Essential Domains of Anaplasma phagocytophilum Invasins Utilized to Infect Mammalian Host Cells

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLex-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLex sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLex but express the structurally similar glycan, 6-sulfo-sLex, required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLex antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLex antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilumbinding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection

Complement Activation-Independent Attenuation of SARS-CoV-2 Infection by C1q and C4b-Binding Protein

Data Availability Statement: Not applicable.Copyright © 2023 by the authors. The complement system is a key component of the innate immune response to viruses and proinflammatory events. Exaggerated complement activation has been attributed to the induction of a cytokine storm in severe SARS-CoV-2 infection. However, there is also an argument for the protective role of complement proteins, given their local synthesis or activation at the site of viral infection. This study investigated the complement activation-independent role of C1q and C4b-binding protein (C4BP) against SARS-CoV-2 infection. The interactions of C1q, its recombinant globular heads, and C4BP with the SARS-CoV-2 spike and receptor binding domain (RBD) were examined using direct ELISA. In addition, RT-qPCR was used to evaluate the modulatory effect of these complement proteins on the SARS-CoV-2-mediated immune response. Cell binding and luciferase-based viral entry assays were utilised to assess the effects of C1q, its recombinant globular heads, and C4BP on SARS-CoV-2 cell entry. C1q and C4BP bound directly to SARS-CoV-2 pseudotype particles via the RBD domain of the spike protein. C1q via its globular heads and C4BP were found to reduce binding as well as viral transduction of SARS-CoV-2 spike protein expressing lentiviral pseudotypes into transfected A549 cells expressing human ACE2 and TMPRSS2. Furthermore, the treatment of the SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes with C1q, its recombinant globular heads, or C4BP triggered a reduction in mRNA levels of proinflammatory cytokines and chemokines such as IL-1β, IL-8, IL-6, TNF-α, IFN-α, and RANTES (as well as NF-κB) in A549 cells expressing human ACE2 and TMPRSS2. In addition, C1q and C4BP treatment also reduced SARS-CoV-2 pseudotype infection-mediated NF-κB activation in A549 cells expressing human ACE2 and TMPRSS2. C1q and C4BP are synthesised primarily by hepatocytes; however, they are also produced by macrophages, and alveolar type II cells, respectively, locally at the pulmonary site. These findings support the notion that the locally produced C1q and C4BP can be protective against SARS-CoV-2 infection in a complement activation-independent manner, offering immune resistance by inhibiting virus binding to target host cells and attenuating the infection-associated inflammatory response.UAEU grant (UK), Wellcome Trust (NT), and Researchers Supporting Project No. RSPD2023R 770, King Saud University, Riyadh (HK)

Predicting motor, cognitive and functional impairment in Parkinson's

Objective We recently demonstrated that 998 features derived from a simple 7‐minute smartphone test could distinguish between controls, people with Parkinson's and people with idiopathic Rapid Eye Movement sleep behavior disorder, with mean sensitivity/specificity values of 84.6‐91.9%. Here, we investigate whether the same smartphone features can be used to predict future clinically relevant outcomes in early Parkinson's. Methods A total of 237 participants with Parkinson's (mean (SD) disease duration 3.5 (2.2) years) in the Oxford Discovery cohort performed smartphone tests in clinic and at home. Each test assessed voice, balance, gait, reaction time, dexterity, rest, and postural tremor. In addition, standard motor, cognitive and functional assessments and questionnaires were administered in clinic. Machine learning algorithms were trained to predict the onset of clinical outcomes provided at the next 18‐month follow‐up visit using baseline smartphone recordings alone. The accuracy of model predictions was assessed using 10‐fold and subject‐wise cross validation schemes. Results Baseline smartphone tests predicted the new onset of falls, freezing, postural instability, cognitive impairment, and functional impairment at 18 months. For all outcome predictions AUC values were greater than 0.90 for 10‐fold cross validation using all smartphone features. Using only the 30 most salient features, AUC values greater than 0.75 were obtained. Interpretation We demonstrate the ability to predict key future clinical outcomes using a simple smartphone test. This work has the potential to introduce individualized predictions to routine care, helping to target interventions to those most likely to benefit, with the aim of improving their outcome

