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Population-based data on COVID-19 are urgently needed. We report on three rounds of probability sample household surveys in the state of Rio Grande do Sul (Brazil), carried out in nine large municipalities using the Wondfo lateral flow point-of-care test for immunoglobulin M and G antibodies against SARS-CoV-2 (https://en.wondfo.com.cn/product/ wondfo-sars-cov-2-antibody-test-lateral-flow-method-2/). Before survey use, the assay underwent four validation studies with pooled estimates of sensitivity (84.8%; 95% confidence interval (CI) = 81.4-87.8%) and specificity (99.0%; 95% CI = 97.8-99.7%). We calculated that the seroprevalence was 0.048% (2/4,151; 95% CI = 0.006-0.174) on 11-13 April (round 1), 0.135% (6/4,460; 95% CI = 0.049-0.293%) on 25-27 April (round 2) and 0.222% (10/4,500; 95% CI = 0.107-0.408) on 9-11 May (round 3), with a significant upward trend over the course of the surveys. Of 37 family members of positive individuals, 17 (35%) were also positive. The epidemic is at an early stage in the state, and there is high compliance with social distancing, unlike in other parts of Brazil. Periodic survey rounds will continue to monitor trends until at least the end of September, and our population-based data will inform decisions on preventive policies and health system preparedness at the state level.

Aims Investigating human heart development and applying this to deviations resulting in disease is incomplete without molecular characterization of the cell types required for normal functioning. We investigated foetal human heart single-cell transcriptomes from mid-gestational healthy and anti-SSA/Ro associated congenital heart block (CHB) samples. Methods and results Three healthy foetal human hearts (19th to 22nd week of gestation) and one foetal heart affected by autoimmune-associated CHB (21st week of gestation) were subjected to enzymatic dissociation using the Langendorff preparation to obtain single-cell suspensions followed by 10x Genomics- and Illumina-based single-cell RNA-sequencing (scRNA-seq). In addition to the myocytes, fibroblasts, immune cells, and other minor cell types, previously uncharacterized diverse sub-populations of endothelial cells were identified in the human heart. Differential gene expression analysis revealed increased and heterogeneous interferon responses in varied cell types of the CHB heart compared with the healthy controls. In addition, we also identified matrisome transcripts enriched in CHB stromal cells that potentially contribute to extracellular matrix deposition and subsequent fibrosis. Conclusion These data provide an information-rich resource to further our understanding of human heart development, which, as illustrated by comparison to a heart exposed to a maternal autoimmune environment, can be leveraged to provide insight into the pathogenesis of disease.

Heritable APOE variants in patients with melanoma influence anti-tumor immunity and modulate metastatic progression and response to immunotherapy. Common germline variants of the APOE gene are major risk modifiers of neurodegenerative and atherosclerotic diseases(1-3), but their effect on cancer outcome is poorly defined. Here we report that, in a reversal of their effect on Alzheimer's disease, the APOE4 and APOE2 variants confer favorable and poor outcomes in melanoma, respectively. Mice expressing the human APOE4 allele exhibited reduced melanoma progression and metastasis relative to APOE2 mice. APOE4 mice exhibited enhanced anti-tumor immune activation relative to APOE2 mice, and T cell depletion experiments showed that the effect of APOE genotype on melanoma progression was mediated by altered anti-tumor immunity. Consistently, patients with melanoma carrying the APOE4 variant experienced improved survival in comparison to carriers of APOE2. Notably, APOE4 mice also showed improved outcomes under PD1 immune checkpoint blockade relative to APOE2 mice, and patients carrying APOE4 experienced improved anti-PD1 immunotherapy survival after progression on frontline regimens. Finally, enhancing APOE expression via pharmacologic activation of liver X receptors, previously shown to boost anti-tumor immunity(4), exhibited therapeutic efficacy in APOE4 mice but not in APOE2 mice. These findings demonstrate that pre-existing hereditary genetics can impact progression and survival outcomes of a future malignancy and warrant prospective investigation of APOE genotype as a biomarker for melanoma outcome and therapeutic response.

Introduction: Despite its limitations, CA125 remains the most widely used biomarker for the diagnosis and treatment monitoring of ovarian cancer. Targeting the unshed portion of serum biomarkers such as CA125/MUC16 may afford more specific imaging and targeting of MUC16-positive tumors in High Grade Serous Ovarian Cancer (HGSOC) patients. Methods: Six monoclonal antibodies raised against the 58 amino acid sequence between the extracellular cleavage site and the transmembrane region of MUC16 were radiolabeled with [Zr-89]Zr4+. The radioimmunoconjugates were evaluated in vitro for molar activities, target binding affinity, cellular internalization and serum stability. In vivo characterization was performed via longitudinal positron emission tomography (PET) imaging and ex vivo biodistribution studies in mice bearing subcutaneous xenografts of SKOV3 cells transfected with the proximal 114 amino-acids of MUC16 carboxy-terminus (SKOV3+). Results: In vitro screening identified 9C9 and 4H11 as the lead antibody candidates based on their comparable binding affinities, serum stability and cellular internalization profiles. Despite an identical molecular footprint for binding to MUC16, [Zr-89]Zr-DFO-4H11 yielded a more favorable in vivo radiopharmacologic profile. Furthermore, a humanized variant of 4H11 capable of binding MUC16 in vitro also yielded excellent in vivo profile in subcutaneous xenograft models of SKOV3+, OVCAR3 tumors and a patient-derived xenograft model representative of HGSOC. Conclusion: Radiopharmacologic screening of antibodies early during their development can provide crucial information pertinent to the in vitro characterization and in vivo pharrnacokinetics. The favorable in vivo profile demonstrated by humanized 4H11 combined with the use of its murine predecessor for immunohistochemical staining of biopsied tumor tissues from HGSOC patients makes a unique pair of antibodies that is poised for clinical translation. (C) 2020 Elsevier Inc. All rights reserved.

Aberrant chromosome numbers in cancer cells may impose distinct constraints on the emergence of drug resistance-a major factor limiting the long-term efficacy of molecularly targeted therapeutics. However, for most anticancer drugs we lack analyses of drug-resistance mechanisms in cells with different karyotypes. Here, we focus on GSK923295, a mitotic kinesin CENP-E inhibitor that was evaluated in clinical trials as a cancer therapeutic. We performed unbiased selections to isolate inhibitor-resistant clones in diploid and near-haploid cancer cell lines. In diploid cells we identified single-point mutations that can suppress inhibitor binding. In contrast, transcriptome analyses revealed that the C-terminus of CENP-E was disrupted in GSK923295-resistant near-haploid cells. While chemical inhibition of CENP-E is toxic to near-haploid cells, knockout of the CENPE gene does not suppress haploid cell proliferation, suggesting that deletion of the CENP-E C-terminus can confer resistance to GSK923295. Together, these findings indicate that different chromosome copy numbers in cells can alter epistatic dependencies and lead to distinct modes of chemotype-specific resistance.

Aims Current treatments of prosthetic joint infection (PJI) are minimally effective against Staphylococcus aureus biofilm. A murine PJI model of debridement, antibiotics, and implant retention (DAIR) was used to test the hypothesis that PlySs2, a bacteriophage-derived lysin, can target S. aureus biofilm and address the unique challenges presented in this periprosthetic environment. Methods The ability of PlySs2 and vancomycin to kill biofilm and colony-forming units (CFUs) on orthopaedic implants were compared using in vitro models. An in vivo murine PJI model of DAIR was used to assess the efficacy of a combination of PlySs2 and vancomycin on periprosthetic bacterial load. Results PlySs2 treatment reduced 99% more CFUs and 75% more biofilm compared with vancomycin in vitro. A combination of PlySs2 and vancomycin in vivo reduced the number of CFUs on the surface of implants by 92% and in the periprosthetic tissue by 88%. Conclusion PlySs2 lysin was able to reduce biofilm, target planktonic bacteria, and work synergistically with vancomycin in our in vitro models. A combination of PlySs2 and vancomycin also reduced bacterial load in periprosthetic tissue and on the surface of implants in a murine model of DAIR treatment for established PJI.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease. Here the authors present an approach that can reveal the full complement of mRNA isoforms encoded by individual genes, and they identify a major isoform of the retinal degeneration gene CRB1 which functions at the cell-cell junctions of the outer limiting membrane to promote photoreceptor survival.

Oxycodone (Oxy) conditioned place preference (CPP) in Sprague Dawley rats results in sex-specific alterations in hippocampal opioid circuits in a manner that facilitates opioid-associative learning processes, particularly in females. Here, we examined if Oxy (3 mg/kg, I.P.) or saline (Sal) injections not paired with behavioral testing similarly affect the hippocampal opioid system. Sal-injected females compared to Sal-injected males had: (1) higher densities of cytoplasmic delta opioid receptors (DOR) in GABAergic hilar dendrites suggesting higher baseline reserve DOR pools and (2) elevated phosphorylated DOR levels, but lower phosphorylated mu opioid receptor (MOR) levels in CA3a suggesting that the baseline pools of activated opioid receptors vary in females and males. In contrast to CPP studies, Oxy-injections in the absence of behavioral tests resulted in few changes in the hippocampal opioid system in either females or males. Specifically, Oxy-injected males compared to Sal-injected males had fewer DORs near the plasma membrane of CA3 pyramidal cell dendrites and in CA3 dendritic spines contacted by mossy fibers, and lower pMOR levels in CA3a. Oxy-injected females compared to Sal-injected females had higher total DORs in GABAergic dendrites and lower total MORs in parvalbumin-containing dendrites. Thus, unlike Oxy CPP, Oxy-injections redistributed opioid receptors in hippocampal neurons in a manner that would either decrease (males) or not alter (females) excitability and plasticity processes. These results indicate that the majority of changes within hippocampal opioid circuits that would promote opioid-associative learning processes in both females and males do not occur with Oxy administration alone, and instead must be paired with CPP.

Although much is known about the interaction of fibrinogen with alpha IIb beta 3, much less is known about the interaction of platelets with cross-linked fibrin. Fibrinogen residue Lys406 plays a vital role in the interaction of fibrinogen with alpha IIb beta 3, but because it participates in fibrin cross-linking, it is not available for interacting with alpha IIb beta 3. We studied the adhesion of platelets and HEK cells expressing normal and constitutively active alpha IIb beta 3 to both immobilized fibrinogen and D-dimer, a proteolytic fragment of cross-linked fibrin, as well as platelet-mediated clot retraction. Nonactivated platelets and HEK cells expressing normal alpha IIb beta 3 adhered to fibrinogen but not D-dimer, whereas activated platelets as well as HEK cells expressing activated alpha IIb beta 3 both bound to D-dimer. Small-molecule antagonists of the alpha IIb beta 3 RGD (Arg-Gly-Asp) binding pocket inhibited adhesion to D-dimer, and an Asp119Ala mutation that disrupts the beta 3 metal ion-dependent adhesion site inhibited alpha IIb beta 3-mediated adhesion to D-dimer. D-dimer and a polyclonal antibody against D-dimer inhibited clot retraction. The monoclonal antibody (mAb) 10E5, directed at alpha IIb and a potent inhibitor of platelet interactions with fibrinogen, did not inhibit the interaction of activated platelets with D-dimer or dot retraction, whereas the mAb 7E3, directed at beta 3, inhibited both phenomena. We conclude that activated, but not nonactivated, alpha IIb beta 3 mediates interactions between platelets and D-dimer, and by extrapolation, to cross-linked fibrin. Although the interaction of alpha IIb beta 3 with D-dimer differs from that with fibrinogen, it probably involves contributions from regions on beta 3 that are close to, or that are affected by, changes in the RGD binding pocket.

The foamy viruses (FV) or spumaviruses are an ancient subfamily of retroviruses that infect a variety of vertebrates. FVs are endemic, but apparently apathogenic, in modern non-human primates. Like other retroviruses, FV replication is inhibited by type-I interferon (IFN). In a previously described screen of IFN-stimulated genes (ISGs), we identified the macaque PHD finger domain protein-11 (PHF11) as an inhibitor of prototype foamy virus (PFV) replication. Here, we show that human and macaque PHF11 inhibit the replication of multiple spumaviruses, but are inactive against several orthoretroviruses. Analysis of other mammalian PHF11 proteins revealed that antiviral activity is host species dependent. Using multiple reporter viruses and cell lines, we determined that PHF11 specifically inhibits a step in the replication cycle that is unique to FVs, namely basal transcription from the FV internal promoter (IP). In so doing, PHF11 prevents expression of the viral transactivator Tas and subsequent activation of the viral LTR promoter. These studies reveal a previously unreported inhibitory mechanism in mammalian cells, that targets a family of ancient viruses and may promote viral latency. Author summary Foamy viruses have a unique replication strategy and long evolutionary relationship with their vertebrate hosts that has resulted in wide-spread infection of modern species without any apparent pathogenic consequence. How foamy virus infections are controlled by their hosts is unknown. Here, we demonstrate that infection of a variety of foamy viruses is inhibited by the interferon-stimulated gene product, PHF11, in a species-dependent manner. We show that PHF11 prevents replication by a previously undescribed mechanism, namely by inhibiting gene expression from an internal viral promoter, a conserved and distinct feature of the foamy viruses. Inhibition of early viral gene expression by PHF11 may promote viral latency and the apathogenicity of foamy viruses.