Quantifying the importance of the key sites on haemagglutinin in determining the selection advantage of influenza virus: Using A/H3N2 as an example Dear Editor , Authors' contributionsSZ and MHW conceived the study. SZ carried out the analysis, and drafted the first manuscript. SZ and MHW discussed the results. All authors read, revised the manuscript, and gave final approval for publication. Declaration of Competing InterestMHW is a shareholder of Beth Bioinformatics Co., Ltd, and BCYZ is a shareholder of Beth Bioinformatics Co., Ltd and Health View Bioanalytics Ltd. Declarations Ethics approval and consent to participateThe ethical approval or individual consent was not applicable. Availability of data and materialsAll influenza viruses sequence data were collected via the influenza virus database (IVD) of the National center for Biotechnology Information (NCBI). Please see the online supporting information for details. Consent for publicationNot applicable.

No abstract

Background Tocilizumab blocks pro-inflammatory activity of interleukin-6 (IL-6), involved in pathogenesis of pneumonia the most frequent cause of death in COVID-19 patients. Methods A multicenter, single-arm, hypothesis-driven trial was planned, according to a phase 2 design, to study the effect of tocilizumab on lethality rates at 14 and 30 days (co-primary endpoints, a priori expected rates being 20 and 35%, respectively). A further prospective cohort of patients, consecutively enrolled after the first cohort was accomplished, was used as a secondary validation dataset. The two cohorts were evaluated jointly in an exploratory multivariable logistic regression model to assess prognostic variables on survival. Results In the primary intention-to-treat (ITT) phase 2 population, 180/301 (59.8%) subjects received tocilizumab, and 67 deaths were observed overall. Lethality rates were equal to 18.4% (97.5% CI: 13.6–24.0, P = 0.52) and 22.4% (97.5% CI: 17.2–28.3, P < 0.001) at 14 and 30 days, respectively. Lethality rates were lower in the validation dataset, that included 920 patients. No signal of specific drug toxicity was reported. In the exploratory multivariable logistic regression analysis, older age and lower PaO2/FiO2 ratio negatively affected survival, while the concurrent use of steroids was associated with greater survival. A statistically significant interaction was found between tocilizumab and respiratory support, suggesting that tocilizumab might be more effective in patients not requiring mechanical respiratory support at baseline. Conclusions Tocilizumab reduced lethality rate at 30 days compared with null hypothesis, without significant toxicity. Possibly, this effect could be limited to patients not requiring mechanical respiratory support at baseline. Registration EudraCT (2020-001110-38); clinicaltrials.gov (NCT04317092).

Hyperglycemia was reported to enhance angiotensin (Ang) II generation in rat cardiomyocytes, and Ang II inhibition reduces cardiovascular morbidity and mortality in diabetic patients. In diabetic patients, the enhanced activation of intracellular pathways related with myocyte hypertrophy and gene expression might enhance the progression of cardiac damage. Therefore, we investigated the effects of glucose on Ang II-mediated activation of Janus-activated kinase (JAK)-2, a tyrosine kinase related with myocyte hypertrophy and cytokine and fibrogenetic growth factor overexpression, in ventricular myocytes isolated from nonfailing human hearts (n ‫؍‬ 5) and failing human hearts (n ‫؍‬ 8). In nonfailing myocytes, JAK2 phosphorylation was enhanced by Ang II only in the presence of high glucose (25 mmol/l) via Ang II type I (AT1) receptors (؉79% vs. normal glucose, P < 0.05). JAK2 activation was prevented by inhibitors of reactive oxygen species (ROS) generation (diphenyleneiodonium [DPI], tiron, and apocynin). In myocytes isolated from failing hearts, JAK2 phosphorylation was enhanced by high glucose alone (؉107%, P < 0.05). High glucose-induced JAK2 activation was blunted by both ACE inhibition (100 nmol/l ramipril) and AT1 antagonism (1 mol/l valsartan), thus revealing that the effects are mediated by autocrine Ang II production. Inhibition of ROS generation also prevented high glucose-induced JAK2 phosphorylation. In conclusion, in human nonfailing myocytes, high glucose allows Ang II to activate JAK2 signaling, whereas in failing myocytes, hyperglycemia alone is able to induce Ang II generation, which in turn activates JAK2 via enhanced oxidative stress. Diabetes 54: 394 -401, 2005

