Biology subjects

Ohashi, K.

Publications and source records attributed to Ohashi, K..

18 recordsLinked to original sources

bioRxiv2026

Density-Dependent Rearrangement of Desmoplakin in Epithelial Junctions: Insights from Expansion Microscopy

Desmosomes mechanically couple neighboring epithelial cells to the intermediate filament cytoskeleton. However, how the nanoscale organization changes with epithelial cell density remains incompletely understood. Here, we used expansion microscopy (ExM) to examine Madin-Darby canine kidney epithelial monolayers. ExM revealed density-associated differences in the relative apicobasal organization of epithelial junctions, with increased separation between occludin-positive tight junctions and desmoglein 2-positive desmosomes in confluent monolayers. A comparison of subconfluent and confluent cultures revealed a density-associated reorganization of desmoplakin (DSP) within opposing desmosomal plaques, with a more pronounced change in C-terminal spacing than in N-terminal spacing. Independent STED analysis of non-expanded samples confirmed the density-associated increase in DSP C-terminal spacing. In keratin filament-deficient cells, this spacing was elevated in subconfluent cultures and did not increase with cell density. Pharmacological inhibition of myosin II or ROCK reduced this elevated spacing, indicating that keratin loss-associated reorganization of DSP is sensitive to actomyosin contractility. These findings show that epithelial cell density is associated with the remodeling of epithelial junction organization and DSP plaque architecture, demonstrating the utility of ExM for quantitative analysis of junctional architecture.

cell biology↗

bioRxiv2025

Astrocytic Cathepsin D truncates α-synuclein and promotes Lewy neurite-like aggregation in neurons

Parkinsons disease (PD) and related synucleinopathies are marked by the accumulation and propagation of -synuclein (-syn) aggregates, a process primarily studied in neurons. However, the contribution of astrocytes to this process remains unclear. Here, using a physiologically relevant neuron-astrocyte co-culture system that recapitulates tripartite synapse architecture, we show that astrocytes actively process and propagate -syn aggregates. Astrocytes internalize -syn pre-formed fibrils (PFFs) and cleave them into C-terminally truncated, seeding-competent species via the lysosomal protease Cathepsin D (CtsD). These truncated species are subsequently transferred to neurons, where they promote the growth of Lewy neurite (LN)-like aggregates. Notably, -syn PFF exposure disrupts lysosomal membrane integrity in astrocytes, leading to CtsD upregulation. These findings reveal a feed-forward mechanism in which astrocytic lysosomal dysfunction amplifies -syn pathogenicity. Our findings identify astrocytes as active contributors to -syn propagation and highlight the astrocytic lysosomal pathway as a potential therapeutic target in PD.

pathology↗

bioRxiv2025

Actin cytoskeletal remodeling requires the interaction between Solo and LARG in response to substrate stiffness

In response to external mechanical stimuli, cells remodel their actin cytoskeleton. Solo, a Rho guanine nucleotide exchange factor (RhoGEF), is involved in mechanical stress responses. Using BioID, we identified PDZ-RhoGEF (PRG), a member of the RGS-RhoGEF family (regulator of G protein signaling domain-containing RhoGEFs, as a Solo-interacting protein. Moreover, we found that Solo regulates PRG during the mechanical stress response. Furthermore, we identified leukemia-associated RhoGEF (LARG), another RGS-RhoGEF member, as a Solo-interacting protein; however, the functional role of this interaction remains unknown. Therefore, in this study, we investigated the interaction between Solo and LARG and found that LARG localizes to Solo accumulation sites at the basal plane and that LARG is required for Solo-induced actin polymerization. Additionally, Solo is required to maintain LARG activity in cells, and this interaction is related to actin regulation in response to substrate stiffness. We further investigated the relationship between LARG and PRG as a function of Solo. We noted that although they did not competitively localize at Solo accumulation sites, knockdown of either PRG or LARG suppressed Solo-induced actin polymerization to the same extent as double knockdown, indicating that these signaling pathways cooperatively regulate Solo-induced actin polymerization.

