of ALS pathology. Amyloid deposits from different proteins such as TDP-43, C9ORF72 dipeptide repeats (DPRs), phosphorylated high molecular weight neurofilament protein (pNFH), rho guanine nucleotide exchange factor (RGNEF), and FUS have been detected in ALS MNs . These aProtein aggregation is the process of aberrant folding of a protein leading to self-association that may cause the formation of oligomers and fibrils via polymerization. The resulting amyloid fibrils are rich in beta sheet structure. Physiologically, proteins undergo folding through chaperones to attain their biologically favored stable conformation. Protein structure is stabilized by covalent and non-covalent interaction between the amino acids . In diseThere are several factors that lead to destabilization of protein structure . GeneticThe neurodegenerative diseases caused by protein aggregation are termed proteinopathies. Protein aggregates are the hallmarks of several neurodegenerative disease, for example, amyloid beta and tau in Alzheimer’s disease, alpha-synuclein in Parkinson’s disease (PD), and huntingtin in Huntington’s disease . ALS is A recombinant human monoclonal antibody (MAb) (α-miSOD1) was generated by screening human memory B cells from a large cohort of healthy elderly subjects. This antibody selectively binds to misfolded SOD1, but not to physiological SOD1 dimers. On postmortem spinal cord sections from 121 patients with ALS, α-miSOD1 antibody identified misfolded SOD1 in a majority of cases, regardless of their SOD1 genotype. In transgenic mice overexpressing disease-causing human SOD1-G37R or SOD1-G93A mutations, treatment with the α-miSOD1 antibody delayed the onset of motor symptoms, extended survival by up to 2 months, and reduced aggregation of misfolded SOD1 and MN degeneration .in utero electroporation without any apparent side effects in postnatal brain pathology or development  bind reversibly to specific amino acid residues of proteins which enables targeting the process of aggregation rather than targeting a specific protein or protein conformation. MTs achieve this activity by hydrophobic and electrostatic interactions involving labile binding to positively charged amino acid residues, primarily lysine and to a lower extent arginine. Hydrophobic and electrostatic interactions are important, particularly in the early stages of the aberrant self-assembly process which are effectively interrupted by MTs . They dot groups . The MT t groups  and coulColchicine is a plant alkaloid that interrupts microtubule formation and other cellular processes. This compound enhances the expression of heat-shock protein B8 (HSPB8) and several other autophagy factors . HSPB8 rAn investigational drug candidate, arimoclomol enhances expression of HSPs. Previous studies of transgenic SOD1 mice showed a large safety margin up to 300 mg/day. In separate studies, the effect of arimoclomol was tested in early and late stages of the disease. The compound showed promising results in both disease stages as survival was improved . A doublThe polyphenolic flavonoid, myricetin, has shown promise in targeting neurodegenerative diseases such as PD . The cleNSC-34 culture studies revealed that macrophage migration inhibitory factor (MIF) can reduce misfolded SOD1 and increase cell survival. One of the functions of MIF involves chaperone-like properties, which appear to change SOD1 amyloid aggregation pathways by forming disordered aggregates, which are less toxic to the cells . Shvil eMutations in SOD1 cause 15–20% of fALS cases. The resulting amino-acid substitutions destabilize SOD1’s protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause MN degeneration. The aggregates are found in the brain and spinal cord of affected individuals as intracellular inclusions. The antisense molecule tofersen (BIIB067) was designed to bind SOD1 mRNA. The artificially created DNA specifically targets the mRNA stage. Tofersen prevents translation of mRNA and ultimately the RNA degrades due to abnormal DNA-RNA strands, thereby reducing mutant SOD1 protein production. A Phase 1 clinical trial assessing the safety, tolerability, and activity of tofersen in SOD1-related fALS patients has been completed. The randomized, double-blind, placebo-controlled safety trial tested four different doses of tofersen  in 33 patients over a 12-h period. No serious adverse effects were observed in their assessment over 28 days post-treatment . This trThe most common cause of fALS is the hexanucleotide repeat expansion in the C9ORF72 gene which is linked to 25–40% of all familial cases. Mutant C9ORF72 forms toxic DPRs . Recent A number of different compounds that target a diverse range of mechanisms of neuronal protein aggregation pathways have demonstrated early favorable results in preclinical and early clinical ALS studies. Those include targeting mRNA—both for degradation and to inhibit translation, clearance of protein aggregates using antibodies, disruption of