A new fluorescent oligonucleotide probe for in-situ identification of Microcystis aeruginosa in freshwater.

contaminated water bodies (freshwater, brackish and marine areas). Among 150 known cyanobacteria genera,>40 species are able to produce toxins, which are natural compounds that differ from both a chemical and toxicological point of view and are responsible for acute and chronic poisoning in animals and humans. Among the main classes of cyanotoxins, microcystins are frequently found in the environment. Fast and accurate methods for unequivocally identifying microcystin-producing cyanobacteria, such as Microcystis aeruginosa in water bodies, are necessary to distinguish them from other non-toxic cyanobacteria and to manage and monitor algal blooms. For this purpose, we designed, developed and validated an oligonucleotide probe for FISH (Fluorescence In Situ Hybridization) analysis to detect Microcystis aeruginosa at the species level even at relatively low concentrations in freshwater. The FISH probe, MicAerD03, was designed using the ARB software with the Silva database within the framework of the MicroCoKit project, also with the intention of adding it to the microarray from the EU project, μAQUA, for freshwater pathogens, which had only genus level probes for Microcystis. We tested various fixative methods to minimize the natural autofluorescence from chlorophyll-a and certain accessory pigments (viz., phycobilins and carotenoids). The FISH probe was tested on pure cultures of Microcystis aeruginosa, and then successfully applied to water samples collected from different sampling points of the Tiber River (Italy), using a laser confocal microscope. Subsequently, the probe was also conjugated at the 5′ end with horse-radish peroxidase (HRP-MicAerD03) to apply the CAtalysed Reported Deposition-FISH (CARD-FISH) for increasing the fluorescence signal of the mono-fluorescently labelled probe and make it possible to detect M. aeruginosa using an epifluorescence microscope. Samples taken within the EU MicroCokit project indicated thatmicroarray signals for Microcystis were coming from single cells and not colonial cells. We confirmed this with the CARD-FISH protocol used here to validate the microarray signals for Microcystis detected at the genus level in MicroCokit. This paper provides a new early warning tool for investigating M. aeruginosa at the species level even at low cell concentrations in surface water, which can be added to the μAqua microarray for all freshwater pathogens to complete the probe hierarchy for Microcystis aeruginosa

A comparative study on the effects of a pesticide (cypermethrin) and two metals (copper, lead) to serum biochemistry of Nile tilapia, Oreochromis niloticus

The present study was designed to compare the responses in freshwater fish Oreochromis niloticus exposed to a synthetic pyrethroid, cypermethrin (CYP); an essential metal, copper (Cu); and a nonessential metal, lead (Pb). Fish were exposed to 0.05 μg/l CYP, 0.05 mg/l Cu, and 0.05 mg/l Pb for 4 and 21 days, and the alterations in serum enzyme activities, metabolite, and ion levels were determined. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities increased in response to CYP, Cu, and Pb exposures at both exposure periods. While elevations in alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities and in cholesterol level were observed in pesticide-exposed fish at 4 and 21 days, they increased in Cu- and Pb-exposed fish at 21 days. Although metal-exposed fish showed increases in cortisol and glucose levels at 4 days followed by a return to control levels at the end of the exposure period, their levels elevated in pesticide-exposed fish at both exposure periods. Total protein levels decreased in Pb- and pesticide-exposed fish at 21 days. Na+ and Cl− levels decreased in pesticide-exposed fish at both exposure periods and in Cu- and Pb-exposed fish at 21 days. The exposures of pesticide and metals caused an elevation in K+ level at the end of the exposure period. The present study showed that observed alterations in all serum biochemical parameters of fish-treated pesticide were higher than those in fish exposed to metals

Sarilumab in patients admitted to hospital with severe or critical COVID-19: a randomised, double-blind, placebo-controlled, phase 3 trial

Background: Elevated proinflammatory cytokines are associated with greater COVID-19 severity. We aimed to assess safety and efficacy of sarilumab, an interleukin-6 receptor inhibitor, in patients with severe (requiring supplemental oxygen by nasal cannula or face mask) or critical (requiring greater supplemental oxygen, mechanical ventilation, or extracorporeal support) COVID-19. Methods: We did a 60-day, randomised, double-blind, placebo-controlled, multinational phase 3 trial at 45 hospitals in Argentina, Brazil, Canada, Chile, France, Germany, Israel, Italy, Japan, Russia, and Spain. We included adults (≥18 years) admitted to hospital with laboratory-confirmed SARS-CoV-2 infection and pneumonia, who required oxygen supplementation or intensive care. Patients were randomly assigned (2:2:1 with permuted blocks of five) to receive intravenous sarilumab 400 mg, sarilumab 200 mg, or placebo. Patients, care providers, outcome assessors, and investigators remained masked to assigned intervention throughout the course of the study. The primary endpoint was time to clinical improvement of two or more points (seven point scale ranging from 1 [death] to 7 [discharged from hospital]) in the modified intention-to-treat population. The key secondary endpoint was proportion of patients alive at day 29. Safety outcomes included adverse events and laboratory assessments. This study is registered with ClinicalTrials.gov, NCT04327388; EudraCT, 2020-001162-12; and WHO, U1111-1249-6021. Findings: Between March 28 and July 3, 2020, of 431 patients who were screened, 420 patients were randomly assigned and 416 received placebo (n=84 [20%]), sarilumab 200 mg (n=159 [38%]), or sarilumab 400 mg (n=173 [42%]). At day 29, no significant differences were seen in median time to an improvement of two or more points between placebo (12·0 days [95% CI 9·0 to 15·0]) and sarilumab 200 mg (10·0 days [9·0 to 12·0]; hazard ratio [HR] 1·03 [95% CI 0·75 to 1·40]; log-rank p=0·96) or sarilumab 400 mg (10·0 days [9·0 to 13·0]; HR 1·14 [95% CI 0·84 to 1·54]; log-rank p=0·34), or in proportions of patients alive (77 [92%] of 84 patients in the placebo group; 143 [90%] of 159 patients in the sarilumab 200 mg group; difference −1·7 [−9·3 to 5·8]; p=0·63 vs placebo; and 159 [92%] of 173 patients in the sarilumab 400 mg group; difference 0·2 [−6·9 to 7·4]; p=0·85 vs placebo). At day 29, there were numerical, non-significant survival differences between sarilumab 400 mg (88%) and placebo (79%; difference +8·9% [95% CI −7·7 to 25·5]; p=0·25) for patients who had critical disease. No unexpected safety signals were seen. The rates of treatment-emergent adverse events were 65% (55 of 84) in the placebo group, 65% (103 of 159) in the sarilumab 200 mg group, and 70% (121 of 173) in the sarilumab 400 mg group, and of those leading to death 11% (nine of 84) were in the placebo group, 11% (17 of 159) were in the sarilumab 200 mg group, and 10% (18 of 173) were in the sarilumab 400 mg group. Interpretation: This trial did not show efficacy of sarilumab in patients admitted to hospital with COVID-19 and receiving supplemental oxygen. Adequately powered trials of targeted immunomodulatory therapies assessing survival as a primary endpoint are suggested in patients with critical COVID-19. Funding: Sanofi and Regeneron Pharmaceuticals

