CD98 Increases Renal Epithelial Cell Proliferation by Activating MAPKs

CD98 heavy chain (CD98hc) is a multifunctional transmembrane spanning scaffolding protein whose extracellular domain binds with light chain amino acid transporters (Lats) to form the heterodimeric amino acid transporters (HATs). It also interacts with β1 and β3 integrins by its transmembrane and cytoplasmic domains. This interaction is proposed to be the mechanism whereby CD98 mediates cell survival and growth via currently undefined signaling pathways. In this study, we determined whether the critical function of CD98-dependent amino acid transport also plays a role in cell proliferation and defined the signaling pathways that mediate CD98-dependent proliferation of murine renal inner medullary collecting duct (IMCD) cells. We demonstrate that downregulating CD98hc expression resulted in IMCD cell death. Utilizing overexpression studies of CD98hc mutants that either lacked a cytoplasmic tail or were unable to bind to Lats we showed that CD98 increases serum-dependent cell proliferation by a mechanism that requires the CD98hc cytoplasmic tail. We further demonstrated that CD98-dependent amino acid transport increased renal tubular epithelial cell proliferation by a mechanism that does not require the CD98hc cytoplasmic tail. Both these mechanisms of increased renal tubular epithelial cell proliferation are mediated by Erk and p38 MAPK signaling. Although increased amino transport markedly activated mTor signaling, this pathway did not alter cell proliferation. Thus, these studies demonstrate that in IMCD cells, the cytoplasmic and extracellular domains of CD98hc regulate cell proliferation by distinct mechanisms that are mediated by common MAPK signaling pathways

Assessment of humoral immunity to SARS-CoV-2 by a sample examination of medical workers in a large specialized multidisciplinary hospital

Introduction. The assessment of specific IgG antibodies to RBD Spike SARS-CoV-2 and their quantitation permit to calculate the intensity of immunity to COVID-19, i.e. to determine the level of immunity to infection, the risk of infection, the severity of the disease, as well as the ability to prevent death. Meanwhile, the protective level of antibodies is not determined. Therefore, determining the nature of immunity and quantitation of IgG antibodies to RBD Spike SARS-CoV-2 make it possible to assess the effectiveness of preventive measures and correct them in a timely manner. The aim is to determine the presence of IgG antibodies to RBD Spike SARS-CoV-2, their concentrations, and the nature of humoral immunity in different age and occupational groups of employees in a closed-type hospital after the completed vaccination with "Gam-Covid-Vac" vaccine. Materials and methods. The blood sera of 310 members of medical staff who received a full course of immunization with the "Gam-Covid-Vac" vaccine were tested using "SARS-CoV-2-ELISA-IgG" kit according to instructions provided in 21.20.23-004-28597318-2020, RU No. RZN 2021/15898. IgG antibodies to RBD Spike SARS-CoV-2 were quantitated against WHO standard NIBSC 20/136. Results. Specific IgG antibodies to RBD Spike SARS-CoV-2 were found in 92.9% of the examined individuals, including 67.4% having hybrid immunity (both vaccine- and infection- induced), and 25.5% having post-vaccination immunity after immunization with the "Gam-Covid-Vac" vaccine; 7.1% participants were nonimmune. A higher level of IgG antibodies to RBD Spike SARS-CoV-2 was detected in the group of individuals with hybrid immunity (p 0.01). Only 11.6% of employees had a protective antibody level of more than 300 BAU/ml. Discussion. Most employees with hybrid immunity were identified in the older age groups and in the junior medical staff. The results of this serological study, taking into account the age and professional aspects, can serve as the basis for adjusting preventive measures in medical institutions

Factor XII and kininogen asymmetric assembly with gC1qR/C1QBP/P32 is governed by allostery

The contact system is composed of Factor XII (FXII), prekallikrein (PK) and co-factor kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+ dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion with residues Arg36 and Arg65 forming contacts with two distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII binding site and a comparison with the ligand free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+ binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with SPR demonstrate the gC1qR Zn2+ site contributes to FXII binding and plasma based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only one high affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher order 500kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors which may be a prelude for initiating the cascades which drive bradykinin generation and the intrinsic pathway of coagulation

