Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide.

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

Measurements of neutrino oscillation parameters from the T2K experiment using 3.6×1021 protons on target.

The T2K experiment presents new measurements of neutrino oscillation parameters using 19.7(16.3)×1020 protons on target (POT) in (anti-)neutrino mode at the far detector (FD). Compared to the previous analysis, an additional 4.7×1020 POT neutrino data was collected at the FD. Significant improvements were made to the analysis methodology, with the near-detector analysis introducing new selections and using more than double the data. Additionally, this is the first T2K oscillation analysis to use NA61/SHINE data on a replica of the T2K target to tune the neutrino flux model, and the neutrino interaction model was improved to include new nuclear effects and calculations. Frequentist and Bayesian analyses are presented, including results on sin2θ13 and the impact of priors on the δCP measurement. Both analyses prefer the normal mass ordering and upper octant of sin2θ23 with a nearly maximally CP-violating phase. Assuming the normal ordering and using the constraint on sin2θ13 from reactors, sin2θ23=0.561-0.032+0.021 using Feldman-Cousins corrected intervals, and Δm322=2.494-0.058+0.041×10-3eV2 using constant Δχ2 intervals. The CP-violating phase is constrained to δCP=-1.97-0.70+0.97 using Feldman-Cousins corrected intervals, and δCP=0,π is excluded at more than 90% confidence level. A Jarlskog invariant of zero is excluded at more than 2σ credible level using a flat prior in δCP, and just below 2σ using a flat prior in sinδCP. When the external constraint on sin2θ13 is removed, sin2θ13=28.0-6.5+2.8×10-3, in agreement with measurements from reactor experiments. These results are consistent with previous T2K analyses.

Exploration of O-GlcNAc transferase glycosylation sites reveals a target sequence compositional bias.

O-GlcNAc transferase (OGT) is an essential glycosylating enzyme that catalyzes the addition of N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. The enzyme glycosylates a broad range of peptide sequences and the prediction of glycosylation sites has proven challenging. The lack of an experimentally verified set of polypeptide sequences that are not glycosylated by OGT has made prediction of legitimate glycosylation sites more difficult. Here, we tested a number of intrinsically disordered protein regions as substrates of OGT to establish a set of sequences that are not glycosylated by OGT. The negative data set suggests an amino acid compositional bias for OGT targets. This compositional bias was validated by modifying the amino acid composition of the protein fused in sarcoma (FUS) to enhance glycosylation. NMR experiments demonstrate that the tetratricopeptide repeat region of OGT can bind FUS and that glycosylation-promoting mutations enhance binding. These results provide evidence that the tetratricopeptide repeat region recognizes disordered segments of substrates with particular compositions to promote glycosylation, providing insight into the broad specificity of OGT.

Bioinformatics analysis of rhinovirus capsid proteins VP1-4 sequences for cross-serotype vaccine development.

BACKGROUND: Rhinoviruses (RV) are associated with the development and exacerbations of asthma and chronic obstructive pulmonary disease. They've also been linked to more severe diseases like pneumonia, acute bronchiolitis, croup, and otitis media. Because of the hypervariable sequences in the same serotypes, no effective vaccine against rhinoviruses has been developed to date. With the availability of new full-length genome sequences for all RV-A and RV-B serotyped strains, this study used bioinformatics to find a suitable RV strain with the highest similarity matrices to the other strains. METHODS: The full genomic sequences of all known different RV-A and -B prototypes were downloaded from the National Centre for Biotechnology Information (NCBI) and divided into minor low-density lipoprotein receptor (LDLR) and major intercellular adhesion molecule groups (ICAM). The sequences were edited using Biological Sequence Alignment Editor, v 7.2.0 (BioEdit software) to study each capsid protein (VP1, VP2, VP3, and VP4) and analyzed using the EMBL-EBI ClustalW server and the more current Clustal Omega tool for the calculation of the identities and similarities. RESULTS: We analyzed and predicted immunogenic motifs from capsid proteins that are conserved across distinct RV serotypes using a bioinformatics technique. The amino acid sequences of VP3 were found to be the most varied, while VP4 was the most conserved protein among all RV-A and RV-B strains. Among all strains studied, RV-74 demonstrated the highest degree of homology to other strains and could be a potential genetic source for recombinant protein production. Nine highly conserved regions with a minimum length of 9-mers were identified, which could serve as potential immune targets against rhinoviruses. CONCLUSION: Therefore, bioinformatics analysis conducted in the current study has paved the way for the selection of immunogenic targets. Bioinformatically, the ideal strain's capsid protein is suggested to contain the most common RVs immunogenic sites.

FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation.

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation.

O-Linked-N-Acetylglucosaminylation of the RNA-Binding Protein EWS N-Terminal Low Complexity Region Reduces Phase Separation and Enhances Condensate Dynamics.

Many membraneless organelles are thought to be biomolecular condensates formed by phase separation of proteins and other biopolymers. Post-translational modifications (PTMs) can impact protein phase separation behavior, although for many PTMs this aspect of their function is unknown. O-linked ß-D-N-acetylglucosaminylation (O-GlcNAcylation) is an abundant form of intracellular glycosylation whose roles in regulating biomolecular condensate assembly and dynamics have not been delineated. Using an in vitro approach, we found that O-GlcNAcylation reduces the phase separation propensity of the EWS N-terminal low complexity region (LCRN) under different conditions, including in the presence of the arginine- and glycine-rich RNA-binding domains (RBD). O-GlcNAcylation enhances fluorescence recovery after photobleaching (FRAP) within EWS LCRN condensates and causes the droplets to exhibit more liquid-like relaxation following fusion. Following extended incubation times, EWS LCRN+RBD condensates exhibit diminished FRAP, indicating a loss of fluidity, while condensates containing the O-GlcNAcylated LCRN do not. In HeLa cells, EWS is less O-GlcNAcylated following OGT knockdown, which correlates with its increased accumulation in a filter retardation assay. Relative to the human proteome, O-GlcNAcylated proteins are enriched with regions that are predicted to phase separate, suggesting a general role of O-GlcNAcylation in regulation of biomolecular condensates.

Non-cooperative 4E-BP2 folding with exchange between eIF4E-binding and binding-incompatible states tunes cap-dependent translation inhibition.

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.

Biological disease-modifying antirheumatic drugs in juvenile idiopathic arthritis of polyarticular course, enthesitis-related arthritis, and psoriatic arthritis: a consensus statement.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is the most common type of inflammatory arthritis in children. Treatment options have been expanded since the introduction of biologics, which are highly effective. The existing local JIA treatment guideline was published more than a decade ago, when use of biologics was not as common. In this article, we review the latest evidence on using biologics in three JIA subtypes: JIA of polyarticular course (pcJIA), enthesitis-related arthritis (ERA), and psoriatic arthritis (PsA). Based on the latest information, an update on eligibility, response assessment, termination, and safety information for using biologics in these patients was performed. CONSENSUS PROCESS: The JIA Work Group, which consisted of nine paediatricians experienced in managing JIA, was convened in 2016. Publications before July 2017 were screened. Eligible articles were clinical trials, extension studies, systemic reviews, and recommendations from international societies and regulatory agencies about the use of biologics in pcJIA, ERA, and PsA. Evidence extraction, appraisal, and drafting of propositions were performed by two reviewers. Extracted evidence and drafted propositions were presented and discussed at the first two meetings. Overwhelming consensus was obtained at the final meeting in May 2018. Seven practice consensus statements were formulated. Regular review should be performed to keep the practice evidence-based and up-to-date.

A review of studies examining the association between genetic biomarkers (short tandem repeats and single-nucleotide polymorphisms) and risk of prostate cancer: the need for valid predictive biomarkers.