Comparison of the safety and tolerance profile of micafungin with that of other echinocandins and azoles in patients with pre-existing child-pugh B or C liver disease: a case-control retrospective study

Introduction: To assess the association between exposure to micafungin, other echinocandins, or azoles and the development of short-term liver injury (STLI) or long-term liver injury (LTLI) in patients with Child-Pugh B or C liver disease. Methods: Multicenter case-control study of patients with Child-Pugh B or C liver disease who received antifungals (AF) for ≥ 72 h (May 2009-May 2015) in six Spanish and Italian hospitals. All micafungin patients were randomly matched with one patient who received another echinocandin and with one patient who received azole treatment. Primary outcome was development of STLI or LTLI (development of any type of liver tumor during the follow-up period). Results: Of 2335 patients with chronic liver disease admitted to the six centers, 20 (0.85%) were found to have Child-Pugh B or C liver disease and received micafungin for ≥ 72 h. During AF treatment, the frequency of STLI was 10% in each group. Most cases of STLI were asymptomatic, and AFs had to be switched to another class of AF in only two patients (one micafungin and one azole). No patients developed acute liver insufficiency, were admitted to the ICU, or had to undergo transplantation. Follow-up data (median of 1.3 years) were available for 30 patients. LTLI was observed in only one patient, who had previously received treatment with azoles. Conclusions: Our study suggests that the administration of micafungin to patients with end-stage liver disease does not imply a higher risk of developing STLI or LTLI

Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases

10.1093/hmg/ddm320Human Molecular Genetics173431-439HMGE

Efficacy and safety of a booster dose of influenza vaccination in solid organ transplant recipients, TRANSGRIPE 1-2: study protocol for a multicenter, randomized, controlled clinical trial

BACKGROUND: Despite administration of annual influenza vaccination, influenza-associated complications in transplant recipients continue to be an important cause of hospitalization and death. Although influenza vaccination has been proven to be the most effective measure to reduce influenza infection after transplantation, transplant recipients are still vulnerable to influenza infections, with lower serological responses to vaccination compared to the general population. In order to assess the efficacy and safety of an alternative immunization scheme for solid organ transplant recipients, the TRANSGRIPE1-2 Study Group aimed to test a booster dose administration 5 weeks after the standard vaccination. The primary objective of this trial was to compare short-term and long-term neutralizing antibody immunogenicity of a booster dose of influenza vaccination to the standard single-dose immunization scheme. Secondary objectives included the evaluation of the efficacy and/or safety, cellular immune response, incidence of influenza infection, graft rejection, retransplant and mortality rates. METHODS/DESIGN: This phase III, randomized, controlled, open-label clinical trial was conducted between October 2012 and December 2013 in 12 Spanish public referral hospitals. Solid organ transplant recipients (liver, kidney, heart or lung), older than 16 years of age more than 30 days after transplantation were eligible to participate. Patients (N = 514) were stratified 1:1 by center, type of organ and time after transplantation and who either received the standard single dose (n = 257) or were treated according to a novel influenza vaccination schedule comprising the administration of a booster dose 5 weeks after standard vaccination (n = 254). Seroconversion rates were measured as a determinant of protection against influenza (main outcome). Efficacy and safety outcomes were followed until 1 year after influenza vaccination with assessment of short-term (0, 5, 10 and 15 weeks) and long-term (12 months) results. Intention-to-treat, per-protocol and safety analyses will be performed. DISCUSSION: This trial will increase knowledge about the safety and efficacy of a booster dose of influenza vaccine in solid organ transplant recipients. At the time the manuscript was submitted for publication, trial recruitment was closed with a total of 499 participants included during a 2-month period (within the seasonal influenza vaccination campaign). TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01761435 (registered 13 December 2012). EudraCT Identifier: 2011-003243-21 (registered 4 July 2011)