D2 Domain Research Articles

Heterozygous FGFR1 mutation may be responsible for an incomplete form of osteoglophonic dysplasia, characterized only by radiolucent bone lesions and teeth retentions

Non-ossifying fibromas are seen in different disorders recognizable by specific features. Indeed, osteoglophonic dysplasia (OD) is characterized by radiolucent bone lesions associated with severe short stature, dysmorphism and failure of dental eruption. This syndrome is caused by heterozygous activating mutations in the immunoglobulin-like D3 domain of the FGFR1 gene, encoding a tyrosine kinase. Here, we report three patients from the same family presenting with radiolucent bone lesions and teeth retentions. Exome sequencing allowed identification of a novel mutation c.917C > T, p. Pro306Leu in exon 7 of the FGFR1 gene. Our patients present with normal stature and no severe dysmorphism. This report describes a mild form of OD and expands the phenotype related to FGFR1 mutations. These findings emphasize the need to consider FGFR1 variants in the case of multiple non-ossifying bone lesions associated with dental eruption anomalies.

Intestinal Gel-Forming Mucins Polymerize by Disulfide-Mediated Dimerization of D3 Domains

The mucin 2 glycoprotein assembles into a complex hydrogel that protects intestinal epithelia and houses the gut microbiome. A major step in mucin 2 assembly is further multimerization of preformed mucin dimers, thought to produce a honeycomb-like arrangement upon hydrogel expansion. Important open questions are how multiple mucin 2 dimers become covalently linked to one another and how mucin 2 multimerization compares with analogous processes in related polymers such as respiratory tract mucins and the hemostasis protein von Willebrand factor. Here we report the x-ray crystal structure of the mucin 2 multimerization module, found to form a dimer linked by two intersubunit disulfide bonds. The dimer structure calls into question the current model for intestinal mucin assembly, which proposes disulfide-mediated trimerization of the same module. Key residues making interactions across the dimer interface are highly conserved in intestinal mucin orthologs, supporting the physiological relevance of the observed quaternary structure. With knowledge of the interface residues, it can be demonstrated that many of these amino acids are also present in other mucins and in von Willebrand factor, further indicating that the stable dimer arrangement reported herein is likely to be shared across this functionally broad protein family. The mucin 2 module structure thus reveals the manner by which both mucins and von Willebrand factor polymerize, drawing deep structural parallels between macromolecular assemblies critical to mucosal epithelia and the vasculature.

In-silico studies on conformational stability of flagellin–receptor complexes

Flagellin is a protein, responsible for virulent activities of bacteria. The host cell surface receptor protein TLR5 is known to interact with flagellin in order to activate immune response. However, the underlying microscopic details of this immune response are still elusive. In this study, we report on conformational stability of flagellin of two different organisms known as fliC and flaD in bilayer with reference to water. We find that both the flagellin is conformationally more stable in bilayer than in water. We also observe that fliC–TLR5 and flaD–TLR5 complexes are conformationally stable when the extracellular domain of the protein binds to conserved D1 domain of both fliC and flaD, although the binding interface between fliC–TLR5 and flaD–TLR5 is not identical. Our studies suggest that this might lead to differences in coreceptor bindings involved in immune response and thus have potential application in pharmaceutical developments.AbbreviationsA2Aadenosine receptorDPPCdipalmitoyl phosphatidylcholineecdextracellular domainecl2extracellular loop 2eLRRextracellular Leucine rich repeat domainflaDflagellin of Vibrio choleraefliCflagellin of Salmonella typhimuriumHPVhyper-variableMDmolecular dynamicsRMSDroot means squared deviationTIRtoll-interleukin receptorTLR5toll like receptor 5VPAC1vasoactive intestinal peptide receptorCommunicated by Ramaswamy H. Sarma

Raindrop Size Distributions and Rain Characteristics Observed by a PARSIVEL Disdrometer in Beijing, Northern China

Fourteen-month precipitation measurements from a second-generation PARSIVEL disdrometer deployed in Beijing, northern China, were analyzed to investigate the microphysical structure of raindrop size distribution and its implications on polarimetric radar applications. Rainfall types are classified and analyzed in the domain of median volume diameter D 0 and the normalized intercept parameter N w . The separation line between convective and stratiform rain is almost equivalent to rain rate at 8.6 mm h−1 and radar reflectivity at 36.8 dBZ. Convective rain in Beijing shows distinct seasonal variations in log 10 N w – D 0 domain. X-band dual-polarization variables are simulated using the T-matrix method to derive radar-based quantitative precipitation estimation (QPE) estimators, and rainfall products at hourly scale are evaluated for four radar QPE estimators using collocated but independent rain gauge observations. This study also combines the advantages of individual estimators based on the thresholds on polarimetric variables. Results show that the blended QPE estimator has better performance than others. The rainfall microphysical analysis presented in this study is expected to facilitate the development of a high-resolution X-band radar network for urban QPE applications.