Crystal structures of the recombinant β-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics

© 2019 International Union of Crystallography. Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (βFXIIaHis) with yields of ~1–2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-βFXIIaHis). Crystal structures were determined of MBP-βFXIIaHis in complex with the inhibitor d-Phe-ProArg chloromethyl ketone (PPACK) and of βFXIIaHis in isolation. The βFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2–P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2–P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and antiinflammatory agents

A randomised trial of intrapericardial bleomycin for malignant pericardial effusion with lung cancer (JCOG9811)

Safety and efficacy of intrapericardial (ipc) instillation of bleomycin (BLM) following pericardial drainage in patients with malignant pericardial effusion (MPE) remain unclear. Patients with pathologically documented lung cancer, who had undergone pericardial drainage for MPE within 72 h of enrolment, were randomised to either arm A (observation alone after drainage) or arm B (ipc BLM at 15 mg, followed by additional ipc BLM 10 mg every 48 h). The drainage tube was removed when daily drainage was 20 ml or less. The primary end point was survival with MPE control (effusion failure-free survival, EFFS) at 2 months. Eighty patients were enrolled, and 79 were eligible. Effusion failure-free survival at 2 months was 29% in arm A and 46% in arm B (one-sided P=0.086 by Fisher's exact test). Arm B tended to favour EFFS, with a hazard ratio of 0.64 (95% confidence interval: 0.40–1.03, one-sided P=0.030 by log-rank test). No significant differences in the acute toxicities or complications were observed. The median survival was 79 days and 119 days in arm A and arm B, respectively. This medium-sized trial failed to show statistical significance in the primary end point. Although ipc BLM appeared safe and effective in the management of MPE, the therapeutic advantage seems modest

Low exposure long-baseline neutrino oscillation sensitivity of the DUNE experiment

The Deep Underground Neutrino Experiment (DUNE) will produce world-leading neutrino oscillation measurements over the lifetime of the experiment. In this work, we explore DUNE's sensitivity to observe charge-parity violation (CPV) in the neutrino sector, and to resolve the mass ordering, for exposures of up to 100 kiloton-megawatt-years (kt-MW-yr). The analysis includes detailed uncertainties on the flux prediction, the neutrino interaction model, and detector effects. We demonstrate that DUNE will be able to unambiguously resolve the neutrino mass ordering at a 3$\sigma$ (5$\sigma$) level, with a 66 (100) kt-MW-yr far detector exposure, and has the ability to make strong statements at significantly shorter exposures depending on the true value of other oscillation parameters. We also show that DUNE has the potential to make a robust measurement of CPV at a 3$\sigma$ level with a 100 kt-MW-yr exposure for the maximally CP-violating values \delta_{\rm CP}} = \pm\pi/2. Additionally, the dependence of DUNE's sensitivity on the exposure taken in neutrino-enhanced and antineutrino-enhanced running is discussed. An equal fraction of exposure taken in each beam mode is found to be close to optimal when considered over the entire space of interest

Low exposure long-baseline neutrino oscillation sensitivity of the DUNE experiment

The Deep Underground Neutrino Experiment (DUNE) will produce world-leading neutrino oscillation measurements over the lifetime of the experiment. In this work, we explore DUNE's sensitivity to observe charge-parity violation (CPV) in the neutrino sector, and to resolve the mass ordering, for exposures of up to 100 kiloton-megawatt-years (kt-MW-yr). The analysis includes detailed uncertainties on the flux prediction, the neutrino interaction model, and detector effects. We demonstrate that DUNE will be able to unambiguously resolve the neutrino mass ordering at a 3σ (5σ) level, with a 66 (100) kt-MW-yr far detector exposure, and has the ability to make strong statements at significantly shorter exposures depending on the true value of other oscillation parameters. We also show that DUNE has the potential to make a robust measurement of CPV at a 3σ level with a 100 kt-MW-yr exposure for the maximally CP-violating values \delta_{\rm CP}} = \pm\pi/2. Additionally, the dependence of DUNE's sensitivity on the exposure taken in neutrino-enhanced and antineutrino-enhanced running is discussed. An equal fraction of exposure taken in each beam mode is found to be close to optimal when considered over the entire space of interest