cell biology↗

bioRxiv2025

Childhood Maltreatment and Brain Aging During Adulthood

ImportanceChildhood maltreatment (CM) is associated with the early onset of psychiatric and medical disorders and accelerated biological aging. ObjectiveTo identify types of maltreatment and developmental sensitive periods that are associated with accelerated adult brain aging. DesignParticipants were mothers of infants recruited from the community into a study assessing the effects of CM on maternal behavior, infant attachment, and maternal and infant neurobiology. Data were collected from July 2015 to November 2019 and were analyzed from July 2023 to October 2024. SettingAcademic medical centers. ParticipantsHigh-quality MRI scans were obtained on 92 of 150 mothers enrolled in the study. The main exclusion criteria for neuroimaging were histories of head trauma with loss of consciousness or concussion, psychotropic use before age 18, pregnancy, and customary MRI exclusions (e.g., metal implant). The primary reasons for non-completion of the neuroimaging study were unwillingness to be scanned, inability to attend the MRI study visit due to work and/or childcare, metal implants, or pregnancy. Main Outcome(s) and Measure(s)The Maltreatment and Abuse Chronology of Exposure scale was used to retrospectively assess the annual severity of exposure to ten types of CM from birth to age 18 years. Brain age was calculated from T1-weighted 3T MRI Scans using a previously published machine learning algorithm. Sensitive periods were identified using random forest regression with conditional inference trees. ResultsForty-nine (53.3%) of the 92 mothers (mean [SD] age, 32.4 [4.3] years) reported experiencing one or more types of CM. Total CM severity was associated with accelerated brain aging ({beta}=0.05, 95% CI, 0.02 to 0.09, p<.005). The most robust type/time risk factors for accelerated brain aging were parental physical abuse between ages 4 to 6 years, witnessing sibling violence between ages 4 to 15 years, parental verbal abuse between ages 10 to 12 years, and parental emotional neglect between ages 16 to 18 years. Conclusions and RelevanceSeveral types of CM between ages 4-18 years were associated with accelerated brain aging. Understanding how these specific types and ages of exposure contribute to accelerated brain aging may provide important insights into preventing key clinical consequences of CM. SUMMARYQuestion: How does childhood adversity relate to brain aging in adulthood, and are there sensitive periods for this association? Findings: In this cohort-based study of adult women, we observed sensitive periods for the association between adult brain aging and five classes of childhood maltreatment: parental physical abuse, parental verbal abuse, parental emotional neglect, and witnessing sibling violence. Meaning: These findings suggest that maltreatment subtype and age at exposure may be important factors contributing to the impact of childhood adversity on brain aging later in life.

neuroscience↗

bioRxiv2024

Tertiary lymphoid structure as a therapeutic target of Crohns disease: modulation by TNFα blockade

Inflammatory diseases, such as Crohns disease (CD), are primarily treated with therapies aimed at alleviating inflammation. Notably, biological agents like TNF inhibitors have transformed the management of these conditions. Previous research has indicated that TNF inhibitors affect the cells in the lamina propria. However, this study demonstrates that infliximab, a type of TNF inhibitor, reduces the presence of tertiary lymphoid structures (TLS) and alleviates inflammation of CD by targeting follicular dendritic cells and macrophages within these structures. TLS could also be a potential target for various biological agents used to treat inflammatory diseases beyond CD. Furthermore, our study suggests that the status of TLS may serve as a predictor for the response to TNF inhibitor treatment. Our research could pave the way for new treatment strategies and the advancement of personalized medicine for inflammatory diseases.

immunology↗

bioRxiv2024

Structural Analyses of a GABARAP~ATG3 Conjugate Uncover a Novel Non-covalent Ubl-E2 Backside Interaction

Members of the ATG8 family of ubiquitin-like proteins (Ubls) are conjugated to phosphatidylethanolamine (PE) in the autophagosomal membrane, where they recruit degradation substrates and facilitate membrane biogenesis. Despite this well-characterized function, the mechanisms underlying the lipidation process, including the action of the E2 enzyme ATG3, remain incompletely understood. Here, we report the crystal structure of human ATG3 conjugated to the mammalian ATG8 protein GABARAP via an isopeptide bond, mimicking the Ubl[~]E2 thioester intermediate. In this structure, the GABARAP[~]ATG3 conjugate adopts an open configuration with minimal contacts between the two proteins. Notably, the crystal lattice reveals non-covalent contacts between GABARAP and the backside of ATG3s E2 catalytic center, resulting in the formation of a helical filament of the GABARAP[~]ATG3 conjugate. While similar filament formations have been observed with canonical Ub[~]E2 conjugates, the E2 backside-binding interface of GABARAP is distinct from those of Ub/Ubl proteins and overlaps with the binding site for LC3 interacting region (LIR) peptides. NMR analysis confirms the presence of this non-covalent interaction in solution, and mutagenesis experiments demonstrate the involvement of the E2 backside in PE conjugation. These findings highlight the critical role of the E2 backside in the lipidation process and suggest evolutionary adaptations in the unique E2 enzyme ATG3.

biochemistry↗

bioRxiv2024

Oligodendrocyte precursor cells exacerbate acute CNS inflammation via macrophage and T cell activation in a mouse model of multiple sclerosis

Oligodendrocyte precursor cells (OPCs) are a type of glial cell that differentiates into mature oligodendrocytes, a cell type that contributes to myelination, but their roles in the pathologies are not fully understood. Activities other than differentiation into oligodendrocytes have recently been reported for OPCs present in the inflammatory milieu, but intervention studies using animal models are lacking. This study aimed to explore the role of OPCs in mouse MS model experimental autoimmune encephalomyelitis (EAE). Using inducible diphtheria toxin receptor-expressing transgenic mice, platelet-derived growth factor receptor A (PDGFR)+ OPCs were depleted in EAE mice. Surprisingly, OPC depletion in the acute phase improved clinical scores and reduced demyelination. Major histocompatibility complex (MHC) class II was reduced in the spinal cord, whereas astrocyte marker and blood-spinal cord barrier tight junction and adhesion molecule expressions were unaffected after OPC depletion. The numbers of T cells and IL17-expressing Th17 cells were decreased in the spinal cords of the OPC-depleted group. MHC class II expression in spinal cord macrophages was consistently decreased by OPC depletion. These data suggest that in the acute phase of EAE, OPCs are involved in activation of infiltrated macrophages and induce subsequent T cell activation and neuroinflammation. Although the precise mechanisms remain unclear, this implies that OPCs exist not only as the source for oligodendrocytes but also play a pivotal role in central nervous system (CNS) autoimmune inflammation. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/596190v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@98162dorg.highwire.dtl.DTLVardef@d7f92dorg.highwire.dtl.DTLVardef@1280619org.highwire.dtl.DTLVardef@a85907_HPS_FORMAT_FIGEXP M_FIG C_FIG MAIN POINTSO_LIOPC depletion in the acute phase of EAE improved clinical scores and reduced demyelination. C_LIO_LIOPC depletion in the spinal cord suppressed Antigen presentation via MHC class II. C_LIO_LIOPCs are involved in activation of infiltrated macrophages and induce subsequent T cell activation and neuroinflammation. C_LI

neuroscience↗

bioRxiv2024

Realized flower constancy in bumble bees: optimal foraging strategy balancing cognitive and travel costs and its possible consequences for floral diversity

O_LIPollinating insects often exhibit flower constancy, i.e., the tendency to make consecutive visits to the same flower species while disregarding others. This behaviour is commonly attributed to the cost of retrieving visual or motor memories from long-term storage while switching between flowers with distinct colours and shapes. Accordingly, researchers often predict co-flowering species to exhibit significantly greater phenotypic diversity than random expectation, thereby minimizing heterospecific pollen transfer. However, field observations have not consistently supported this notion. C_LIO_LIThe observed inconsistencies may arise from variations in travel costs, which depend on the interaction between the foragers constancy level and the spatial mixing of plant species. If species are evenly mixed, constant pollinators incur higher levels of travel cost due to frequent skipping of neighbouring flowers. In contrast, if species are patchily distributed, constant pollinators experience lower levels of travel cost, as most neighbours are of the same species. Considering this, realized flower constancy may be determined as an optimal strategy for balancing cognitive and travel costs, which dynamically vary across different degrees of spatial species mixing. Here we test this possibility in indoor experiments with bumble bees foraging from two differently coloured artificial flowers (species) arranged at three mixing levels. C_LIO_LIFirst, bees dramatically reduced flower constancy as species mixing increased, irrespective of flower spacing. Second, bees were less inclined to switch species after accumulating consecutive visits to one species, suggesting a rapid decay of another species information in short-term memory back to long-term storage. This effect may have additionally contributed to the increased flower constancy observed in species with patchy distributions. Third, bees showed minimal constancy for similarly coloured, evenly mixed flower species, suggesting that these flowers were operated with shared short-term memory. The constancy level was hardly affected by colour similarity when species were patchily distributed. C_LIO_LIResults support our initial hypothesis that realized flower constancy reflects an optimal foraging strategy rather than a fixed outcome of cognitive limitation. Notably, bees constancy increased significantly with greater colour difference only when species were evenly mixed, suggesting a novel perspective: spatial mixing promotes the evolution and maintenance of floral diversity. C_LI

animal behavior and cognition↗

bioRxiv2023

Interaction between Solo and PDZ-RhoGEF is involved in actin cytoskeletal remodeling and response to substrate stiffness

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study was aimed at identifying Solo-interacting proteins using the BioID, a proximal-dependent labeling method and elucidating the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG co-localized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling and RhoA activation. Moreover, overexpression of the binding domains of Solo and PRG had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

cell biology↗

bioRxiv2023

MARK3-mediated Slingshot-1 phosphorylation is essential for polarized lamellipodium formation

Cofilin acts as a key regulator of actin cytoskeletal remodeling via stimulating actin filament disassembly. Cofilin is inactivated by Ser-3 phosphorylation and reactivated by cofilin-phosphatase Slingshot-1 (SSH1). SSH1 is activated upon binding to F-actin, and this activation is inhibited by its phosphorylation at Ser-937 and Ser-978 and the subsequent binding of 14-3-3 proteins. In this study, we identified MARK3 (also named Par-1a and C-TAK1) as a kinase responsible for Ser-937/Ser-978 phosphorylation of SSH1. MARK3-mediated phosphorylation promoted SSH1 binding to 14-3-3 proteins and suppressed its F-actin-assisted cofilin-phosphatase activity. When Jurkat cells were stimulated with SDF-1, actin filaments formed multidirectional F-actin-rich lamellipodia around the cells in the initial stage, and thereafter, they were rearranged as a single polarized lamellipodium to the direction of cell migration. Upon SDF-1 stimulation, SSH1 was translocated into F-actin-rich lamellipodia, but its Ser-937/Ser-978 non-phosphorylatable mutant SSH1(2SA) was retained at the location of the original cortical F-actin. Knockdown of MARK3 or overexpression of SSH1(2SA), similar to SSH1 knockdown, impaired the conversion of multiple lamellipodia to a single polarized lamellipodium. These results indicate that MARK3-mediated Ser-937/Ser-978 phosphorylation is required for SSH1 liberation from F-actin and translocation to lamellipodia, and hence, facilitates the formation of a single polarized lamellipodium for directional cell migration. Our results suggest that the phosphorylation-dephosphorylation cycle of SSH1 is crucial for its localization to lamellipodia via promoting the dissociation-reassociation cycle of SSH1 to F-actin, and thereby the stimulus-induced lamellipodium formation to the direction of cell movement.

cell biology↗

bioRxiv2023

Negative selection on a SOD1 mutation limits canine degenerative myelopathy while avoiding inbreeding

Several hundred disease-causing mutations are currently known in domestic dogs. Breeding management is therefore required to minimize their spread. Recently, genetic methods such as direct-to-consumer testing have gained popularity; however, their effects on dog populations are unclear. Here, we aimed to evaluate the influence of genetic testing on the frequency of mutations responsible for canine degenerative myelopathy (DM) and assess the changes in the genetic structure of a Pembroke Welsh corgi population from Japan. Genetic testing of 5,512 dogs for the causative mutation in superoxide dismutase 1 (SOD1) (c.118G>A (p.E40K)) uncovered a recent decrease in frequency, plummeting from 14.5% (95/657) in 2019 to 2.9% (24/820) in 2022. Weir and Cockerham population differentiation (FST) and simulation-based genome-wide single-nucleotide polymorphism (SNP) analysis of 117 selected dogs revealed 143 candidate SNPs for selection. The SNP with the highest FST value was located in the intron of SOD1 adjacent to the c.118G>A mutation, supporting a strong selection signature on SOD1. Further genome-wide SNP analyses revealed no obvious changes in inbreeding levels and genetic diversity between the 2019 and 2022 populations. Our study highlights that genetic testing can help inform improved mating choices in breeding programs to reduce the frequency of risk variants and avoid inbreeding. This combined strategy could decrease the genetic risk of canine DM, a fatal disease, within only a few years. Significance statementGenetic breeding methods using direct-to-consumer testing have gained popularity, but their effects on dog populations remain unclear. In this study, the effect of direct-to-consumer genetic testing on SOD1 mutation, the causative element of canine degenerative myelopathy, in a domestic dog population (Pembroke Welsh corgi) from Japan was investigated. Our analyses revealed that since the expansion of genetic testing in 2019, breeders used these tests to artificially select against the SOD1 mutation, considerably decreasing its occurrence in the corgi population within only a few years (2019 versus 2022). Our study makes a substantial contribution to existing literature by providing empirical evidence that direct-to-consumer genetic testing can have rapid influence on pet genetics, noticeable in a span of 2-3 years.

genetics↗

bioRxiv2022

The effect of floral symmetry and orientation on the consistency of pollinator's entry angle

Since the publication of Sprengels (1793) observations, it has been considered that flowers with zygomorphic (or bilaterally symmetrical) corollas evolved to restrict the movement of pollinators into the flower by limiting the pollinators direction of approach. However, little empirical support has been accumulated so far, except Culbert and Forrest (2016) who found that zygomorphy reduced variance in pollinators flower entry angle. Our aim was to build on this work and observe whether floral symmetry or orientation had an effect on pollinator entry angle in a laboratory experiment using bumble bees, Bombus ignitus. Using nine different combinations of artificial flowers created from three symmetry types (radial, bilateral and disymmetrical) and three orientation types (upward, horizontal and downward), we tested the effects of these two floral aspects on the consistency of bees entry angle. Our results show that horizontal orientation significantly reduced the variance in entry angle, while symmetry had little effect. We also found no significant interactions between angle and symmetry in their effect on entry angle. Thus, our results suggest that horizontal orientation forces the bees to orient themselves relative to gravity rather than the corolla and stabilizes their flower entry. This stabilizing effect may have been mistaken for the effect of zygomorphic corolla as it is presented horizontally in most species. Consequently, we suggest that the evolution of horizontal orientation preceded that of zygomorphy as indicated by some authors, and that the reason behind the evolution of zygomorphy should be revisited.

plant biology↗

bioRxiv2022

Diagnosis and early prediction of lymphoma using high-throughput clonality analysis of bovine leukemia virus-infected cells

Bovine leukemia virus (BLV), a retrovirus, infects into B cells of ruminants and causes aggressive leukemia or lymphoma in cattle, enzootic bovine leukosis (EBL). Clonal expansion of BLV-infected cells is a promising marker for early detection and diagnosis of EBL. Recently, we developed rapid amplification of the integration site without interference by genomic DNA contamination (RAISING) and CLOVA, a software to analyze clonality. RAISING-CLOVA could assess the risk of adult T-cell leukemia/lymphoma development in human T-cell leukemia virus-I-infected individuals through its clonality analysis. Thus, we herein examined the performance of RAISING-CLOVA for the clonality analysis of BLV-infected cells and conducted a comprehensive clonality analysis by RAISING-CLOVA in EBL and non-EBL cattle. RAISING-CLOVA successfully distinguished EBL from non-EBL cattle with high sensitivity and specificity. A longitudinal clonality analysis in BLV-infected sheep, an EBL model, also confirmed the effectiveness of BLV clonality analysis with RAISING-CLOVA for early detection of EBL development. Therefore, our study emphasizes the usefulness of RAISING-CLOVA as a routine clinical test for monitoring virus-related cancers.

microbiology↗

bioRxiv2021

Laboratory analysis of glucose, fructose, and sucrose contents in Japanese common beverages for the exact assessment of beverage-derived sugar intake

BackgroundThe adverse health effects of sugar-sweetened beverage consumption have been studied worldwide. There are several reports on actual sugar contents in sugar-sweetened beverages. However, there is no recent report on actual sugar contents in Japanese sugar-sweetened beverages. Therefore, we attempted to analyze glucose, fructose, and sucrose contents in Japanese common beverages. MethodsGlucose, fructose, and sucrose contents in 49 beverages including 8 energy drinks, 11 sodas, 4 fruit juices, 7 probiotic drinks, 4 sports drinks, 5 coffee drinks, 6 green tea drinks, and 4 tea drinks were determined using the enzymatic methods. ResultsTow zero calorie drinks, 2 sugarless coffee drinks, and 6 green tea drinks contained no sugar. Three coffee drinks contained only sucrose. The orders of median glucose, fructose, and sucrose contents in categorized beverages containing sugars were as follows: for glucose, fruit juice > energy drink [≥] soda >> probiotic drink > black tea drink > sports drink; for fructose, probiotic drink [≥] energy drink > fruit juice > soda >> sports drink > black tea drink; and for sucrose, black tea drink > energy drink [≥] probiotic drink > fruit juice > soda > coffee drink >> sports drink. The rate of total fructose content in total sugar content in 38 sugar-containing beverages was approximately 40-60%. The total sugar content analyzed was not always equivalent to carbohydrate content indicated on the nutrition label. ConclusionsThese results indicate that actual sugar content in Japanese common beverages is necessary for the exact assessment of beverage-derived sugar intake.