protein–protein interactions that lead to aggregation, preventing the formation of aggregates using vaccines, and maintaining proteostasis by activating protein clearance mechanism. This diverse list of approaches highlights the complexity and the investigational challenges of targeting protein aggregation in ALS. Although there is growing evidence that the process of protein aggregation is an important driver of neurodegeneration, a proven structure–proteotoxicity relationship based on specific protein abnormalities such as folding or aggregate state is lacking. Further basic research into the role of protein abnormalities in ALS disease mechanism is needed to guide preclinical studies toward disease specific treatments.RM contributed to writing the manuscript. MW contributed to writing and reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Understanding the pathogenesis and preventing long term complications have been major goals of research in diabetes mellitus (DM). Research in the past few years has linked oxidative stress and inflammation to beta cell dysfunction. Aim of this study is to evaluate serum gamma-glutamyl transferase (GGT) activity (marker of oxidative stress) and high sensitivity C reactive protein (hsCRP) level (an inflammatory marker) in type 2 DM subjects with good and poor glycemic control. Further, we investigated correlation between serum GGT and hsCRP level with glycemic control  in subjects.Diabetes is undoubtedly one of the most challenging health problems in 212BS, HbA1c, and other biochemical investigations include serum liver enzymes and lipids were measured.A cross sectional study consists of 150 patients out of them 50 patients having type 2 DM with good control (Group II), 50 patients with type 2 DM with poor control (Group III) and 50 normal healthy control (Group I) were selected. Serum GGT, serum hsCRP, FBS, PP1c, FBS, and PP2BS.Mean serum GGT and hsCRP concentration were statistically significantly higher in group III patients compared to group I and group II subjects as well as increased in group II compared to group I (p < 0.001). Further significant positive correlation was observed between GGT and hsCRP concentration as well as both with HbAOxidative stress and inflammation appears to be a key component and also associated with poor glycemic control and further pathogenesis of diabetes and its complications. All our finding suggesting a link between oxidative stress, inflammation and glycemic control in patient with type 2 diabetes mellitus.The online version of this article (doi:10.1186/2251-6581-12-56) contains supplementary material, which is available to authorized users. Hyperglt century. Researct century.Gamma-glutamyl transferase  is a cell-surface protein contributing to the extracellular catabolism of glutathione (GSH). The enzyme is produced in many tissues, but most GGT in serum is derived from the liver. GGT hasHigh sensitivity C-reactive protein (hsCRP) is a C-reactive protein measured by a highly sensitive assay. CRP represents the classical acute-phase protein produced in the liver in response to inflammatory stimuli, and plasma levels of hsCRP provide a sensitive marker of increased inflammatory activity in the arterial wall. Chronic1c). These measurements are complementary: the patient’s glucose measurements provide a picture of short-term glycemic control, whereas HbA1c reflects average glycemic control over the previous 3 months[Because the development of complications is linked to the accumulation of glycation adducts in tissue proteins. The core of the issue is glycemic control. Optimal monitoring of glycemic control involves plasma glucose measurements  and measurement of glycated hemoglobin HbAc. These Since, oxidative stress and inflammation appears to be a key component of many reactions associated with poor glycemic control and further pathogenesis of diabetes and its complications; we found it interesting to study serum GGT activity (marker of oxidative stress) and hsCRP level (an inflammatory marker) in diabetic subjects. Further, we investigated correlation between serum GGT and hsCRP with glycemic control in subjects.This study was a hospital based cross sectional study conducted at shree sayajirao general hospital and medical college, Vadodara (India) between May 2009 to June 2010. A cross sectional study consists of 150 subjects out of them 50 patients having type 2 DM with good glycemic control (Group II), 50 patients with type 2 DM with poor glycemic control (Group III) and 50 normal healthy control (Group I) were selected. Subjects were recruited according to simple random sampling method meeting the selection criteria.The subjects selected for study were grouped as follows:This group consisted of age and sex matched healthy subjects. They were free from any ailment which could affect the parameters under study. They were not on any medication. They were taken from general population.1c level less than 7%. They were on life style modifications and oral hypoglycemic drugs and free from clinical evidence of any complication of diabetes mellitus.This group consisted of patients with type 2 DM with duration less than 8 years, HbA1c level more than 7%. They were on life style modifications, oral hypoglycemic drugs, insulin or combination of all three and associated with one or more microvascular or macrovascular complication of diabetes mellitus for e.g. diabetic retinopathy, heart disease, diabetic neuropathy.This group consisted of patients with type 2 DM with duration more than 8 years, HbAThe patients with type 1 diabetes mellitus, high (>30 g/d) alcohol consumption, with known liver or gastrointestinal diseases, with liver enzyme concentrations higher than three times the upper limit, on corticosteroids, methotrexate, amiodarone, tamoxifen or other hepatotoxic drugs, any chronic infection like tuberculosis, sarcoidosis etc.,, hemolytic anaemia, hemoglobin variants were excluded from this study.The objectives of study were explained to all eligible subjects for this study. Informed written consent of all subjects included in the study was obtained for involvement in study groups and for venipuncture. Emphasis was given that participation in this study was voluntary.A questionnaire was specifically designed to obtain information which helps to select individuals according to the selection criteria of the study. The questions also focused on baseline information on socio-demographic variables  and background characteristics of diabetes  during a standardized interview. In addition, all participants underwent an extensive standardized medical examination including the collection of blood sample.2BS estimation. Serum or plasma separated within half an hour and stored at 2-8°C temperature till analysis was done.A 5 ml of venous blood was drawn from each volunteer using a disposable vacutainer system in fasting condition . Post prandial (2 hour) sample collected in fluoride vacutainer for PP2BS) estimated by glucose oxidase-peroxidase (GOD-POD) enzymatic end point method (Kit: Quantitative determination blood sugar by glucose oxidase peroxidase method mfg by Spinreact). Glycated hemoglobin (HbA1c) concentration was measured by immuno turbidimetric method (Kit: Quantitative determination of glycated hemoglobin (HbA1c) in human blood by latex turbidimetry mfg by Spinreact). Serum GGT activity was determined by carboxy substrate kinetic method. . Serum hsCRP level is measured by immuno turbidimetric method (Kit: Quantitative determination of hsCRP in human blood by latex turbidimetry assay mfg by Spinreact). All other biochemical investigation includes serum liver enzymes, lipids, and other biochemical blood measurements were determined using standard laboratory procedures on fully automatic analyzer I.S.E. srl MIURA. Hemogram and urine examination were done in pathology laboratory. Fundoscopy and electrocardiogram were done in respective department.Fasting and post prandial (2 hour) blood sugar . Also a significant positive association was observed between serum hsCRP level with glycemic control  (Table Our study shows mean serum GGT concentration in group III was significantly higher compared to control (group I) and group II cases . Also serum GGT concentration in group II was significantly higher compared to group I . Further mean hsCRP levels in group III and group II were significantly higher compared to group I . Also hsCRP level in group III was higher than group II  (Table S) Table .Table 1Baseline characteristics of control and subjects as well other biochemical parameter are given in Table 2BS. These findings suggest a link between oxidative stress (indicated by increased serum GGT concentration), inflammation (raised hsCRP concentration) and glycemic control in patients with type 2 DM and related complications. Also at levels of GGT and hsCRP considered well within the normal range, there was a substantial and significant increased concentration in patients with type 2 DM with good glycemic control compared to healthy subjects. This suggests a role of oxidative stress and chronic low grade inflammation in pathogenesis of type 2 diabetes patients.Our study shows statistical significantly increased concentration of GGT and hsCRP in serum in patient with type 2 DM with poor glycemic control compared to healthy persons as well as subjects having type 2 DM with good glycemic control. Also we found a significant positive linear relationship between GGT and hsCRP concentration as well as both with HbA1C, FBS, and PPSeveral possible mechanisms which explain increased serum GGT activity and hsCRP level in patients with type 2 DM with good and poor control and its correlation with glycemic control.Elevation of serum GGT could be the expression of an excess deposition of fat in the liver, termed non-alcoholic fatty liver disease. Fatty liver is thought to cause hepatic insulin resistance and to contribute to the development