Prostate cancer (PCa) is a challenging polygenic disease because the genes that cause PCa remain largely elusive and are affected by several causal factors. Consequently, research continuously strives to identify a genetic marker which could be used as an indicator to predict the most vulnerable (i.e., predisposed) segments of the population to the disease or for the gene which may be directly responsible for PCa. To enhance the genetic etiology of PCa, this research sought to discover the key studies conducted in this field using data from the main journal publication search engines, as it was hoped that this could shed light on the main research findings from these studies, which in turn could assist in determining these genes or markers. From the research highlighted, the studies primarily used two kinds of markers: short tandem repeats or single-nucleotide polymorphisms. These markers were found to be quite prevalent in all the chromosomes within the research carried out. It also became apparent that the studies differed in both quantity and quality, as well as being conducted in a variety of societies. Links were also determined between the degree and strength of the relationship between these markers and the occurrence of the disease. From the studies identified, most recommended a larger and more diverse survey for the parameters which had not been studied before, as well as an increase in the size of the community (i.e., the population) being studied. This is an indication that work in this field is far from complete, and thus, current research remains committed toward finding genetic markers that can be used clinically for the diagnosis and screening of patients with PCa.

Antifungal susceptibilities, biofilms, phospholipase and proteinase activities in the Candida rugosa complex and Candida pararugosa isolated from tertiary teaching hospitals.

PURPOSE: Non-albicansCandida species have emerged as fungal pathogens that cause invasive infections, with many of these species displaying resistance to commonly used antifungal agents. This study was confined to studying the characteristics of clinical isolates of the C. rugosa complex and C. pararugosa species. METHODOLOGY: Seven isolates of the C. rugosa complex and one isolate of C. pararugosa were obtained from two tertiary referral hospitals in Malaysia. Their antifungal susceptibilities, biofilm, proteinase, phospholipase, esterase and haemolysin activities were characterized. Biofilms were quantified using crystal violet (CV) and tetrazolium (XTT) reduction assays at 1.5, 6, 18, 24, 48 and 72 h.Results/Key findings. The E-test antifungal tests showed that both species have elevated MICs compared to C. albicans and C. tropicalis. The highest biomass was observed in one of the C. rugosa isolates (0.237), followed by C. pararugosa (0.206) at 18 h of incubation. However, the highest bioactivity was observed in the C. rugosa ATCC 10571 strain at 24 h (0.075), followed by C. pararugosa at 48 h (0.048) and the same C. rugosa strain at 24 h (0.046), with P<0.05. All isolates exhibited high proteinase activity (+++) whereas six isolates showed very strong esterase activity (++++). All the isolates were alpha haemolytic producers. None of the isolates exhibited phospholipase activity. CONCLUSION: Elevated MICs were shown for the C. rugosa complex and C. pararugosa for commonly used antifungal drugs. Further studies to identify virulence genes involved in the pathogenesis and genes that confer reduced drug susceptibility in these species are proposed.

Selection of reference genes for quantitative studies in acute myeloid leukaemia.

INTRODUCTION: Quantitative polymerase chain reaction (qPCR) is commonly used in the investigation of acute myeloid leukaemias (AML). Stable reference genes (RG) are essential for accurate and reliable reporting but no standard method for selection has been endorsed. MATERIALS AND METHODS: We evaluated simple statistics and published model-based approaches. Multiplex-qPCR was conducted to determine the expression of 24 candidate RG in AMLs (N=9). Singleplex-qPCR was carried out on selected RG (SRP14, B2M and ATP5B) and genes of interest in AML (N=15) and healthy controls, HC (N=12). RESULTS: RG expression levels in AML samples were highly variable and coefficient of variance (CV) ranged from 0.37% to 10.17%. Analysis using GeNorm and Normfinder listed different orders of most stable genes but the top seven (ACTB, UBE2D2, B2M, NF45, RPL37A, GK, QARS) were the same. In singleplex-qPCR, SRP14 maintained the lowest CV in AML samples. B2M, one of most stable reference genes in AML, was expressed near significantly different in AML and HC. GeNorm selected ATP5B+SRP14 while Normfinder chose SRP14+B2M as the best two RG in combination. The median expressions of combined RG genes in AML compared to HC were less significantly different than individually implying smaller expression variation after combination. Genes of interest normalised with RG in combination or individually, displayed significantly different expression patterns. CONCLUSIONS: The selection of best reference gene in qPCR must consider all sample sets. Model-based approaches are important in large candidate gene analysis. This study showed combination of RG SRP14+B2M was the most suitable normalisation factor for qPCR analysis of AML and healthy individuals.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Candida glabrata, Candida parapsilosis and Candida rugosa from two major hospitals in Kuala Lumpur, Malaysia.