Molecular Dynamics Simulations of p97 Including Covalent, Allosteric and ATP-Competitive Inhibitors

Binary (nucleotide-protein dimer and hexamer complexes) and ternary (nucleotide-protein-inhibitor complexes) p97 complexes were subjected to molecular dynamics simulations in an attempt to further our understanding of the p97 protein oligomer domain stability and, more importantly, of the recently reported diverse molecular mechanisms of inhibition including allosteric, ATP-competitive and covalent inhibitors. Analysis of stable states following equilibration phases indicated a higher intrinsic stability of the homohexamer as opposed to the dimer, and of N-D1 domains as opposed to the D2 domain. The molecular dynamics of the proposed allosteric binding model reproduced important molecular interactions identified experimentally with high frequency throughout the trajectory. Observed conformational changes occurring in the D2 nucleotide binding site provided a novel bind-rearrange-react hypothesis of stepwise molecular events involved in the specific covalent inhibitor mode of action.

Molecular Dynamics Simulations of p97 Including Covalent, Allosteric and ATP-competitive Inhibitors

Binary (nucleotide-protein dimer and hexamer complexes) and ternary (nucleotide-protein-inhibitor complexes) p97 complexes were subjected to molecular dynamics simulations in an attempt to further our understanding of the p97 protein oligomer domain stability and, more importantly, of the recently reported diverse molecular mechanisms of inhibition including allosteric, ATP-competitive and covalent inhibitors. Analysis of stable states following equilibration phases indicated a higher intrinsic stability of the homohexamer as opposed to the dimer, and of N-D1 domains as opposed to the D2 domain. The molecular dynamics of the proposed allosteric binding model reproduced important molecular interactions identified experimentally with high frequency throughout the trajectory. Observed conformational changes occurring in the D2 nucleotide binding site provided a novel bind-rearrange-react hypothesis of stepwise molecular events involved in the specific covalent inhibitor mode of action.

LEKTI domains D6, D7 and D8+9 serve as substrates for transglutaminase 1: implications for targeted therapy of Netherton syndrome.

Transglutaminase (TG)1 plays a key role in the formation of the cornified envelope and thus in the maintenance of the epidermal barrier. Patients with Netherton syndrome (LEKTI deficiency) have increased activity of both TG1 and serin proteases. To determine whether there is a functional biochemical link between TG1 and LEKTI and whether LEKTI domains could possibly serve as substrates for TG1. We analysed the protein sequence of LEKTI for possible TG1 recognition sites using bioinformatics. Synthetic peptides and recombinant LEKTI domains D6, D7 and D8+9 were examined invitro and insitu for possible substrate specificity. The recombinant LEKTI domains were studied for inhibitory activity in a kallikrein (KLK)5 activity test. We identified possible TG1 consensus sequences in LEKTI domains D6, D7 and D8+9, pointing to a novel biological link between these two proteins. Indeed, synthesized short peptides from these consensus sequences were incorporated into the TG1 activity zone of the epidermis. In vitro the entire recombinant domains of LEKTI showed substrate specificity for TG1, which was again confirmed insitu. The inhibitory activity of the recombinant LEKTI domains was confirmed by a KLK5 inhibition test. The strongest inhibition was observed for domains D8+9. There are specific domains of LEKTI that are recognized and processed by TG1. LEKTI domains D6, D7 and D8+9 contribute to the formation and protection of the cornified envelope. These results impact the development of protein replacement therapy approaches for Netherton syndrome. What's already known about this topic? LEKTI and transglutaminase (TG)1 are key proteins involved in the terminal differentiation of the epidermis. Lack of LEKTI causes Netherton syndrome; TG1 deficiency causes lamellar ichthyosis. The serine protease inhibitor LEKTI is processed into different functional units. Among different target proteases, kallikrein (KLK)5 appears to be a key player in disease pathology. It has been demonstrated that LEKTI domain 6 inhibits KLK5 and KLK7; LEKTI domains 8-11 also inhibit KLK14. What does this study add? The single LEKTI domains 6, 7 and the functional unit of domains 8 and 9 contain recognition motifs for TG1. We show that these domains and unit are crosslinked into the epidermis by TG1. Functional analyses of the recombinant LEKTI domains revealed that LEKTI D8+9 has the strongest inhibitory effect on KLK5. What is the translational message? The novel functional link between LEKTI and TG1 should be taken into account when considering the development of a targeted topical protein therapy for Netherton syndrome. The unit of domains D8+9 may be sufficient for this purpose.

