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The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1–CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.Cryo-EM structure of the LAT1–CD98hc heterodimer in complex with two antibodies offers insights into the assembly and function of LAT1–CD98hc, and reveals the epitopes targeted by the potentially therapeutic antibodies with an antitumor activity.

Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Red-light-activated channelrhodopsins are of particular interest, because red light penetrates deeper into biological tissues and also enables dual-color experiments in combination with blue-light-activated optogenetic tools. Here we report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama , Chrimson, at 2.6 Å resolution. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for Chrimson’s red light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, we engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics. Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Here, the authors report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama at 2.6 Å resolution.

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

Background Sugar-protein glycocalyx coats healthy endothelium, but its ultrastructure is not well described. Our aim was to determine the three-dimensional ultrastructure of capillary endothelial glycocalyx in the heart, kidney, and liver, where capillaries are, respectively, continuous, fenestrated, and sinusoidal. Methods Tissue samples were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx. Results Scanning and transmission electron microscopy revealed that the endothelial glycocalyx layer in continuous and fenestrated capillaries was substantially thicker than in sinusoids. In the heart, the endothelial glycocalyx presented as moss- or broccoli-like and covered the entire luminal endothelial cell surface. In the kidney, the glycocalyx appeared to nearly occlude the endothelial pores of the fenestrated capillaries and was also present on the surface of the renal podocytes. In sinusoids of the liver, glycocalyx covered not only the luminal side but also the opposite side, facing the space of Disse. In a mouse lipopolysaccharide-induced experimental endotoxemia model, the capillary endothelial glycocalyx was severely disrupted; that is, it appeared to be peeling off the cells and clumping. Serum concentrations of syndecan-1, a marker of glycocalyx damage, were significantly increased 24 h after administration of lipopolysaccharide. Conclusions In the present study, we visualized the three-dimensional ultrastructure of endothelial glycocalyx in healthy continuous, fenestrated, and sinusoidal capillaries, and we also showed their disruption under experimental endotoxemic conditions. The latter may provide a morphological basis for the microvascular endothelial dysfunction associated with septic injury to organs.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Endothelial disorders are related to various diseases. An initial endothelial injury is characterized by endothelial glycocalyx injury. We aimed to evaluate endothelial glycocalyx injury by measuring serum syndecan-1 concentrations in patients during comprehensive medical examinations. A single-center, prospective, observational study was conducted at Asahi University Hospital. The participants enrolled in this study were 1313 patients who underwent comprehensive medical examinations at Asahi University Hospital from January 2018 to June 2018. One patient undergoing hemodialysis was excluded from the study. At enrollment, blood samples were obtained, and study personnel collected demographic and clinical data. No treatments or exposures were conducted except for standard medical examinations and blood sample collection. Laboratory data were obtained by the collection of blood samples at the time of study enrolment. According to nonlinear regression, the concentrations of serum syndecan-1 were significantly related to age (p = 0.016), aspartic aminotransferase concentration (AST, p = 0.020), blood urea nitrogen concentration (BUN, p = 0.013), triglyceride concentration (p < 0.001), and hematocrit (p = 0.006). These relationships were independent associations. Endothelial glycocalyx injury, which is reflected by serum syndecan-1 concentrations, is related to age, hematocrit, AST concentration, BUN concentration, and triglyceride concentration.

Background Direct hemoperfusion with polymyxin B-immobilized fiber column (PMX-DHP) could improve the hemodynamic status of septic shock patients. As PMX-DHP is an invasive and costly procedure, it is desirable to estimate the therapeutic effect before performing the therapy. However, it is still unclear when this therapy should be started and what type of sepsis it should be employed for. In this study, we retrospectively examined the clinical effect of patients treated with PMX-DHP by using central venous pressure (CVP). Methods Seventy patients who received PMX-DHP for septic shock during the study period were recruited and divided into a low CVP group ( n  = 33, CVP < 12 mmHg) and a high CVP group ( n  = 37, CVP≧12 mmHg). The primary endpoint was vasopressor dependency index at 24 hours after starting PMX-DHP, and the secondary endpoint was the 28-day survival rate. Additionally, we performed a multivariate linear regression analysis on the difference in the vasopressor dependency index. Results The vasopressor dependency index significantly improved at 24 h in the low CVP group (0.33 to 0.16 mmHg −1 ; p  < 0.01) but not in the high CVP group (0.43 to 0.34 mmHg −1 ; p  = 0.41), and there was a significant difference between the two groups in the index at 24 h ( p  = 0.02). The 28-day survival rate was higher in the low CVP group (79 vs. 43 %; p  < 0.01). Multivariate linear regression analysis showed that CVP ( p  = 0.04) was independently associated with the difference in the vasopressor dependency index. Conclusions Our study indicates that the clinical effect of PMX-DHP for septic shock patients with higher CVP (≧12 mmHg) might be limited and that the initial CVP when performing PMX-DHP could function as an independent prognostic marker for the hemodynamic improvement.

The L-type amino acid transporter 1 (LAT1) transports large neutral amino acids and drugs across the plasma membrane and is crucial for nutrient uptake, brain drug delivery and tumor growth. LAT1 is a unique solute carrier that forms a disulfide-linked heterodimer with the cell-surface glycoprotein CD98 heavy chain (CD98hc), but the mechanisms of its molecular assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.4 Å resolution, revealing the hitherto unprecedented architecture of a solute carrier-glycoprotein heterocomplex. LAT1 features a canonical LeuT-fold while exhibiting an unusual loop structure on transmembrane helix 6, creating an extended cavity to accommodate bulky hydrophobic amino acids and drugs. CD98hc engages with LAT1 through multiple interactions, not only in the extracellular and transmembrane domains but also in the interdomain linker. The heterodimer interface features multiple sterol molecules, corroborating previous biochemical data on the role of cholesterols in heterodimer stabilization. We also visualized the binding modes of two anti-CD98 antibodies and show that they recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities despite the glycan heterogeneity. Furthermore, we mapped disease-causing mutations onto the structure and homology models, which rationalized some of the phenotypes of SLC3- and SLC7-related congenital disorders. Together, these results shed light on the principles of the structural assembly between a glycoprotein and a solute carrier, and provide a template for improving preclinical drugs and therapeutic antibodies targeting LAT1 and CD98.

Chemotherapy drugs, such as gemcitabine and cisplatin (GC), are frequently administered to patients with advanced urothelial carcinoma, however the influence of the gut microbiota on their action is unclear. Thus, we investigated the effects of GC on the gut microbiome and determined whether oral supplementation with a probiotics mixture of Lactobacillus casei Shirota and Bifidobacterium breve enhanced the anti‐tumor immune response. After subcutaneous inoculation with MBT2 murine bladder cancer cells, syngenic C3H mice were randomly allocated into eight groups. The gut microbiome cluster pattern was altered in both the GC and oral probiotics groups (p = 0.025). Both tumor‐bearing conditions (no treatment) and GC chemotherapy influenced Pseudoclostridium, Robinsoniella, Merdimonas, and Phocea in the gut. Furthermore, comparison of the GC‐treated and GC + probiotics groups revealed an association of four methyltransferase family enzymes and two short‐change fatty acid‐related enzymes with oral probiotics use. A significant difference in tumor volume was observed between the GC and GC + probiotics groups at week 2 of treatment. Additionally, decreased recruitment of cancer‐associated fibroblasts and regulatory T cells, and activation of CD8+ T cells and dendritic cells were observed in the tumor microenvironment. Our findings reveal the positive effects of a probiotics mixture of Lactobacillus and Bifidobacterium in enhancing anti‐tumor effects through the gut–tumor immune response axis. Future clinical trials are needed to evaluate the full benefits of this novel supplement with oral probiotics in patients with advanced urothelial carcinoma. We investigated the effects of gemcitabine plus cisplatin (GC) chemotherapy on the gut microbiome and determined whether oral supplementation with a probiotics mixture of Lactobacillus casei Shirota and Bifidobacterium breve could improve the anti‐tumor response induced by GC chemotherapy. We found altered levels of gut microbiota and increased infiltration of CD8+ T cells and dendritic cells in the tumor microenvironment of GC + probiotics group compared with the GC group. We believe that our study makes a significant contribution to the literature, because our findings highlight the anti‐tumor responses induced by a probiotics mixture of Lactobacillus and Bifidobacterium during GC chemotherapy.

Although tumor-infiltrating regulatory T (Treg) cells play a pivotal role in tumor immunity, how Treg cell activation are regulated in tumor microenvironments remains unclear. Here, we found that mice deficient in the inhibitory immunoreceptor CD300a on their dendritic cells (DCs) have increased numbers of Treg cells in tumors and greater tumor growth compared with wild-type mice after transplantation of B16 melanoma. Pharmacological impairment of extracellular vesicle (EV) release decreased Treg cell numbers in CD300a-deficient mice. Coculture of DCs with tumor-derived EV (TEV) induced the internalization of CD300a and the incorporation of EVs into endosomes, in which CD300a inhibited TEV-mediated TLR3–TRIF signaling for activation of the IFN-β-Treg cells axis. We also show that higher expression of CD300A was associated with decreased tumor-infiltrating Treg cells and longer survival time in patients with melanoma. Our findings reveal the role of TEV and CD300a on DCs in Treg cell activation in the tumor microenvironment.