Virological investigation on aerosol from waste depuration plants

Aerosol from activated mud decontamination plants used for the treatment of urban sewage can represent a vehicle for bacteria, virus and fungi. As a result, they become an infective hazard for plant personnel, the general population residing in the surrounding area and the occasional visitor. The present investigation focuses on the identification of enteric-type viruses in this kind of aerosol. The following methods were employed on 214 samples collected in the 1999-2000 period: cell culture (BGM, RD, Hep-2), electron microscopy, and polymerase chain reaction (PCR). Cytopathic effect was mild in 180 samples, and severe in 14, upon their first passage in culture. Virus identification was based on positivity to both electron microscopy (EM) and PCR. Thus, one positive sample was recognized to be of enteric-type virus and two positive samples were recognized as reovirus-type. All samples were negative for Norwalk-type virus or HAV. There was considerable discrepancy between electron microscopy and PCR concerning the number of enteric-type viruses recognized. A possible explanation is contamination with animal-type enterovirus

Short Vi-polysaccharide abrogates T-independent immune response and hyporesponsiveness elicited by long Vi-CRM197 conjugate vaccine

Publisher Copyright: © 2020 National Academy of Sciences. All rights reserved.Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG+ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines.Peer reviewe

Differential diagnosis of neurodegenerative dementias with the explainable MRI based machine learning algorithm MUQUBIA

Biomarker-based differential diagnosis of the most common forms of dementia is becoming increasingly important. Machine learning (ML) may be able to address this challenge. The aim of this study was to develop and interpret a ML algorithm capable of differentiating Alzheimer's dementia, frontotemporal dementia, dementia with Lewy bodies and cognitively normal control subjects based on sociodemographic, clinical, and magnetic resonance imaging (MRI) variables. 506 subjects from 5 databases were included. MRI images were processed with FreeSurfer, LPA, and TRACULA to obtain brain volumes and thicknesses, white matter lesions and diffusion metrics. MRI metrics were used in conjunction with clinical and demographic data to perform differential diagnosis based on a Support Vector Machine model called MUQUBIA (Multimodal Quantification of Brain whIte matter biomArkers). Age, gender, Clinical Dementia Rating (CDR) Dementia Staging Instrument, and 19 imaging features formed the best set of discriminative features. The predictive model performed with an overall Area Under the Curve of 98%, high overall precision (88%), recall (88%), and F1 scores (88%) in the test group, and good Label Ranking Average Precision score (0.95) in a subset of neuropathologically assessed patients. The results of MUQUBIA were explained by the SHapley Additive exPlanations (SHAP) method. The MUQUBIA algorithm successfully classified various dementias with good performance using cost-effective clinical and MRI information, and with independent validation, has the potential to assist physicians in their clinical diagnosis

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased fromone in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons

COVID-19 in children and adolescents in Europe: a multinational, multicentre cohort study

Background To date, few data on paediatric COVID-19 have been published, and most reports originate from China. This study aimed to capture key data on children and adolescents with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection across Europe to inform physicians and health-care service planning during the ongoing pandemic. Methods This multicentre cohort study involved 82 participating health-care institutions across 25 European countries, using a well established research network—the Paediatric Tuberculosis Network European Trials Group (ptbnet)—that mainly comprises paediatric infectious diseases specialists and paediatric pulmonologists. We included all individuals aged 18 years or younger with confirmed SARS-CoV-2 infection, detected at any anatomical site by RT-PCR, between April 1 and April 24, 2020, during the initial peak of the European COVID-19 pandemic. We explored factors associated with need for intensive care unit (ICU) admission and initiation of drug treatment for COVID-19 using univariable analysis, and applied multivariable logistic regression with backwards stepwise analysis to further explore those factors significantly associated with ICU admission. Findings 582 individuals with PCR-confirmed SARS-CoV-2 infection were included, with a median age of 5·0 years (IQR 0·5–12·0) and a sex ratio of 1·15 males per female. 145 (25%) had pre-existing medical conditions. 363 (62%) individuals were admitted to hospital. 48 (8%) individuals required ICU admission, 25 (4%) mechanical ventilation (median duration 7 days, IQR 2–11, range 1–34), 19 (3%) inotropic support, and one (<1%) extracorporeal membrane oxygenation. Significant risk factors for requiring ICU admission in multivariable analyses were being younger than 1 month (odds ratio 5·06, 95% CI 1·72–14·87; p=0·0035), male sex (2·12, 1·06–4·21; p=0·033), pre-existing medical conditions (3·27, 1·67–6·42; p=0·0015), and presence of lower respiratory tract infection signs or symptoms at presentation (10·46, 5·16–21·23; p<0·0001). The most frequently used drug with antiviral activity was hydroxychloroquine (40 [7%] patients), followed by remdesivir (17 [3%] patients), lopinavir–ritonavir (six [1%] patients), and oseltamivir (three [1%] patients). Immunomodulatory medication used included corticosteroids (22 [4%] patients), intravenous immunoglobulin (seven [1%] patients), tocilizumab (four [1%] patients), anakinra (three [1%] patients), and siltuximab (one [<1%] patient). Four children died (case-fatality rate 0·69%, 95% CI 0·20–1·82); at study end, the remaining 578 were alive and only 25 (4%) were still symptomatic or requiring respiratory support. Interpretation COVID-19 is generally a mild disease in children, including infants. However, a small proportion develop severe disease requiring ICU admission and prolonged ventilation, although fatal outcome is overall rare. The data also reflect the current uncertainties regarding specific treatment options, highlighting that additional data on antiviral and immunomodulatory drugs are urgently needed. Funding ptbnet is supported by Deutsche Gesellschaft für Internationale Zusammenarbeit

Ap1s2-truncating variant in a patient with severe neurodevelopmental disorder and cerebral folate deficiency

Not abstract availabl

The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins-2

AmphIISH3) or the carboxy-terminal SH3 of Grb2 (Grb2SH3-C) expressed in bacteria as GST fusions. GST alone is used as a negative control. Anti-GST antibodies were utilized to identify bound proteins. The peptide sequences are indicated. The best binding peptides are shown in bold. The three positive control peptides are: GST binding peptides (positive control for GST and secondary antibody) and two peptides containing SH3 interacting motifs. Each binding experiment was carried out in duplicate. The sequence in bold correspond to the best binders. The best peptide 338PPTPPPR344 shows an intensity above 11.000 arbitrary units. Another peptide containing the sequence 214LKARPS219 binds to the SH3 of Amphiphysin, albeit producing a spot intensity around 9000 arbitrary units. Quantitative data are available in additional file . ) The POB1 fifteenmer peptide (PPTPPPRPQKTHSRA), containing the 338PPTPPPR344 binding motif, was systematically mutagenized by introducing an alanine at each of the indicated 15 positions. The upper membrane was probed with GST as a control, the middle one with the SH3 domain of Amphiphysin II, the bottom one with the carboxy-terminal SH3 of Grb2 fused to GST. The substitution of arginine 344 with alanine reduces the binding of Amphiphysin-SH3 down to 2.5%; a substitution of proline 339 with alanine reduces the binding to 31%. A reduction in Grb2 binding is observed when arginine 344 is substituted with alanine (13%) and when Pro343 or Pro 435 are substituted with Ala (30%). Supplemental quantitative data are provided in additional file . ) HEK293 cells were transiently transfected with plasmids expressing GFP fused to the proline rich domain PRD1 of POB1 (lane 1, 2, 3) or with an equivalent construct directing the synthesis of the POB1 PRD1 region mutated in Arg344 (PRD1 R344A), in lanes 4, 5, 6. Cell extracts were adsorbed to glutathione resins containing either the SH3 of Amphiphysin II (top gel) or the C-terminal SH3 of Grb2 (bottom gel). Adsorbed proteins were identified with anti GFP antibodies (for POB1 PRD1). Input is 3% of the lysate utilized in the GST pull down.<p><b>Copyright information:</b></p><p>Taken from "The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins"</p><p>http://www.biomedcentral.com/1471-2091/9/21</p><p>BMC Biochemistry 2008;9():21-21.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2494995.</p><p></p

The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins-1

L of Grb2 (Grb2SH3-N), GST-SH3 carboxy-terminal of Grb2 (Grb2SH3-C) and GST alone, adsorbed to glutathione-Sepharose 4B beads. The retained proteins were separated by SDS-PAGE and probed with an anti-Myc antibody (for POB1). The input is 10% of the affinity-selected lysate. The AmphiphysinII SH3 binds 25% of the input while the carboxy-terminal SH3 of Grb2 binds 4%. The ammino-terminal SH3 of Grb2 does not bind POB1. The Ponceau staining shows the GST fusions and the input lysates. . HEK293 cells were transiently transfected with the indicated POB1 constructs expressed as fusions to GFP protein: GFP (lanes 1, 2, 3), GFP-POB1 full length (lanes 4, 5, 6) and GFP-PRD1, the poly-proline rich PRD1 region of POB1 from residue 308 to 365 (7, 8, 9). Cellular extracts were incubated with GST-SH3 of AmphiphysinII (left) or GST-SH3 carboxy-terminal of Grb2 (right) and GST alone. The retained proteins were subjected to SDS-PAGE and probed with an anti-GFP antibody (for POB1 or PRD1). The sample loaded in the input lane is 5% of the material used for affinity selection. A faint unspecific band is visible between the POB1 full length and the PRD1. POB1 PRD1 (308–365) is bound by both the SH3 tested: Amphiphysin II (last lane in leftmost gel) and Grb2 (last lane in rightmost gel). The Ponceau staining shows the GST fusions and the input lysates. . Left: POB1-GFP was co-expressed in HEK293 cells together with Grb2-HA or Amphiphysin II-FLAG (Amph-FLAG). Cellular extracts were immunoprecipitated (IP) with anti-HA antibody (for Grb2) or anti-FLAG resin (for Amphiphysin II), respectively and the co-immunoprecipitated POB1 was revealed with an anti-GFP antibody. Two lanes for each immuno-precipitation correspond to the same experiment repeated twice but at different washing stringency (without or with 1% Triton). Mock HA and FLAG lanes represent controls transfected with empty vectors. In the input lane we have loaded 1.5% of the cell lysate used for the immunoprecipitation. Right: An empty vector (lane 1) and two independent transfections of POB1-Myc were carried out in HEK293 cells (lanes 2, 3). Upper panel: POB1-Myc in cell lysate was revealed with an anti-Myc antibody (5% of the total cell lysate). Second panel: Cells extracts were immunoprecipitated with anti-Myc (for POB1) and the immunoprecipitated POB1 (an amount corresponding to 20% of the total lysate) were revealed with an anti-Myc antibody; Third panel: the cell lysate was probed with anti-Grb2 antibody, to detect endogenous Grb2 (1% of the total lysate). Panel below: Cells extracts were immunoprecipitated (IP) with anti-Myc (for POB1) and the co-precipitated material was blotted (WB) with an anti-Grb2 antibody to detect endogenous Grb2 co-immunoprecipitated by POB1.<p><b>Copyright information:</b></p><p>Taken from "The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins"</p><p>http://www.biomedcentral.com/1471-2091/9/21</p><p>BMC Biochemistry 2008;9():21-21.</p><p>Published online 22 Jul 2008</p><p>PMCID:PMC2494995.</p><p></p