Pilus-1 Backbone Protein RrgB of Streptococcus pneumoniae Binds Collagen I in a Force-Dependent Way.

Attachment to host tissue is a prerequisite for successful host colonization and invasion of pathogens. Many pathogenic bacteria use surface appendices, called pili, to bind and firmly attach to host tissue surfaces. Although it has been speculated that the laterally positioned D3 domain of the pilus-1 backbone protein RrgB of Streptococcus pneumoniae may promote bacterial-host interaction, via adhesion to extracellular matrix molecules, such as collagen, earlier studies showed no affinity of RrgB to collagen I. Using atomic force microscopy-based single molecule force spectroscopy combined with lateral force microscopy, we show that under mechanical load, RrgB in fact binds to human collagen I in a force-dependent manner. We observe exceptionally strong interactions, with interaction forces reaching as much as 1500 pN, and we show that high force loading and shearing rates enhance and further strengthen the interaction. In addition, the affinity of RrgB to collagen I under mechanical load not only depends on the orientation of the D3 domain but also on the orientation of the collagen fibrils, relative to the pulling direction. Both exceptionally high binding forces and force-induced bond strengthening resemble the behavior of so-called catch bonds, which have recently been observed in bacterial adhesins, but have not been reported for multimeric backbone subunits of virulence related pili.

The effect of differences in the third domain of the glycoprotein E of tick-borne encephalitis virus of the Far Eastern, Siberian and European subtypes on the binding of recombinant D3 proteins with a chimeric antibody

Currently, a therapeutic drug based on recombinant antibodies for the prevention and treatment of tick-borne encephalitis virus (TBEV) is developed in ICBFM SB RAS, and the chimeric antibody ch14D5 is considered as one of the key components of this drug. It was previously shown that this antibody is directed to the domain D3 of the glycoprotein E of TBEV. It was previously shown that this antibody is able to protect mice from the European subtype of TBEV, strain “Absettarov”, and the presence of virus-neutralizing activity against the Far Eastern subtype of TBEV, strain 205 was also shown for this antibody. However, it remains unclear whether this antibody exhibits selectivity for different subtypes of TBEV. The aim of this study was to investigate the effect of amino acid sequence differences of recombinant D3 domains derived from the glycoprotein E of TBEV of the Far Eastern, Siberian and European subtypes on the binding of the protective antibody ch14D5 to these proteins. Using Western blot analysis and surface plasmon resonance, it was shown that ch14D5 antibody has the highest affinity (KD= 1.7±0.5 nM) for the D3 domain of the TBEV of the “Sofjin-Ru” strain belonging to the Far Eastern subtype of the virus. At the same time, the affinity of ch14D5 antibody for similar D3 proteins derived from “Zausaev”, “1528-99” and “Absettarov” strains of the Siberian and European subtypes of TBEV was noticeably lower (KD= 25±4, 300±50, 250±50 nM, respectively). In addition, information about the spatial arrangement of amino acid residues that are different for the studied recombinant proteins indicates that the epitope recognized by the ch14D5 antibody is in close proximity to the lateral ridge of D3 domain of E glycoprotein.

Two new species of Phylloporus (Fungi, Boletales) from tropical Quercus forests in eastern Mexico.

We present a proposal of two new species of Phylloporus discovered in tropical oak forests from central Veracruz, Mexico. Both species were distinguished based on macro and micro-morphologic features and supported with a molecular phylogenetic analysis, based on sequences of nuc rDNA ITS, D1, D2 and D3 domains of nuc 28S rDNA (LSU), and transcription elongation factor 1-alpha (tef-1α). In the phylogenetic reconstruction inferred, the new species clustered in two different clades related to species from USA, Costa Rica and Panama. The recollection of fructifications in monodominant stands of either Quercusoleoides or Q.sapotifolia, allowed recognizing the distribution of one of the Phylloporus species under both Quercus species, and the other under Q.oleoides only. Detailed macro and microscopic descriptions accompanied by illustrations, photos and a taxonomic discussion are provided.