biochemistry↗

bioRxiv2020

Association between the extent of DNA methylation at the CpG sites of HIF3A and parameters of obesity in the general Japanese population

Obesity is a major public health problem worldwide owing to the substantial increase in risk of metabolic diseases. Hypoxia-inducible factors (HIFs) regulate transcriptional responses to hypoxic stress. DNA methylation in the CpG sites of intron 1 of HIF3A is associated with body mass index in the whole blood and adipose tissue. This study investigates the correlation between DNA methylation of HIF3A and parameters of obesity, including thickness of visceral (VAT) and subcutaneous adipose tissues, in the general Japanese population. Participants (220 men and 253 women) who underwent medical examination were enrolled in this cross-sectional study. We used pyrosequencing to quantify DNA methylation (CpG sites of cg16672562, cg22891070, and cg27146050) in HIF3A. DNA methylation of HIF3A was only different in women. Multiple regression analysis showed that DNA methylation level at cg27146050 was associated with thickness of VAT in women. DNA methylation level at cg27146050 also correlated with body mass index and percentage of body fat in women after excluding smokers and non-smokers who quit smoking with the last 5 years. DNA methylation in the CpG site (cg27146050) of HIF3A correlated with parameters of obesity in Japanese women.

biochemistry↗

bioRxiv2020

PD-L1 expression in equine malignant melanoma and functional effects of PD-L1 blockade

Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on exhausted T cells during chronic illness. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We reported that the PD-1/PD-L1 pathway is closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Moreover, we found that blocking antibodies targeting PD-1 and PD-L1 restore T-cell functions and may be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway remains unclear for chronic equine diseases, including tumors. In this study, we identified nucleotide sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 monoclonal antibodies (mAbs) against EqPD-L1 using in vitro assays. We also evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a high identity and similarity with homologs from other mammalian species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Importantly, PD-L1 expression was confirmed in EMM tumor tissues by immunohistochemistry. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These results suggest that our anti-PD-L1 mAbs may be useful for investigating the expression and role of the equine PD-1/PD-L1 pathway. Further research is required to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horse.

immunology↗

bioRxiv2017

Structural analyses reveal that the ATG2A-WIPI4 complex functions as a membrane tether for autophagosome biogenesis

Autophagy is an enigmatic cellular process in which double-membrane compartments, called autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the phosphatidylinositol-3-phosphate (PI3P)-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

biochemistry↗

bioRxiv2017

Bidirectional Changes Of Hepatic And Peripheral Insulin Clearance Characterize Abnormal Temporal Patterns Of Serum Insulin Concentration In Diabetic Subjects

Insulin plays a central role in glucose homeostasis, and impairment of insulin action causes glucose intolerance and leads to type 2 diabetes mellitus (T2DM). A decrease in the transient peak and sustained increase of circulating insulin by glucose infusion accompany T2DM pathogenesis. However, the mechanism underlying this abnormal temporal pattern of circulating insulin concentration remains unknown. Here we show that bidirectional changes of hepatic and peripheral insulin clearance characterize this abnormal temporal pattern of circulating insulin concentration during the progression of T2DM. We developed a mathematical model using a hyperglycemic and hyperinsulinemic-euglycemic clamp in 111 subjects, including healthy normoglycemic and diabetic subjects. The hepatic and peripheral insulin clearance significantly increase and decrease, respectively, during the progression of glucose intolerance. The increased hepatic insulin clearance reduces the amplitude of circulating insulin concentration, whereas the decreased peripheral insulin clearance changes the temporal patterns of circulating insulin concentration from transient to sustained. These results provide insight that may be useful in treating T2DM associated with aberrant insulin clearance.

systems biology↗