D2 Domain Research Articles

Binding of the Mycobacterium tuberculosis EccCb1 ATPase double hexameric ring to the EsxAB virulence factor is enhanced by ATP.

The EccC enzyme of Mycobacterium tuberculosis ESX-1 secretion system is involved in EsxAB virulence factor secretion and offers an attractive target for antivirulence inhibitors development against M. tuberculosis. The EccCb1 polypeptide of the EccC enzyme contains two Ftsk/SpoIIIE type ATPase domains (D2 and D3) and binds to the EsxAB factor at the C-terminal region of the D3 domain. In the current study, we have determined a low-resolution structure of EccCb1, and its mechanism involved in ATPase activity and EsxAB factor binding. Small-angle X-ray scattering data yielded a double hexameric ring structure of EccCb1 in solution and was further confirmed by SEC-MALS and dynamic light scattering. ATPase activity of wild-type, D2, and D3 mutants showed that D2-K90A and D3-K382A mutations led to a complete loss of enzyme activity. The full-length EccCb1 showed ∼3.7-fold lower catalytic efficiency than D2 domain and ∼1.7 fold lower than D3 domain. The EsxAB factor binds EccCb1 with Kd ∼ 11.3 ± 0.6 nM and its affinity is enhanced ∼2 fold in presence of ATP + Mg2+. These data indicate the involvement of ATPase activity in EsxAB factor translocation. Molecular dynamics simulation on wild-type, ATP + Mg2+, and EsxAB + ATP + Mg2+ bound EccCb1 double-ring structure showed enhanced stability of enzyme upon ATP + Mg2+ and EsxAB binding. Overall, our study showed a low-resolution structure of EccCb1, and the mechanism involved in ATPase activity and EsxAB factor recognition, which can be targeted for the development of antivirulence drugs against M. tuberculosis.

On the Influences of Urbanization on the Extreme Rainfall over Zhengzhou on 20 July 2021: A Convection-Permitting Ensemble Modeling Study

This study investigates the influences of urban land cover on the extreme rainfall event over the Zhengzhou city in central China on 20 July 2021 using the Weather Research and Forecasting model at a convection-permitting scale [1-km resolution in the innermost domain (d3)]. Two ensembles of simulation (CTRL, NURB), each consisting of 11 members with a multi-layer urban canopy model and various combinations of physics schemes, were conducted using different land cover scenarios: (i) the real urban land cover, (ii) all cities in d3 being replaced with natural land cover. The results suggest that CTRL reasonably reproduces the spatiotemporal evolution of rainstorms and the 24-h rainfall accumulation over the key region, although the maximum hourly rainfall is underestimated and displaced to the west or southwest by most members. The ensemble mean 24-h rainfall accumulation over the key region of heavy rainfall is reduced by 13%, and the maximum hourly rainfall simulated by each member is reduced by 15–70 mm in CTRL relative to NURB. The reduction in the simulated rainfall by urbanization is closely associated with numerous cities/towns to the south, southeast, and east of Zhengzhou. Their heating effects jointly lead to formation of anomalous upward motions in and above the planetary boundary layer (PBL), which exaggerates the PBL drying effect due to reduced evapotranspiration and also enhances the wind stilling effect due to increased surface friction in urban areas. As a result, the lateral inflows of moisture and high-θe (equivalent potential temperature) air from south and east to Zhengzhou are reduced.

Prior Experience of Physiotherapy Treatment Among the General Public

Background: Physiotherapy services continue to be an essential component in patient recovery throughout hospital care, from acute to subacute, palliative extended care and outpatient services. Physiotherapists are also concerned as their role in working with a physical or mental condition that limits a person's movement as they deal through active rehabilitation and health promotions. The physiotherapists gain experience through volunteer work with local senior care centre or local clinic. This study aims to find out the Prior experience of Physiotherapy treatment among General public. Methodology A qualitative study conducted with 105 willing participants aged more than 40 years, of both sexes who had a prior experience of physiotherapy treatment at least once in recent 2 years. A self-reported questionnaire was given and the responses recorded. Results It shows that Domain (D1-45, D2 -59, D4-53, D5-59, D6-61, D7-60, D8-57, D9-55) participants reported maximum responses towards agreeing, whereas Domain D3 received maximum responses of 46 participants towards disagreeing. Conclusion The study concluded that the Prior experience of Physiotherapy treatment among General public using a self-administered questionnaire showed more positive responses towards the Physiotherapy treatment and Physiotherapists.

Genetic distance and phylogenetic relationships of some Echinostoma species (E. malayanum, E. revolutum, E. miyagawai) and Hypoderaeum conoideum (family Echinostomatidae) inferred from partial 28S rDNA sequence analysis

Echinostomiasis is a neglected disease caused by the intestinal flukes (family Echinostomatidae, suborder Echinostomata) and is common in communities in Asian countries, such as India, Indonesia, the Philippines, China, Malaysia, Singapore, Korea, Japan, Thailand, Laos, Cambodia and Vietnam. The genetic markers from the nuclear ribosomal transcription units are commonly used in genetic studies and phylogenetic analyses. A portion of the 28S rDNA sequence (domains D1–D3, of 1062–1067 bp for the final use) was obtained from the zoonotic Echinostoma malayanum (strain E/Amala-EMI3-TH), E. revolutum (strain Erevo-MSD15-TH), E. miyagawai (Emiya-RED11-TH), and Hypoderaeum conoideum (Hcono-RED42-TH) species; and used to perform an alignment for genetic distance estimation and phylogenetic analysis. The alignment was performed using 62 strains of 42 species from 19 genera of the family Echinostomatidae, including Echinoparyphium, Echinostoma, Artyfechinostomum, Patagifer, Neoacanthoparyphium, Hypoderaeum, Echinoparyphium, Drepanocephalus, Euparyphium, Chaunocephalus, Neopetasiger, Ribeiroia, Cathaemasia, Rhopalias, Isthmiophora, Petasiger, Moliniella, Pegosomum, and Schistosoma (family Schistosomatidae). The genetic distance estimation among 16 strains/10 species has shown a low intra-specific divergence level between strains within the same species, such as E. miyagawai (0–0.10%), E. revolutum (0.10–0.50%), and H. conoideum (0–0.10%), while between strains within the same genus it was higher (normally over 1.0%) and among strains/species between genera it was the highest (3.06–4.12%). The 28S rDNA sequence analysis and phylogenetic relationship well supported the Echinostoma/ Artyfechinostomum malayanum intergeneric taxonomy and the topology indicated clear, well-supported positions of member species in different genera in the family Echinostomatidae of the suborder Echinostomata. More sensu lato samples of the genera, are required for sequencing, particularly those of zoonotic species in the five genera: Artyfechinostomum, Echinostoma, Hypoderaeum, Echinoparyphium, and Isthmiophora. The resultant mitochondrial and nuclear data obtained from these species will be a good source to use to clearly assess the taxonomic and generic relationships.

PDGF-D Prodomain Differentially Inhibits the Biological Activities of PDGF-D and PDGF-B

As a member of PDGF/VEGF (Platelet-derived growth factor/ Vascular endothelial growth factor) growth factors, PDGF-D regulates blood vessel development, wound healing, innate immunity, and organogenesis. Unlike PDGF-A and PDGF-B, PDGF-D has an additional CUB (Complement C1r/C1s, Uegf, Bmp1) domain at the N-terminus of its growth factor domain, and thus it is secreted in a latent, inactive complex, which needs to be proteolytically activated for its biological activities. However, how the CUB domain contributes to the latency and activation of the growth factor remains elusive. In this study, we modeled the dimeric structure of PDGF-D pro-complex and studied the inhibitory functions of PDGF-D prodomain on PDGF-B and PDGF-D signaling. In our model, the growth factor domain of PDGF-D forms a VEGF-D-like dimer through their β1 and β3 interactions. The hinge and CUB domains of PDGF-D bind at the opposite sides of the growth factor domain and exclude the PDGFR-β (PDGF Receptor β) D2 and D3 domains from recognizing the growth factor. In addition, we verified that PDGF-D prodomain could inhibit both PDGF-B and PDGF-D mediated PDGFR-β transphosphorylation in a dose-dependent manner. However, PDGF-D prodomain could only inhibit the proliferation of NIH 3T3 cells stimulated by PDGF-D but not by PDGF-B, indicating its differential inhibitory activities toward PDGF-B and PDGF-D signaling.