HIV-1-specific B cell immune responses in Rhesus Macaques induced by early region deleted-Ad5 vectors

Abstract As a vaccine delivery vector, Adenovirus (Ad) is recognized for its ability to prime immune responses against HIV and other infectious agents. We reported that early region 1B55k (E1B55k) deleted vector induced cellular immunity, transgene-specific antibodies and innate immune signals including chemokines, cytokines and NKG2D ligands MIC A/B in immunized mice. However, the immune priming ability of E3-E1B55k-E4orf1 and E3-E1B55k- E4orf1-4deleted Ad vectors is unknown. Here we compared HIV-specific immune responses generated by E3deleted, E3-E1B55k, E3-E1B55k-E4orf1and E3-E1B55k-E4orf1-4deleted Ad5 vectors encoding full-length single-chain HIVBalgp120 linked to the D1 and D2 domains of rhesus macaque (RM) CD4 (rhFLSC). RMs were immunized twice mucosally with each vector. Blood and rectal tissue were collected at pre and 2-week post-immunization time points. The frequency of plasmablasts (PB)/plasma cells (PC), memory B cells, and antibody secreting cells and antibody titers were assessed by flow cytometry, ELISpot and ELISA, respectively. All vectors induced increased frequencies of rectal PB, PC and rhFLSC specific memory B cells. rhFLSC specific IgA and IgG secreting cells were detected in all immunized macaques; the IgA secreting cells were significantly increased compared to pre values. This was accompanied by elevated levels of IgG, IgG1 and IgA serum antibodies. In summary all four vectors induced B cells responses while retaining replicability. Moreover, the largest deletion, E3-E1B55k-E4orf1-4, improved the transgene carrying capacity of the Ad vector while maintaining the ability to induce B cell immunity important for protective efficacy.

Phylogenetic and functional analysis of ADAMTS13 identifies highly conserved domains essential for allosteric regulation

AbstractThe metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats member 13) prevents microvascular thrombosis by cleaving von Willebrand factor (VWF) within platelet-rich thrombi, and cleavage depends on allosteric activation of ADAMTS13 by the substrate VWF. Human ADAMTS13 has a short propeptide, metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains (distal domains). Distal domains inhibit the catalytic proximal domains; binding of distal T8-CUB domains to the VWF D4 domain relieves autoinhibition and promotes cleavage of the nearby VWF A2 domain. However, the role of specific ADAMTS13 distal domains in this allosteric mechanism is not established. Assays of plasma ADAMTS13 from 20 placental mammals, birds, and amphibians show that allosteric regulation is broadly conserved, and phylogenetic analysis of 264 vertebrates shows the long propeptide, T3, T4, T6, and T6a domains have been deleted several times in placental mammals, birds, and fish. Notably, pigeon ADAMTS13 has only 3 distal T domains but was activated normally by human VWF D4 and cleaved VWF multimers, preferentially under fluid shear stress. Human ADAMTS13 constructed to resemble pigeon ADAMTS13 retained normal allosteric regulation and shear-dependent cleavage of VWF. Thus, the T3-T6 domains of human ADAMTS13 are dispensable. Conversely, deletion of T7 or T8 abolished allosteric activation. For most species, some sequence changes in the VWF substrate can markedly increase the rate of cleavage, suggesting that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties may reflect evolutionary pressure to balance the risk for VWF-dependent bleeding and thrombosis.

Exploring the “minimal” structure of a functional ADAMTS13 by mutagenesis and small-angle X-ray scattering

AbstractHuman ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains, followed by 7 additional T domains and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. ADAMTS13 inhibits the growth of von Willebrand factor (VWF)–platelet aggregates by cleaving the cryptic Tyr1605-Met1606 bond in the VWF A2 domain. ADAMTS13 is regulated by substrate-induced allosteric activation; without shear stress, the distal T8-CUB domains markedly inhibit VWF cleavage, and binding of VWF domain D4 or selected monoclonal antibodies (MAbs) to distal ADAMTS13 domains relieves this autoinhibition. By small angle X-ray scattering (SAXS), ADAMTS13 adopts a hairpin-like conformation with distal T7-CUB domains close to the proximal MDTCS domains and a hinge point between T4 and T5. The hairpin projects like a handle away from the core MDTCS and T7-CUB complex and contains distal T domains that are dispensable for allosteric regulation. Truncated constructs that lack the T8-CUB domains are not autoinhibited and cannot be activated by VWF D4 but retain the hairpin fold. Allosteric activation by VWF D4 requires T7, T8, and the 58–amino acid residue linker between T8 and CUB1. Deletion of T3 to T6 produced the smallest construct (delT3-6) examined that could be activated by MAbs and VWF D4. Columba livia (pigeon) ADAMTS13 (pADAMTS13) resembles human delT3-6, retains normal activation by VWF D4, and has a SAXS envelope consistent with amputation of the hairpin containing the dispensable T domains of human ADAMTS13. Our findings suggest that human delT3-6 and pADAMTS13 approach a “minimal” structure for allosterically regulated ADAMTS13.

Structural analysis of the flagellar capping protein FliD from Helicobacter pylori

Helicobacter pylori is a pathogenic flagellated bacterium that infects the gastroduodenal mucosa and causes peptic ulcers in humans. FliD caps the distal end of the flagellar filament and is essential in filament growth. Moreover, FliD has been studied to diagnose and prevent H. pylori infection. Here, we report structure-based molecular studies of H. pylori FliD (hpFliD). A crystal structure of hpFliD at 2.6 Å resolution presents a four-domain (D2-D5) structure, where the D3 domain forms a central platform surrounded by the other three domains (D2, D4, and D5). hpFliD domains D2 and D3 structurally resemble those of FliD orthologs, whereas the D4 and D5 domains are exclusive to hpFliD. Moreover, our ELISA analysis using anti-H. pylori antibodies demonstrated that the hpFliD-specific D4 and D5 domains are highly antigenic compared to the D2 and D3 domains. Collectively, our structural and serological analyses underscore the structural role of hpFliD domains and provide a molecular basis for vaccine and diagnosis development.

Phylogeny of the genus Fuscoporia and taxonomic assessment of the F. contigua group

ABSTRACTThe genus Fuscoporia of the Hymenochaetaceae is characterized by resupinate to pileate basidiocarps, a dimitic hyphal system with fine crystal aggregates and encrusted generative hyphae in dissepiment edge and tube trama, the presence of hymenial setae, and hyaline, thin-walled, smooth basidiospores. Members of the F. contigua group are easy to distinguish from other species of Fuscoporia because of the moderately large pores, presence of mycelial setae, and large hymenial setae. Here, we explore phylogenetic relationships among 20 species of Fuscoporia based on examination of some 90 collections sampled worldwide. Seven new species are recognized in the F. contigua group—F. americana, F. centroamericana, F. costaricana, F. latispora, F. monticola, F. septiseta, and F. sinica—described from China, Costa Rica, Mexico, and the United States. Phylogenetic analyses inferred from DNA sequences of the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), D1–D2 domains of nuc 28S rDNA (28S), and translation elongation factor EF-1 alpha (tef1) support the F. contigua group as one of two major clades within Fuscoporia comprising nine species worldwide.

Dynamics Properties of Photosynthetic Microorganisms Probed by Incoherent Neutron Scattering

Studies on the dynamical properties of photosynthetic membranes of land plants and purple bacteria have been previously performed by neutron spectroscopy, revealing a tight coupling between specific photochemical reactions and macromolecular dynamics. Here, we probed the intrinsic dynamics of biotechnologically useful mutants of the green alga Chlamydomonas reinhardtii by incoherent neutron scattering coupled with prompt chlorophyll fluorescence experiments. We brought to light that single amino acid replacements in the plastoquinone (PQ)-binding niche of the photosystem II D1 protein impair electron transport (ET) efficiency between quinones and confer increased flexibility to the host membranes, expanding to the entire cells. Hence, a more flexible environment in the PQ-binding niche has been associated to a less efficient ET. A similar function/dynamics relationship was also demonstrated in Rhodobacter sphaeroides reaction centers having inhibited ET, indicating that flexibility at the quinones region plays a crucial role in evolutionarily distant organisms. Instead, a different functional/dynamical correlation was observed in algal mutants hosting a single amino acid replacement residing in a D1 domain far from the PQ-binding niche. Noteworthy, this mutant displayed the highest degree of flexibility, and besides having a nativelike ET efficiency in physiological conditions, it acquired novel, to our knowledge, phenotypic traits enabling it to preserve a high maximal quantum yield of photosystem II photochemistry in extreme habitats. Overall, in the nanosecond timescale, the degree of the observed flexibility is related to the mutation site; in the picosecond timescale, we highlighted the presence of a more pronounced dynamic heterogeneity in all mutants compared to the native cells, which could be related to a marked chemically heterogeneous environment.

