The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels ...of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64'752 unique siRNAs targeting 21'584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51'128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.
The use of monoclonal antibodies to the SARS-Cov-2 spike protein for early treatment of COVID-19 disease is being evaluated, with only phase 2 studies available to date. The emergency authorization ...of bamlanivimab monotherapy was obtained in November 2020 by the FDA and in March 2021 by Italian agency AIFA. Its use was then revoked in April 2021 by both. This study reports the results of bamlanivimab utilization in monotherapy in Umbria (Italian region) to verify whether, in a population with multiple risk factors, comparable results to the phase 2 BLAZE1 trial had been obtained.
Between March and April 2021, a retrospective observational study was performed on patients treated with bamlanivimab. Demographic and clinical characteristics before and after infusion were evaluated. Moreover, a telephone interview was conducted about 30 days after the infusion to evaluate the overall course.
All patients had an early infection (mean 4±1.73 days), almost all by alpha variant (97%). No adverse events to treatment were observed. Altogether within 30 days, the hospitalization rate was 20%, 15% for COVID-19 related pathologies, versus 4% at 11 days in the BLAZE1 phase 2 study. In addition, worsening of some symptoms observed at baseline such as asthenia (77 vs. 51.3%), shortness of breath (38 vs. 23%) was registered, as well as the onset of non-restorative sleep (41%).
The clinical outcome after bamlanivimab monotherapy was far below the expectation despite the patients had been infected by a theoretically sensitive viral variant.
The cellular prion protein PrP.sup.C is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the ...levels of PrP.sup.C may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrP.sup.C expression. We cultured human U251-MG glioblastoma cells in the presence of 64'752 unique siRNAs targeting 21'584 annotated human genes, and measured PrP.sup.C using a one-pot fluorescence-resonance energy transfer immunoassay in 51'128 individual microplate wells. This screen yielded 743 candidate regulators of PrP.sup.C . When downregulated, 563 of these candidates reduced and 180 enhanced PrP.sup.C expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrP.sup.C, 9 of which were confirmed by CRISPR interference as robust regulators of PrP.sup.C biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrP.sup.C expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrP.sup.C levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.
The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels ...of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64'752 unique siRNAs targeting 21'584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51'128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.
Nasal mucosal explant (NEs) cultured at an air–liquid interface mimics in vivo conditions more accurately than monolayer cultures of respiratory cell linesor primary cells cultured in flat-bottom ...microtiter wells. NEs might be relevant for studies of host-pathogen interactions and antiviral immune responses after infection with respiratory viruses, including influenza and corona viruses.
Pigs are natural hosts for swine influenza A virus (IAV) but are also susceptible to IAV from humans, emphasizing the relevance of porcine NEs in the study of IAV infection. Therefore, we performed fundamental characterization and study of innate antiviral responses in porcine NEs using microfluidic high-throughput quantitative real-time PCR (qPCR) to generate expression profiles of host genes involved in inflammation, apoptosis, and antiviral immune responses in mock inoculated and IAV infected porcine NEs.
Handling and culturing of the explants ex vivo had a significant impact on gene expression compared to freshly harvested tissue. Upregulation (2–43 fold) of genes involved in inflammation, including IL1A and IL6, and apoptosis, including FAS and CASP3, and downregulation of genes involved in viral recognition (MDA5 (IFIH1)), interferon response (IFNA), and response to virus (OAS1, IFIT1, MX1) was observed. However, by comparing time-matched mock and virus infected NEs, transcription of viral pattern recognition receptors (RIG-I (DDX58), MDA5 (IFIH1), TLR3) and type I and III interferons (IFNB1, IL28B (IFNL3)) were upregulated 2–16 fold in IAV-infected NEs. Furthermore, several interferon-stimulated genes including MX1, MX2, OAS, OASL, CXCL10, and ISG15 was observed to increase 2–26 fold in response to IAV inoculation. NE expression levels of key genes involved in antiviral responses including IL28B (IFNL3), CXCL10, and OASL was highly comparable to expression levels found in respiratory tissues including nasal mucosa and lung after infection of pigs with the same influenza virus isolate.
The inelastic portion of the tunnel current through an individual magnetic atom grants unique access to read out and change the atom’s spin state, but it also provides a path for spontaneous ...relaxation and decoherence. Controlled closure of the inelastic channel would allow for the latter to be switched off at will, paving the way to coherent spin manipulation in single atoms. Here, we demonstrate complete closure of the inelastic channels for both spin and orbital transitions due to a controlled geometric modification of the atom’s environment, using scanning tunneling microscopy (STM). The observed suppression of the excitation signal, which occurs for Co atoms assembled into chains on a Cu2N substrate, indicates a structural transition affecting the d z 2 orbital, effectively cutting off the STM tip from the spin-flip cotunneling path.
Data concerning the transmission of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) in paucisymptomatic patients are lacking. We report an Italian paucisymptomatic case of ...coronavirus disease 2019 with multiple biological samples positive for SARS-CoV-2. This case was detected using the World Health Organization protocol on cases and contact investigation. Current discharge criteria and the impact of extra-pulmonary SARS-CoV-2 samples are discussed.
Data concerning the transmission of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) in paucisymptomatic patients are lacking. We report an Italian paucisymptomatic case of ...coronavirus disease 2019 with multiple biological samples positive for SARS-CoV-2. This case was detected using the World Health Organization protocol on cases and contact investigation. Current discharge criteria and the impact of extra-pulmonary SARS-CoV-2 samples are discussed.
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