The purpose of this study is to characterize 3 non-albicans Candida spp. that were collected from two major hospitals in a densely populated area of Kuala Lumpur for their susceptibilities to azole and genetic background. Fifteen non-albicans Candida clinical isolates in two major hospitals in Kuala Lumpur area of Malaysia were collected by convenience sampling during 2007 and 2010. The genetic diversity of 15 non-albicans Candida species comprising C. glabrata (n = 5), C. parapsilosis (n = 5) and C. rugosa (n = 5) were assessed by RAPD-PCR typing. Strains were initially identified using biochemical tests and CHROMagar Candida medium. Fluconazole and voriconazole susceptibilities were determined by E-test method. Commercial kits were used for DNA extraction and amplification with RAPD primers (OPA02, OPA03 and OPA08). PCR conditions were optimized and simultaneous identification was possible by agarose gel electrophoresis of PCR products and the bands obtained were analyzed using BioNumerics Applied Maths v.6.6 software. The RAPD primers used in this study generated 100% polymorphic profile. Cluster analysis using the RAPD-PCR profile showed 12.5-25% similarity among the strains. The genetic diversity was based on the strain susceptibility towards both the azoles, site of isolation and place according to their unique banding patterns. In contrast, strains susceptible to azoles were found to be genetically similar with clonal dissimilarity. The use of OPA02, OPA03 and OPA08 primers in differentiating non-albicans Candida spp. underscores the higher resolution of RAPD-PCR as a reliable tool for strain/species level differentiation.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Candida glabrata, Candida parapsilosis and Candida rugosa from two major hospitals in Kuala Lumpur, Malaysia

@#The purpose of this study is to characterize 3 non-albicans Candida spp. that were collected from two major hospitals in a densely populated area of Kuala Lumpur for their susceptibilities to azole and genetic background. Fifteen non-albicans Candida clinical isolates in two major hospitals in Kuala Lumpur area of Malaysia were collected by convenience sampling during 2007 and 2010. The genetic diversity of 15 non-albicans Candida species comprising C. glabrata (n = 5), C. parapsilosis (n = 5) and C. rugosa (n = 5) were assessed by RAPD-PCR typing. Strains were initially identified using biochemical tests and CHROMagar Candida medium. Fluconazole and voriconazole susceptibilities were determined by E-test method. Commercial kits were used for DNA extraction and amplification with RAPD primers (OPA02, OPA03 and OPA08). PCR conditions were optimized and simultaneous identification was possible by agarose gel electrophoresis of PCR products and the bands obtained were analyzed using BioNumerics Applied Maths v.6.6 software. The RAPD primers used in this study generated 100% polymorphic profile. Cluster analysis using the RAPD-PCR profile showed 12.5-25% similarity among the strains. The genetic diversity was based on the strain susceptibility towards both the azoles, site of isolation and place according to their unique banding patterns. In contrast, strains susceptible to azoles were found to be genetically similar with clonal dissimilarity. The use of OPA02, OPA03 and OPA08 primers in differentiating non-albicans Candida spp. underscores the higher resolution of RAPD-PCR as a reliable tool for strain/species level differentiation.