Immunogenicity and protective efficacy of recombinant proteins consisting of multiple epitopes of foot-and-mouth disease virus fused with flagellin.

Many recent studies have shown that flagellin fused to heterologous antigens can induce significantly enhanced humoral and cellular immune responses through its adjuvant activity. Therefore, in this study, two key B cell epitopes and a truncated VP1 (ΔVP1) protein from foot-and-mouth disease virus (FMDV) were expressed as flagellin fusion proteins in different patterns. Specifically, ΔVP1 and two duplicates of two key B cell epitopes (2×B1B2) were fused separately to the C-terminus of flagellin with a universal exogenous T cell epitope to construct FT (Flagellin-Truncated VP1) and FME (Flagellin-Multiple Epitopes). In addition, the D3 domain of flagellin was replaced by ΔVP1 in FME, yielding FTME (Flagellin-Truncated VP1-Multiple Epitopes). The immunogenicity and protective efficacy of the three fusion proteins as novel FMDV vaccine candidates were evaluated. The results showed that FT, FME, and FTME elicited significant FMDV-specific IgG responses at 10μg/dose compared with the mock group (P < 0.05), with FTME producing the highest response. No significant differences in the antibody response to FTME were observed between different immunization routes or among adjuvants (ISA-206, poly(I·C), MPLA, and CpG-ODN) in mice. In addition, at 30μg/dose, all three fusion proteins significantly induced neutralizing antibody production and upregulated the levels of some cytokines, including TNF-α, IFN-γ, and IL-12, in guinea pigs. Importantly, all three fusion proteins provided effective protective immunity against FMDV challenge in guinea pigs, though different protection rates were found. The results presented in this study indicate that the FTME fusion protein is a promising novel vaccine candidate for the future prevention and control of foot-and-mouth disease.

Changes in the asymmetric distribution of cholesterol in the plasma membrane influence streptolysin O pore formation

ATP-binding cassette A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL) and preventing atherosclerosis. ABCA1 exports cholesterol and phospholipid to apolipoprotein A-I (apoA-I) in serum to generate HDL. We found that streptolysin O (SLO), a cholesterol-dependent pore-forming toxin, barely formed pores in ABCA1-expressing cells, even in the absence of apoA-I. Neither cholesterol content in cell membranes nor the amount of SLO bound to cells was affected by ABCA1. On the other hand, binding of the D4 domain of perfringolysin O (PFO) to ABCA1-expressing cells increased, suggesting that the amount of cholesterol in the outer leaflet of the plasma membrane (PM) increased and that the cholesterol dependences of these two toxins differ. Addition of cholesterol to the PM by the MβCD–cholesterol complex dramatically restored SLO pore formation in ABCA1-expressing cells. Therefore, exogenous expression of ABCA1 causes reduction in the cholesterol level in the inner leaflet, thereby suppressing SLO pore formation.

Numerical methodology to predict and analyze cavitating flows in a Kaplan turbine

A computational methodology to predict the appearance and the evolution of the cavitation phenomena in a scale model of a 5–blades Kaplan turbine was developed. The cavitating flow was modeled by a homogeneous approach with a barotropic state law and the k − ω SST turbulence model. RANS simulations were performed, near the Best Efficiency Point, on two computational domains (D1 and D2) comprising one periodic interblades channel consisting of the guide vanes, the runner blade and the cone (domain D1) or taking into account the entire draft tube (domain D2). At first, non–cavitating simulations were carried out in domain D1 in order to match numerical conditions to the experimental ones (similar mass flow Q, head H and mainly the torque T) via an iterative procedure. From this reference operating point obtained in free–cavitation regime, cavitating simulations were performed on both computational domains by reducing the Thoma number, σ. Two different inlet boundary conditions were tested: the classical one, imposing the mass flow rate Q; the new one, fixing the total pressure and, consequently, keeping the machine head H constant during the cavitation drop, as in experiments. In all the performed calculations, the outlet boundary condition is given by a constant static pressure. Computed torque and efficiency drop curves were compared to available experimental data. The best results were obtained with the computational domain D2 applying a constant total pressure on the inlet. The torque and efficiency evolutions were well–predicted with the proposed calculation methodology, and the numerical cavitation structures agreed with experimental observations. Analyses of the blade loading during cavitation breakdown are also proposed in the article. Unsteady simulations are under investigation to improve the prediction and the analyses of more developed cavitating regimes.

Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.