of systemic insulin resistance and hyperinsulinemia. Thus, GGT could serve as a marker of the insulin resistance syndrome in the pathogenesis of diabetes, 13.There is now growing evidence to suggest that GGT is not only a marker of fatty liver but also a marker of oxidative stress. Experimental studies have reported that GGT has a central role in the maintenance of intracellular antioxidant defences through its mediation of extracellular glutathione transport into most types of cells. It is aOxidative stress is currently suggested as mechanism underlying diabetes and diabetic complications. In recent years, much attention has been focused on the role of oxidative stress, and it has been reported that oxidative stress may constitute the key and common event in the pathogenesis of secondary diabetic complications. Implication of oxidative stress in the pathogenesis of diabetes is suggested, not only by oxygen free-radical generation, but also due to nonenzymatic protein glycosylation, auto-oxidation of glucose, impaired glutathione metabolism, alteration in antioxidant enzymes, lipid peroxides formation and decreased ascorbic acid levels. In addition to GSH, there are other defence mechanisms against free radicals like enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) whose activities contribute to eliminate superoxide, hydrogen peroxide and hydroxyl radicals. Raised Various studies suggested that elevated serum GGT could be the expression of subclinical inflammation which also contributes to the development of type 2 DM, 17. As 1c, hsCRP, total cholesterol, nitrate and GGT as compared to controls. HbA1c showed a positive correlation with hsCRP, total cholesterol, nitrate and GGT. Oxidative stress and inflammatory markers should be used in addition to HbA1c for assessment of increased cardiac risk in uncontrolled diabetic patients because of accelerated atherosclerosis due to free radical injury[1c levels. Mean HbA1c levels were significantly higher in patients who had hsCRP levels of 1 mg/L or more. Other factors such as age, blood pressure, BMI, LDL cholesterol, serum creatinine were not correlated with hsCRP level[1c, fasting insulin and HOMA-IR, inflammation, insulin resistance and hyperglycemia jointly contribute to the cardiovascular risk in type 2 DM men[There are various studies which support our results. R Sharma et al. shows rise in levels of hsCRP and GGT in diabetic subjects and their significant association which might be a result of inflammation and oxidative stress in diabetes mellitus. Dilshadal injury. SarinnaCRP level. Also Ba 2 DM men.Other lines of evidence support a relationship between elevated serum GGT and poor glycemic control and metabolic syndrome are also found. Higher GGT levels are accompanied by more insulin resistance and greater risk for developing type 2 DM and poor glycemic control–27. The In general, serum GGT concentration is closely related with other enzymes more specific to the liver, serum ALT or AST concentration, so we did parallel analyses with ALT and AST to further explore the possible role of liver damage in the association of GGT with diabetes. Within their normal ranges, ALT and AST showed significant increase in type 2 diabetes patients with poor control . Moreover, markers of hepatic fat content, such as serum GGT activity and other liver enzymes, have been shown in large prospective studies to predict the incidence of type 2 diabetes, insulin resistance, and cardiovascular disease independently of obesity, 35, 36.Our results also suggest that liver enzymes are closely associated with the risk of metabolic syndrome and type 2 diabetes and that among these enzymes serum GGT is the most powerful risk indicator for developing the metabolic syndrome and type 2 diabetes. Another possible pathophysiological mechanism is that elevated liver enzymes may reflect inflammation, which impairs insulin signalling both in the liver and systemically, 37, 38.Our study had several limitations. First, it is cross sectional study with no causality effect to report. Also serum GGT and hsCRP during follow-up was not included in the analysis. Further, we could not include several confounding variables in this study, such as fasting insulin concentration. Therefore, fasting insulin concentration should be included in future studies.1c, FBS and PP2BS (short and long term glycemic control). So far, the underlying pathophysiological mechanisms are not entirely clear. It seems that insulin resistance, oxidative stress and chronic low grade systemic inflammation may be involved. All these finding suggesting a link between oxidative stress, inflammation and glycemic control in patient with type 2 diabetes mellitus. Further studies are needed to investigate the biological mechanisms underlying this association.Despite these potential limitations, our findings, which were obtained from a cross sectional study shows that serum GGT activity and hsCRP level is significantly increased in patients with type 2 diabetes mellitus compared to healthy control. Both are further increased in diabetic patients with complications and poor glycemic control. Also there is a significant positive correlation between serum GGT activity and hsCRP. Both are also independently positively correlated with HbAIn conclusion, the present study suggests that serum GGT and hsCRP concentration is significantly increased in type 2 diabetes mellitus. Both are further increased in diabetic patients with complications and poor glycemic control. There is a significant positive correlation between serum GGT activity and hsCRP. Serum GGT level and hsCRP concentration was independently and positively correlated with FBS, PP2BS and HbA1c (markers of glycemic control). All these finding suggesting a link between oxidative stress, inflammation and glycemic control in patient with type 2 diabetes mellitus.


Pancreatic islets are clusters of endocrine cells that secrete different hormones to regulate blood glucose levels. Efficient hormone secretion requires a close interaction of endocrine cells with their vascular system. Islets receive blood through feeding arteriole(s) that branch into capillaries made of endothelial cells covered by pericytes. While a lot is known about rodent islet blood vessels, the structure and function of the human islet microvasculature has been less investigated. In this study, we used living pancreas slices from non-diabetic human donors to examine the function of human islet blood vessels. Living human pancreas slices were incubated with a membrane permeant calcium indicator and pericytes/smooth muscle cells were visualized with a fluorescent antibody against the mural cell marker NG2 proteoglycan. By confocal microscopy, we simultaneously recorded changes in the diameter of lectin-labeled blood vessels and cytosolic calcium levels in mural cells in islets. We tested several stimuli with vasoactive properties, such as norepinephrine, endothelin-1 and adenosine and compared human vascular responses with those previously published for mouse islet blood vessels. Norepinephrine and endothelin-1 significantly constricted human islet feeding arterioles, while adenosine dilated them. Islet capillaries were less responsive and only 15–20% of the mouse and human islet capillary network showed vasomotion. Nevertheless, in these responsive regions, norepinephrine and endothelin-1 decreased both mouse and human islet capillary diameter. Changes in islet blood vessel diameter were coupled to changes in cytosolic calcium levels in adjacent mouse and human islet mural cells. Our study shows that mural cells in islets are the targets of different regulatory mechanisms of islet blood perfusion. Several alterations of the human islet microvasculature occur during diabetes progression. Elucidating their functional consequences in future studies will be critical for our understanding of disease pathogenesis. Pancreatic islets are endocrine mini-organs rich in blood vessels , 2. ClosThe mouse is an extensively used and valuable animal model in medical research. However, previous studies have shown that in human islets the microvascular niche differs significantly from that of the mouse. Not only human islets have less and shorter capillaries than mouse islets , 20, butex vivo  and ethidium homodimer-1 (labels dead cells in red). Addition of both reagents was done according to the manufacturer’s recommendations as part of the Live/Dead viability/cytotoxicity kit for mammalian cells . EndocrLycopersicon Esculentum  for 1 h in 3 mM glucose solution prepared in HEPES buffer  supplemented with aprotinin  at room temperature and in the dark. To identify pericytes in situ, slices were incubated for 2 h with a fluorescent-conjugated antibody against NG2 . After incubation, living pancreas slices were placed on a coverslip in an imaging chamber  and imaged under an upright confocal microscope . The chamber was continuously perfused with HEPES-buffered solution containing 3 mM glucose and confocal images were acquired with LAS AF software (Leica Microsystems) using a 40X water immersion objective (NA 0.8). We used a resonance scanner for fast image acquisition to produce time-lapse recordings spanning 50–100 μm of the slice  at 5 seconds resolution (xyzt imaging). Fluo-4 fluorescence was excited at 488 nm and emission detected at 510–550 nm, DyLight 649 labeled tomato lectin was excited at 638 nm and emission detected at 648–690 nm.Living human pancreas slices were incubated with Fluo4-AM  and DyLight 649 lectin from From each human donor, a group of slices was incubated with Fluo4 and lectin and another group of slices was incubated with Fluo4 and NG2-alexa647, to account for potential effects of antibody binding and change in cell physiology. Mural cells responded similarly with or without antibody labeling.2+]i and blood vessel diameter induced by norepinephrine , endothelin-1  and adenosine . To quantify changes in [Ca2+]i, we drew regions of interest around individual islet pericytes and measured the mean Fluo4 fluorescence intensity using ImageJ software (http://imagej.nih.gov/ij/). Changes in fluorescence intensity were expressed as percentage over baseline (ΔF/F). The baseline was defined as the mean of the three first values of the control period of each recording . Blood vessels were labeled with DyLight-649 and we could image vessel borders in slices. Quantification of vessel diameter was done as previously described (A”) and used the “reslice” z-function in ImageJ to generate a single image showing the changes in vessel diameter over time  for 3 h. Thereafter, slices were incubated for 48 h (20°C) with primary antibodies diluted in blocking solution. We immunostained pericytes , endothelial cells , smooth muscle cells , beta cells . Immunostaining was visualized conjugated secondary antibodies . Cell nuclei were stained with dapi. Slides were mounted with Vectashield mounting medium (Vector Laboratories) and imaged on an inverted laser-scanning confocal microscope  with LAS AF software using a 63X oil immersion objective (NA 1.4). To quantify colocalization between αSMA and NG2 immunostainings, we calculated Mander’s coefficients in confocal images using the ImageJ plugin “JACoP”: Just Another Co-localization Plugin.p values < 0.05 were considered statistically significant (indicated with an *). Throughout the manuscript we present data as mean ± SEM.For statistical comparisons we used Prism 7  and performed Student’s t tests (paired and unpaired) or One sample t test. Pericytes, the mural cells of the microcirculation, are part of the mouse and human islet microenvironment , 29, 30.Pericytes in mouse islets have contractile properties . To deteIsolated islets are the most common research material used to study the physiology of pancreatic islet cells in humans. However, islets, similarly to other endocrine organs , lose a Lycopersicon esculentum) that bind to glycoproteins located in the endothelial basement and plasma membranes can be used to visualize blood vessels ex vivo. While in mice these lectins can be injected intravenously before sacrificing the animals, living human pancreas slices have to be incubated with them after tissue slicing. In any case, lectin labeling allows us to visualize islet vascular trees (2+ levels ([Ca2+]i) in different cells in human pancreas slices  at the islet border (islet capillaries those lectin-labeled vessels surrounded by endocrine cells (cells with backscatter) with a diameter around 5–7 μm i in “SMC” pericytes that coincide with a reduction of the arteriole diameter , we had previously determined that pericytes control capillary diameter in mouse islets . To detediameter . NE induIn this study, we performed the first detailed functional characterization of the human islet microvasculature, comparing it side-by-side with what we know about the physiology of mouse islet blood vessels. In human islets, a subset of islet pericytes and smooth muscle cell-like (“SMC”) pericytes express the contractile protein αSMA. We measured vasomotion in different types of islet blood vessels in response to exogenous administration of norepinephrine, endothelin-1, and adenosine. We found that islet feeding arterioles and a small subset of capillaries constrict upon norepinephrine and endothelin-1, but only arterioles dilate upon adenosine. We further determined that changes in blood vessel diameter are coupled to changes in cytosolic calcium levels in adjacent pericytes or “SMC” pericytes. Our study points to pericytes as targets of several regulatory mechanisms of islet blood perfusion, similarly to their role in mouse islets.Our data support that blood flow in the human islet can be regulated locally at the level of the feeding arteriole and of a subset (~15–20%) of islet capillaries, similar to what has been described in rodents , 48–50. In this study we perform a functional and anatomical cross comparison of the islet microvasculature between mice and humans. Previous studies had already shown that the architecture and density of islet vascular trees differed significantly between these two species , 20. OurA receptors expressed on smooth muscle cells  with those from the nervous system or the systemic circulation , 16. Altn islets . Using lThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRB approval, University of Miami. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.LG and JA designed the study, and acquired and analyzed data. Both authors discussed the results and interpreted the data. JA wrote the manuscript. All authors contributed to the article and approved the submitted version.https://hirnetwork.org; UC4 DK104162, New Investigator Pilot Award to Joana Almaça).This work was funded by NIH grants K01DK111757 (JA) and by the NIDDK-supported Human Islet Research Network (HIRN, RRID : SCR_014393; 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.