Mutational Correlates of Virological Failure in Individuals Receiving a WHO-Recommended Tenofovir-Containing First-Line Regimen: An International Collaboration

enofovir disoproxil fumarate (TDF) genotypic resistance defined by K65R/N and/or K70E/Q/G occurs in 20% to 60% of individuals with virological failure (VF) on a WHO-recommended TDF-containing first-line regimen. However, the full spectrum of reverse transcriptase (RT) mutations selected in individuals with VF on such a regimen is not known. To identify TDF regimen-associated mutations (TRAMs), we compared the proportion of each RT mutation in 2873 individuals with VF on a WHO-recommended first-line TDF-containing regimen to its proportion in a cohort of 50,803 antiretroviral-naïve individuals. To identify TRAMs specifically associated with TDF-selection pressure, we compared the proportion of each TRAM to its proportion in a cohort of 5805 individuals with VF on a first-line thymidine analog-containing regimen. We identified 83 TRAMs including 33 NRTI-associated, 40 NNRTI-associated, and 10 uncommon mutations of uncertain provenance. Of the 33 NRTI-associated TRAMs, 12 – A62V, K65R/N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F – were more common among individuals receiving a first-line TDF-containing compared to a first-line thymidine analog-containing regimen. These 12 TDF-selected TRAMs will be important for monitoring TDF-associated transmitted drug-resistance and for determining the extent of reduced TDF susceptibility in individuals with VF on a TDF-containing regimen

MRCP compared to diagnostic ERCP for diagnosis when biliary obstruction is suspected: a systematic review

BACKGROUND: Magnetic resonance cholangiopancreatography (MRCP) is an alternative to diagnostic endoscopic retrograde cholangiopancreatography (ERCP) for investigating biliary obstruction. The use of MRCP, a non-invasive procedure, may prevent the use of unnecessary invasive procedures. The aim of the study was to compare the findings of MRCP with those of ERCP by the computation of accuracy statistics. METHODS: Thirteen electronic bibliographic databases, covering biomedical, science, health economics and grey literature were searched. A systematic review of studies comparing MRCP to diagnostic ERCP in patients with suspected biliary obstruction was conducted. Sensitivity, specificity, likelihood ratios, acceptability and adverse events were reported. RESULTS: 25 studies were identified reporting several conditions including choledocholithiasis (18 studies), malignancy (four studies), obstruction (three studies), stricture (two studies) and dilatation (five studies). Three of the 18 studies reporting choledocholithiasis were excluded from the analysis due to lack of data, or differences in study design. The sensitivity for the 15 studies of choledocholithiasis ranged from 0.50 to 1.00 while specificity ranged from 0.83 to 1.00. The positive likelihood ratio ranged: from 5.44–47.72 and the negative likelihood ratio for the 15 studies ranged from 0.00–0.51. Significant heterogeneity was found across the 15 studies so the sensitivities and specificities were summarised by a Receiver Operating Characteristic (ROC) curve. For malignancy, sensitivity ranged from 0.81 to 0.94 and specificity from 0.92 to 1.00. Positive likelihood ratios ranged from 10.12 to 43 and negative likelihood ratios ranged from 0.15 to 0.21, although these estimates were less reliable. CONCLUSION: MRCP is a comparable diagnostic investigation in comparison to ERCP for diagnosing biliary obstruction

Genetic divergence of rubber tree estimated by multivariate techniques and microsatellite markers

Genetic diversity of 60 Hevea genotypes, consisting of Asiatic, Amazonian, African and IAC clones, and pertaining to the genetic breeding program of the Agronomic Institute (IAC), Brazil, was estimated. Analyses were based on phenotypic multivariate parameters and microsatellites. Five agronomic descriptors were employed in multivariate procedures, such as Standard Euclidian Distance, Tocher clustering and principal component analysis. Genetic variability among the genotypes was estimated with 68 selected polymorphic SSRs, by way of Modified Rogers Genetic Distance and UPGMA clustering. Structure software in a Bayesian approach was used in discriminating among groups. Genetic diversity was estimated through Nei's statistics. The genotypes were clustered into 12 groups according to the Tocher method, while the molecular analysis identified six groups. In the phenotypic and microsatellite analyses, the Amazonian and IAC genotypes were distributed in several groups, whereas the Asiatic were in only a few. Observed heterozygosity ranged from 0.05 to 0.96. Both high total diversity (HT' = 0.58) and high gene differentiation (G st' = 0.61) were observed, and indicated high genetic variation among the 60 genotypes, which may be useful for breeding programs. The analyzed agronomic parameters and SSRs markers were effective in assessing genetic diversity among Hevea genotypes, besides proving to be useful for characterizing genetic variability

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Abstract Background Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results 8489 transcripts were detected across the two oocyte groups, of which ~25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of α-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology