Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some ...patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.
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•SARS-CoV-2 minimally infects human neurons and astrocytes in 2D and brain organoids•Model developed for hiPSC-derived choroid plexus organoids (CPOs)•SARS-CoV-2 productively infects CPOs and increases cell death•SARS-CoV-2 CPO infection leads to transcriptional upregulation of inflammatory genes
SARS-CoV-2 causes neurological symptoms in a significant portion of patients with COVID-19. Ming and colleagues tested SARS-CoV-2 neurotropism by using monolayer neural cells and brain organoids generated from human pluripotent stem cells and show minimal neuron and astrocyte infection but efficient choroid plexus infection, leading to cell death and functional deficits.
During persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease ...progression. We demonstrated that blockade of type I interferon (IFN-I) signaling using an IFN-I receptor neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules, and restored lymphoid architecture in mice persistently infected with lymphocytic choriomeningitis virus. IFN-I blockade before and after establishment of persistent virus infection resulted in enhanced virus clearance and was CD4 T cell-dependent. Hence, we demonstrate a direct causal link between IFN-I signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and virus persistence. Our results suggest that therapies targeting IFN-I may help control persistent virus infections.
•Bunyavirus and arenavirus are important public health threats.•Bunyavirus and arenavirus molecular biology, common and differential features.•Implications of LACV L protein structure for ...understanding viral RNA synthesis.•Current state and future perspectives on bunya- and arenavirus antivirals.
Bunyaviridae and Arenaviridae virus families include an important number of highly pathogenic viruses for humans. They are enveloped viruses with negative stranded RNA genomes divided into three (bunyaviruses) or two (arenaviruses) segments. Each genome segment is coated by the viral nucleoproteins (NPs) and the polymerase (L protein) to form a functional ribonucleoprotein (RNP) complex. The viral RNP provides the necessary context on which the L protein carries out the biosynthetic processes of RNA replication and gene transcription. Decades of research have provided a good understanding of the molecular processes underlying RNA synthesis, both RNA replication and gene transcription, for these two families of viruses. In this review we will provide a global view of the common features, as well as differences, of the molecular biology of Bunyaviridae and Arenaviridae. We will also describe structures of protein and protein-RNA complexes so far determined for these viral families, mainly focusing on the L protein, and discuss their implications for understanding the mechanisms of viral RNA replication and gene transcription within the architecture of viral RNPs, also taking into account the cellular context in which these processes occur. Finally, we will discuss the implications of these structural findings for the development of antiviral drugs to treat human diseases caused by members of the Bunyaviridae and Arenaviridae families.
Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic ...medications. Given evidence of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation and exerts its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1β production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. Metformin blocked LPS-induced ATP-dependent synthesis of the NLRP3 ligand mtDNA independently of AMP-activated protein kinase (AMPK) or NF-κB. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.
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•Metformin inhibits macrophage IL-1β and IL-6 production and blunts ARDS severity•Metformin inhibits cytosolic Ox-mtDNA production and NLRP3 inflammasome activation•By targeting ETCCI, metformin blocks macrophage ATP-dependent mtDNA synthesis•Myeloid targeted inhibition of mtDNA synthesis blunts IL-1β production and ARDS
The exact mechanism by which metformin exerts its anti-inflammatory effects is still not known. Xian et al. shows that metformin’s inhibition of ETCCI blocks ATP-dependent mtDNA synthesis, cytoplasmic ox-mtDNA generation and NLRP3 inflammasome activation in macrophages, independent of AMPK and NF-κB. By limiting IL-1β production, metformin blunts pulmonary inflammation.
Lassa virus (LASV) cell entry is mediated by the interaction of the virus glycoprotein complex (GPC) with alpha-dystroglycan at the cell surface followed by binding to LAMP1 in late endosomes. ...However, LAMP1 is not absolutely required for LASV fusion, as this virus can infect LAMP1-deficient cells. Here, we used LASV GPC pseudoviruses, LASV virus-like particles and recombinant lymphocytic choriomeningitis virus expressing LASV GPC to investigate the role of human LAMP1 (hLAMP1) in LASV fusion with human and avian cells expressing a LAMP1 ortholog that does not support LASV entry. We employed a combination of single virus imaging and virus population-based fusion and infectivity assays to dissect the hLAMP1 requirement for initiation and completion of LASV fusion that culminates in the release of viral ribonucleoprotein into the cytoplasm. Unexpectedly, ectopic expression of hLAMP1 accelerated the kinetics of small fusion pore formation, but only modestly increased productive LASV fusion and infection of human and avian cells. To assess the effects of hLAMP1 in the absence of requisite endosomal host factors, we forced LASV fusion with the plasma membrane by applying low pH. Unlike the conventional LASV entry pathway, ectopic hLAMP1 expression dramatically promoted the initial and full dilation of pores formed through forced fusion at the plasma membrane. We further show that, while the soluble hLAMP1 ectodomain accelerates the kinetics of nascent pore formation, it fails to promote efficient pore dilation, suggesting the hLAMP1 transmembrane domain is involved in this late stage of LASV fusion. These findings reveal a previously unappreciated role of hLAMP1 in promoting dilation of LASV fusion pores, which is difficult to ascertain for endosomal fusion where several co-factors, such as bis(monoacylglycero)phosphate, likely regulate LASV entry.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Ebola virus (EBOV) has been reported to enter cultured cell lines via a dynamin-2-independent macropinocytic pathway or clathrin-mediated endocytosis. The route(s) of productive EBOV ...internalization into physiologically relevant cell types remain unexplored, and viral-host requirements for this process are incompletely understood. Here, we use electron microscopy and complementary chemical and genetic approaches to demonstrate that the viral glycoprotein, GP, induces macropinocytic uptake of viral particles into cells. GP's highly-glycosylated mucin domain is dispensable for virus-induced macropinocytosis, arguing that interactions between other sequences in GP and the host cell surface are responsible. Unexpectedly, we also found a requirement for the large GTPase dynamin-2, which is proposed to be dispensable for several types of macropinocytosis. Our results provide evidence that EBOV uses an atypical dynamin-dependent macropinocytosis-like entry pathway to enter Vero cells, adherent human peripheral blood-derived monocytes, and a mouse dendritic cell line.
Previously, we reported the frequency of epidermal growth factor receptor (EGFR) and KRAS mutations in nonsmall-cell lung cancer (NSCLC) patients in Latin America. The EGFR mutation frequency was ...found between Asian (40%) and Caucasian (15%) populations. Here, we report the updated distribution of NSCLC mutations.
A total of 5738 samples from NSCLC patients from Argentina (1713), Mexico (1417), Colombia (1939), Peru (393), Panama (174), and Costa Rica (102) were genotyped for EGFR and KRAS.
The median patient age was 62.2 ± 12.3 years; 53.5% were women, 46.7% had a history of smoking, and 95.2% had adenocarcinoma histology. The frequency of EGFR mutations was 26.0% (95% confidence interval CI, 24.9–27.1; Argentina, 14.4% 12.8–15.6; México, 34.3% 31.9–36.7; Colombia, 24.7% 22.8–26.6; Peru, 51.1% 46.2–55.9; Panamá, 27.3 20.7–33.9; and Costa Rica, 31.4% 22.4–40.4). The frequency of KRAS mutations was 14.0% (9.1–18.9). In patients with adenocarcinoma, EGFR mutations were independently associated with gender (30.7% females vs. 18.4% males; p < 0.001), nonsmoker status (27.4% vs. 17.1%, p < 0.001), ethnicity (mestizo/indigenous, 35.3% vs. Caucasian, 13.7%, p < 0.001), and the absence of KRAS mutation (38.1% vs. 4.7%; p < 0.001). The overall response rate to EGFR tyrosine kinase inhibitors was 60.6% (95% CI, 52–69), with a median progression-free survival and overall survival of 15.9 (95% CI, 12.420.6) and 32 months (95% CI, 26.5–37.6), respectively.
Our findings support the genetic heterogeneity of NSCLC in Latin America, confirming that the frequency of EGFR mutations is intermediate between that observed in the Asian and Caucasian populations.
AbstractAlcohol-related liver disease (ALD) is a major cause of advanced chronic liver disease in Latin-America, although data on prevalence is limited. Public health policies aimed at reducing the ...alarming prevalence of alcohol use disorder in Latin-America should be implemented. ALD comprises a clinical-pathological spectrum that ranges from steatosis, steatohepatitis to advanced forms such as alcoholic hepatitis (AH), cirrhosis and hepatocellular carcinoma. Besides genetic factors, the amount of alcohol consumption is the most important risk factor for the development of ALD. Continuous consumption of more than 3 standard drinks per day in men and more than 2 drinks per day in women increases the risk of developing liver disease. The pathogenesis of ALD is only partially understood and recent translational studies have identified novel therapeutic targets. Early forms of ALD are often missed and most clinical attention is focused on AH, which is defined as an abrupt onset of jaundice and liver-related complications. In patients with potential confounding factors, a transjugular biopsy is recommended. The standard therapy for AH (i.e. prednisolone) has not evolved in the last decades yet promising new therapies (i.e. G-CSF, N-acetylcysteine) have been recently proposed. In both patients with early and severe ALD, prolonged abstinence is the most efficient therapeutic measure to decrease long-term morbidity and mortality. A multidisciplinary team including alcohol addiction specialists is recommended to manage patients with ALD. Liver transplantation should be considered in the management of patients with end-stage ALD that do not recover despite abstinence. In selected cases, increasing number of centers are proposing early transplantation for patients with severe AH not responding to medical therapy.
Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic ...activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features
similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.
The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate
and
This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.
This paper presents the finding of a singular environment polluted by acid mine drainage in the Iberian Pyrite Belt. This situation is regulated by particular conditions, thus the analysed values can ...be considered as extreme, not only because of the high concentrations of toxic elements, but also due to the extreme low pH, reaching an average negative pH of −1.56, never found before in open-air environments contaminated by acid mine drainage. Concentrations up to 59 g/L of Fe, 2.4 g/L of Al, 740 mg/L of As, 4.3 mg/L of Co, 5.3 mg/L of Ge, 4.8 mg/L of Sb, inter alia, can be found dissolved in these polluted waters. The main aims of the present work are the physicochemical characterization and the toxicity assessment of these radical polluted waters. In addition, a hydrogeochemical model of the system will be proposed, which justifies the extreme pH value and the extraordinarily high concentrations of toxic elements, even for acid mine drainage polluted environments. Extreme acidity and metal and sulphate concentrations in the Radical Environment are due to several processes of different nature, mainly driven by the geochemistry of the minerals presents in the endorheic character of the basin. The extremely acidic nature of these waters control the Fe species present in them, being FeHSO4+ the mainly Fe specie representing 94% of total. High toxicity of these waters has been detected due to the absence of any diatoms species.
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•Negative pH values analysed in an open air environment•Extreme pollution by acid mine drainage in the Iberian Pyrite Belt•High toxicity assessed by the presence of diatoms species•Radical pollution values in an endorheic basin
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