Insights into the atypical autokinase activity of the Pseudomonas aeruginosa GacS histidine kinase and its interaction with RetS

Virulence in Pseudomonas aeruginosa (PA) depends on complex regulatory networks, involving phosphorelay systems based on two-component systems (TCSs). The GacS/GacA TCS is a master regulator of biofilm formation, swarming motility, and virulence. GacS is a membrane-associated unorthodox histidine kinase (HK) whose phosphorelay signaling pathway is inhibited by the RetS hybrid HK. Here we provide structural and functional insights into the interaction of GacS with RetS. The structure of the GacS-HAMP-H1 cytoplasmic regions reveals an unusually elongated homodimer marked by a 135Å long helical bundle formed by the HAMP, the signaling helix (S helix) and the DHp subdomain. The HAMP and S helix regions are essential for GacS signaling and contribute to the GacS/RetS binding interface. The structure of the GacS D1 domain together with the discovery of an unidentified functional ND domain, essential for GacS full autokinase activity, unveils signature motifs in GacS required for its atypical autokinase mechanism.

A novel nanoluciferase transgenic reporter measures proteinuria in zebrafish

The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies.

A conformational transition of the D′D3 domain primes von Willebrand factor for multimerization

AbstractVon Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D′D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D′D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D′D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

VCP interaction with HMGB1 promotes hepatocellular carcinoma progression by activating the PI3K/AKT/mTOR pathway

BackgroundHepatocellular carcinoma (HCC) is the most common pathological type of liver cancer. Valosin-containing protein (VCP) is a member of the AAA-ATPase family associated with multiple molecular functions and involved in tumor metastasis and prognosis. However, the role of VCP in HCC progression is still unclear.MethodsWe examined the expression of VCP in HCC using the RNA sequencing and microarray data from public databases and measured it in clinical samples and cell lines by western blot, and immunohistochemistry (IHC). We also evaluated the correlation between VCP and clinical features. The VCP-interacting proteins were identified by co-immunoprecipitation combined with mass spectrometry (CoIP/MS). The underlying molecular mechanisms were investigated using in vitro and in vivo models of HCC.ResultsWe found that VCP expression is significantly increased in tumor tissues and is associated with advanced TNM stages and poorer prognosis in HCC patients. In vitro analyses revealed that VCP overexpression promoted HCC cell proliferation, migration, and invasion via PI3K/AKT/mTOR pathway activation. Conversely, VCP knockdown resulted in the reverse phenotypes. In vivo studies indicated that up-regulated VCP expression accelerated tumor growth in a subcutaneous HCC model. The D1 domain of VCP and A box of HMGB1 were identified as the critical regions for their interaction, and D1 area was required for the tumor-promoting effects induced by VCP expression. VCP enhanced the protein stability of HMGB1 by decreasing its degradation via ubiquitin–proteasome process. Inhibition of HMGB1 markedly attenuated VCP-mediated HCC progression and downstream activation of PI3K/AKT/mTOR signals.ConclusionCollectively, these findings demonstrate that VCP is a potential prognostic biomarker in HCC and exhibits oncogenic roles via PI3K/AKT/mTOR pathway activation. HMGB1 played an essential role in VCP-mediated HCC progression, indicating that VCP and HMGB1 are potential therapeutic targets in human HCC.

Bending stiffness characterization of Bacillus subtilis’ flagellar filament

The filament of a bacterial flagellum is a tube-like organelle made of a single protein—flagellin—and assembled into multiple polymorphic forms. The filament can be further discretized into four subunit domains (D0, D1, D2, and D3) along the radial direction. However, it remains unclear which subunit domain plays an important role in regulating the rigidity of the filament. In this article, we address how the absence of two outer subunit domains (D2 and D3) affects the bending stiffness of the bacterium B. subtilis’ flagellar filament. We first shear off flagellar filaments from the cell body, anchor one of its ends to the wall of a microfluidic channel, and correlate the elongation of the filament with the driving background flow. A numerical model is then applied to determine the bending stiffness of the filament. We find that the bending stiffness does not change drastically when the filament transforms from normal to hyperextended forms, which is estimated to be 2–3 pN⋅μm2. Furthermore, B. subtilis’ flagellar filament has similar bending stiffness to Salmonella’s, although the radius of the former is almost half of that of the latter, suggesting that the rigidity comes from the inner D0 and D1 subunit domains.

Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium

ABSTRACT The genus Pythium (nom. cons.) sensu lato (s.l.) is composed of many important species of plant pathogens. Early molecular phylogenetic studies suggested paraphyly of Pythium, which led to a formal proposal by Uzuhashi and colleagues in 2010 to split the genus into Pythium sensu stricto (s.s.), Elongisporangium, Globisporangium, Ovatisporangium (= Phytopythium), and Pilasporangium using morphological characters and phylogenies of the mt cytochrome c oxidase subunit 2 (cox2) and D1–D2 domains of nuc 28S rDNA. Although the split was fairly justified by the delineating morphological characters, there were weaknesses in the molecular analyses, which created reluctance in the scientific community to adopt these new genera for the description of new species. In this study, this issue was addressed using phylogenomics. Whole genomes of 109 strains of Pythium and close relatives were sequenced, assembled, and annotated. These data were combined with 10 genomes sequenced in previous studies. Phylogenomic analyses were performed with 148 single-copy genes represented in at least 90% of the taxa in the data set. The results showed support for the division of Pythium s.l. The status of alternative generic names that have been used for species of Pythium in the past (e.g., Artotrogus, Cystosiphon, Eupythium, Nematosporangium, Rheosporangium, Sphaerosporangium) was investigated. Based on our molecular analyses and review of the Pythium generic concepts, we urge the scientific community to adopt the generic names Pythium, Elongisporangium, Globisporangium, and their concepts as proposed by Uzuhashi and colleagues in 2010 in their work going forward. In order to consolidate the taxonomy of these genera, some of the recently described Pythium spp. are transferred to Elongisporangium and Globisporangium.

Immunohistochemical localization of receptor protein tyrosine phosphatase γ in mouse renal proximal tubule, thick ascending limb and distal convoluted tubule

Most normal physiological processes require that both intracellular (i) and extracellular (o) pH be maintained within a tight range. pHo is contingent on arterial (a) pH, which is determined by the ratio of the arterial carbon dioxide to the arterial bicarbonate concentration, [CO2]a/[HCO3−]a. Pulmonary ventilation controls [CO2]a. The kidney controls [HCO3−]a by adjusting HCO3− transport rates (JHCO3) across the basal membranes of particular nephron epithelia into the blood to maintain normal pHa, or compensate for an acid‐base insult. In an effort to identify the sensor proteins for pHa, [CO2]a, or [HCO3−]a, our laboratory previously reported that, in isolated proximal tubules (PTs) from receptor protein tyrosine phosphatase γ (RPTPγ) knockout (KO) mice, JHCO3 is insensitive to changes in the basolateral [CO2] or [HCO3−]. Furthermore, RPTPγ‐KO mice have a deficiency in defending against a whole‐body metabolic acidosis. These data implicate RPTPγ as a major [CO2] or [HCO3−] sensor. RPTPγ possesses an extracellular catalytically inactive carbonic‐anhydrase‐like domain (CALD) and fibronectin type III (FNIII) domain, a single transmembrane helix, and intracellular domains D1 and D2. Dimerization of RPTPγ presumably allows the inhibitory D2 of one monomer to inhibit the otherwise catalytically active D1 of the other monomer. Thus, the dimerization/monomerization state controls downstream signaling. We hypothesize that the binding of CO2 or HCO3− to the CALD alters the CALD conformation, thereby transmitting signals that control the dimerization status of RPTPγ and thereby regulate downstream effects on JHCO3. We hypothesize that RPTPγ is localized in nephron segments important for regulating HCO3− transport/reabsorption, in the basal membranes that face the blood. To determine the location of RPTPγ in wild‐type mouse kidney cryosections, we use a chicken IgY anti‐RPTPγ antibody (Ab) raised against an epitope within the FNIII domain, and co‐stain with rabbit polyclonal Abs against the Na/HCO3 cotransporter (NBCe1‐A) as a PT marker, the Na/K/Cl cotransporter 2 (NKCC2) as a thick ascending limb (TAL) marker, and the Na/Cl cotransporter (NCC) as a distal convoluted tubule (DCT) marker. Imaging sections by confocal microscopy, we observe in 3 WT mice that RPTPγ localizes primarily to the PT basal membrane. However, 54% of PTs (NBCe1‐A positive) also exhibit apical RPTPγ staining of comparable intensity to the RPTPγ staining observed in the basal membrane. We also observe both apical and basal RPTPγ staining in Bowman’s capsules. We stained additional sections from the same animals with a rabbit polyclonal primary Ab that targets the RPTPγ CALD domain. In these sections, co‐stained with the anti‐FNIII Ab, we similarly observe both apical and basal staining in PTs. We observe RPTPγ staining in a minority of TALs, and at much lower intensity than observed in the PT. We observe minimal to no RPTPγ staining in the DCT. We validated the specificity of the RPTPγ Abs on RPTPγ‐KO kidney sections, which exhibit low background levels of fluorescence for both the anti‐FNIII and anti‐CALD Abs. In conclusion, these data confirm RPTPγ expression in PT basal membranes, but also describe the first examples of apical RPTPγ expression in renal epithelia.

Flagellin/Virus-like Particle Hybrid Platform with High Immunogenicity, Safety, and Versatility for Vaccine Development.

Hepatitis B core (HBc) virus-like particles (VLPs) and flagellin are highly immunogenic and widely explored vaccine delivery platforms. Yet, HBc VLPs mainly allow the insertion of relatively short antigenic epitopes into the immunodominant c/e1 loop without affecting VLP assembly, and flagellin-based vaccines carry the risk of inducing systemic adverse reactions. This study explored a hybrid flagellin/HBc VLP (FH VLP) platform to present heterologous antigens by replacing the surface-exposed D3 domain of flagellin. FH VLPs were prepared by the insertion of flagellin gene into the c/e1 loop of HBc, followed by E. coli expression, purification, and self-assembly into VLPs. Using the ectodomain of influenza matrix protein 2 (M2e) and ovalbumin (OVA) as models, we found that the D3 domain of flagellin could be replaced with four tandem copies of M2e or the cytotoxic T lymphocyte (CTL) epitope of OVA without interfering with the FH VLP assembly, while the insertion of four tandem copies of M2e into the c/e1 loop of HBc disrupted the VLP assembly. FH VLP-based M2e vaccine elicited potent anti-M2e antibody responses and conferred significant protection against multiple influenza A viral strains, while FljB- or HBc-based M2e vaccine failed to elicit significant protection. FH VLP-based OVA peptide vaccine elicited more potent CTL responses and protection against OVA-expressing lymphoma or melanoma challenges than FljB- or HBc-based OVA peptide vaccine. FH VLP-based vaccines showed a good systemic safety, while flagellin-based vaccines significantly increased serum interleukin 6 and tumor necrosis factor α levels and also rectal temperature at increased doses. We further found that the incorporation of a clinical CpG 1018 adjuvant could enhance the efficacy of FH VLP-based vaccines. Our data support FH VLPs to be a highly immunogenic, safe, and versatile platform for vaccine development to elicit potent humoral and cellular immune responses.

Phenotypic plasticity, genetic structure, and systematic position of Neoechinorhynchus emyditoides Fisher, 1960 (Acanthocephala: Neoechinorhynchidae) a parasite of emydid turtles from the Nearctic and Neotropical regions.

AbstractThe taxonomy of the 10 recognizedNeoechinorhynchusspecies associated with emydid turtles is complex due to the morphological conservatism. In the present study, specimens ofN. emyditoidesfrom northern and southeastern Mexico exhibit great phenotypic plasticity on its diagnostic characteristics. We sequenced three molecular markers: the internal transcribed spacers ITS1, ITS2 and 5.8S gene, the D2 + D3 domains of the large subunit from nuclear DNA and cytochromecoxidase subunit I (cox1) from mitochondrial DNA. Sequences of the nuclear molecular markers were aligned and compared with other congeneric species associated with emydids available in GenBank. Phylogenetic analyses supported the polyphyly ofNeoechinorhynchus. The species from emydids formed a clade, which was subdivided into five subclades that correspond with each species analysed (N. pseudemydis,N. chrysemydis,N. emydis,N. schmidtiandN. emyditoides). To understand better the genetic structure ofN. emyditoidesa haplotype network was inferred with 29cox1sequences, revealing the presence of 13 haplotypes, two of which were shared and 11 were unique. The high values of fixation index,Fst(0.4227–0.8925) detected between the two populations from southeastern and the two from northern Mexico indicated low genetic flow among the populations. Our data suggest that theNeoechinorhynchusspecies associated with emydid turtles diversified in the eastern USA and that ofN. emyditoidesexpanded its distribution range reached southeastern Mexico.

Notes on the genus Micropsalliota (Agaricales, Basidiomycota) and the description of a new species from Southern Oman

The genus Micropsalliota is a group of saprotrophic mushrooms, characterized by small to medium-sized basidiomata with fibrillose-squamulose or glabrous pilei, ellipsoid to amygdaliform or cymbiform basidiospores and cheilocystidia that vary in shape from clavate to ventricose or lageniform with more or less a capitate apex. In this study, we described a new species of Micropsalliota from southern Oman. Species description is based on morphological features of basidiomata and phylogenetic analyses of nuc ribosomal DNA internal transcribed spacer (ITS) and D1–D2 domains of the 28S region. The new species Micropsalliota ventricocystidiata is characterized by a medium-sized basidiomata, with plano-convex pileus, covered with reddish-brown squamules and basidiospores are ellipsoid to amygdaliform with acute apex in side view and oblong in face view, measuring 7.5–8.5 × 4.5–5 µm. Morphological comparison of M. ventricocystidiata with respect to other species of the genus is provided. We discussed that species of the genus can be divided into two groups based on their pileus morphology and basidiospores size.

Identification of von Willebrand factor D4 domain mutations in patients of Afro‐Caribbean descent: In vitro characterization

BackgroundVon Willebrand disease was diagnosed in two Afro‐Caribbean patients and sequencing of the VWF gene (VWF) revealed the presence of multiple variants located throughout the gene, including variants located in the D4 domain of VWF: p.(Pro2145Thrfs*5) in one patient and p.(Cys2216Phefs*9) in the other patient. Interestingly, D4 variants have not been studied often. ObjectivesOur goal was to characterize how the D4 variants p.(Pro2145Thrfs*5) and p.(Cys2216Phefs*9) influenced VWF biosynthesis/secretion and functions using in vitro assays. MethodsRecombinant VWF (rVWF), mutant or wild‐type, was produced via transient transfection of the human embryonic kidney cell line 293T. The use of different tags for the wild‐type and the mutant allele allowed us to distinguish between the two forms when measuring VWF antigen in medium and cell lysates. Binding of rVWF to its ligands, collagen, factor VIII, ADAMTS13, and platelet receptors was also investigated. ResultsHomozygous expression of the p.(Cys2216Phefs*9)‐rVWF mutation resulted in an almost complete intracellular retention of the protein. Heterozygous expression led to secretion of almost exclusively wild‐type‐rVWF, logically capable of normal interaction with the different ligands. In contrast, the p.(Pro2145Thrfs*5)‐rVWF exhibited reduced binding to type III collagen and αIIbβ3 integrin compared to wild‐type‐rVWF. ConclusionsWe report two mutations of the D4 domains that induced combined qualitative and quantitative defects.

Galectin‐9 activates platelet ITAM receptors glycoprotein VI and C‐type lectin‐like receptor‐2

BackgroundPlatelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate‐binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets. ObjectiveIn this study, we investigated the role of galectin‐9 (Gal‐9) as a novel ligand for platelet glycoprotein VI (GPVI) and C‐type lectin‐like receptor 2 (CLEC‐2). MethodsPlatelet spreading, aggregation, and P‐selectin expression in response to Gal‐9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid‐phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal‐9 and GPVI, and immunoprecipitation for detecting CLEC‐2 phosphorylation. Wild‐type (WT), GPVI‐knockout (Gp6−/−), and GPVI and CLEC‐2‐double knockout (Gp6−/−/Gp1ba‐Cre‐Clec1bfl/fl) mice were used. ResultsWe have shown that recombinant Gal‐9 stimulates aggregation in human and mouse washed platelets dose‐dependently. Platelets from both species adhere and spread on immobilized Gal‐9 and express P‐selectin. Gal‐9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal‐9 stimulated tyrosine phosphorylation of CLEC‐2 and proteins known to lie downstream of GPVI and CLEC‐2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI‐deficient murine platelets exhibited significantly impaired aggregation in response to Gal‐9, which was further abrogated in GPVI and CLEC‐2‐double‐deficient platelets. ConclusionsWe have identified Gal‐9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC‐2.

Structural insights into collagen binding by platelet receptor glycoprotein VI

AbstractGlycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is an important contributor to the onset of thrombosis, heart attack, and stroke. Animal models of thrombosis have identified GPVI as a promising target for antithrombotic therapy. Although for many years the crystal structure of GPVI has been known, the essential details of its interaction with collagen have remained elusive. Here, we present crystal structures of the GPVI ectodomain bound to triple-helical collagen peptides, which reveal a collagen-binding site across the β-sheet of the D1 domain. Mutagenesis and binding studies confirm the observed binding site and identify Trp76, Arg38, and Glu40 as essential residues for binding to fibrillar collagens and collagen-related peptides (CRPs). GPVI binds a site on collagen comprising two collagen chains with the core formed by the sequence motif OGPOGP. Potent GPVI-binding peptides from Toolkit-III all contain OGPOGP; weaker binding peptides frequently contain a partial motif varying at either terminus. Alanine-scanning of peptide III-30 also identified two AGPOGP motifs that contribute to GPVI binding, but steric hindrance between GPVI molecules restricts the maximum binding capacity. We further show that no cooperative interactions could occur between two GPVI monomers binding to a stretch of (GPO)5 and that binding of ≥2 GPVI molecules to a fibril-embedded helix requires non-overlapping OGPOGP motifs. Our structure confirms the previously suggested similarity in collagen binding between GPVI and leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) but also indicates significant differences that may be exploited for the development of receptor-specific therapeutics.

Morphological and Phylogenetic Evidence Reveal Five New Telamonioid Species of Cortinarius (Agaricales) from East Asia.

Five new Cortinarius species, C. neobalaustinus, C. pseudocamphoratus, C. subnymphatus, C. wuliangshanensis and C. yanjiensis spp. nov., are proposed based on a combination of morphological and molecular evidence. Cortinarius neobalaustinus is characterized by a very weakly hygrophanous and yellowish-brown to brown pileus and small and weakly verrucose basidiospores. Cortinarius pseudocamphoratus can be characterized by a viscid pileus, a strongly unpleasant smell, amygdaloid to somewhat ellipsoid basidiospores and lageniform to subfusiform cheilocystidia. Cortinarius subnymphatus is identified by a strongly hygrophanous pileus that is reddish-brown with a black-brown umbo, a yellowish universal veil and ellipsoid to subamygdaloid basidiospores. Cortinarius wuliangshanensis is characterized by a moderately to strongly hygrophanous, translucently striated and yellowish to reddish-brown pileus and rather weakly and moderately verrucose basidiospores. Cortinarius yanjiensis is distinguished by a weakly to moderately hygrophanous and yellowish to brown pileus and moderately to rather strongly verrucose basidiospores. The phylogenetic analyses were performed with maximum likelihood and Bayesian inference methods based on the data set of nuc rDNA ITS1-5.8S-ITS2 (ITS), D1–D2 domains of nuc 28S rDNA (28S) and RNA polymerase II second largest subunit (rpb2), and the results show that C. neobalaustinus, C. wulianghsanensis and C. yanjiensis cluster in sect. Illumini, C. pseudocamporatus belongs to sect. Camphorati and C. subnymphatus belongs to sect. Laeti. In addition, a study of basidiospores under field emission scanning electron microscopy (FESEM) was conducted. An identification key for the five new species and related species from China is also provided.

Cryo-EM structure of dodecamer human p97 in complex with NMS-873 reveals S765-G779 peptide plays critical role for D2 ring oligomerization

The AAA + ATPase p97 is a well-known hexametric enzyme that is evolutionary conserved in eukaryotes. p97 contains an amino-terminal N domain, two tandem ATPase domains (D1 and D2 domain) and a C-terminal unstructured extensive tail, involved in many cellular processes and plays important biological functions, but the structural basis of p97 for its biological roles still remain unclear. Here we report the Cryo-EM structure of full-length human p97 dodecamer in 3.0 Å resolution, the structure was captured in ADP-bound form but only D1 ATPase sites were well occupied by nucleotide and D2 sites are empty, furthermore, 12 non-ATP-competitive inhibitors of NMS-873 bound in the interface between each p97 monomer. We also found that the C-terminal S765-G779 (765-‘SRGFGSFRFPSGNQG’-779) peptide plays critical roles for the D2 ring oligomerization, biochemical and electron microscopy studies confirm that the S765-G779 peptide could induce the D2 ring itself to form the heptamer, this give new insights how p97 protomers assemble to the biological functional multimers.