Objective: To validate a clinical diagnostic tool, used by emergency physicians (EPs), to diagnose the central cause of patients presenting with vertigo, and to determine interrater reliability of this tool. Methods: A convenience sample of adult patients presenting to a single academic ED with isolated vertigo (i.e. vertigo without other neurological deficits) was prospectively evaluated with STANDING (SponTAneous Nystagmus, Direction, head Impulse test, standiNG) by five trained EPs. The first step focused on the presence of spontaneous nystagmus, the second on the direction of nystagmus, the third on head impulse test and the fourth on gait. The local standard practice, senior audiologist evaluation corroborated by neuroimaging when deemed appropriate, was considered the reference standard. Sensitivity and specificity of STANDING were calculated. On the first 30 patients, inter-observer agreement among EPs was also assessed. Results: Five EPs with limited experience in nystagmus assessment volunteered to participate in the present study enrolling 98 patients. Their average evaluation time was 9.9 ± 2.8 min (range 6-17). Central acute vertigo was suspected in 16 (16.3%) patients. There were 13 true positives, three false positives, 81 true negatives and one false negative, with a high sensitivity (92.9%, 95% CI 70-100%) and specificity (96.4%, 95% CI 93-38%) for central acute vertigo according to senior audiologist evaluation. The Cohen's kappas of the first, second, third and fourth steps of the STANDING were 0.86, 0.93, 0.73 and 0.78, respectively. The whole test showed a good inter-observer agreement (k = 0.76, 95% CI 0.45-1). Conclusions: In the hands of EPs, STANDING showed a good interobserver agreement and accuracy validated against the local standard of care.

With demands and reliance on aquaculture still growing, there are various challenges to allow sustainable growth and the shift from fishmeal (FM) to other protein sources in aquafeed formulations is one of the most important. In this regard, interest in the use of insect meal (IM) in aquafeeds has grown rapidly. Accordingly, the aim of the present study was to assess the effects of dietary IM from Hermetia illucens (Hi) larvae included in a low-FM diet on gut microbial communities of rainbow trout (Oncorhynchus mykiss), in terms of both composition and function of microbiome. A feeding trial was conducted using 192 trout of about 100-g mean initial weight. Fish were fed in quadruplicate (4 tanks/diet) for 131 days with two diets: the control (Ctrl) contained 20% of FM as well as other protein sources, whereas the Hi diet contained 15% of Hi larvae meal to replace 50% of the FM contained in the Ctrl diet. High-throughput sequencing of 16S rRNA gene was used to identify the major feed and gut bacterial taxa, whereas Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis was performed on gut bacterial genomes to identify the major active biological pathways. The inclusion of IM led to an increase in Firmicutes, mainly represented by Bacilli class and to a drastic reduction of Proteobacteria. Beneficial genera, such as Lactobacillus and Bacillus, were enriched in the gut of fish fed with the Hi diet, whereas the number of bacteria assigned to the pathogenic Aeromonas genus was drastically reduced in the same fish group. The metagenome functional data provided evidence that dietary IM inclusion can shape the metabolic activity of trout gut microbiota. In particular, intestinal microbiome of fish fed with IM may have the capacity to improve dietary carbohydrate utilization. Therefore, H. illucens meal is a promising protein source for trout nutrition, able to modulate gut microbial community by increasing the abundance of some bacteria taxa that are likely to play a key role in fish health.

Background Aquaculture must continue to reduce dependence on fishmeal (FM) and fishoil in feeds to ensure sustainable sector growth. Therefore, the use of novel aquaculture feed ingredients is growing. In this regard, insects can represent a new world of sustainable and protein-rich ingredients for farmed fish feeds. Accordingly, we investigated the effects of full replacement of FM with Tenebrio molitor (TM) larvae meal in the diet of rainbow trout (Oncorhynchus mykiss) on fish gut and skin microbiota. Methods A feeding trial was conducted with 126 trout of about 80 g mean initial weight that were fed for 22 weeks with two isonitrogenous, isolipidic, and isoenergetic extruded experimental diets. Partially defatted TM meal was included in one of the diets to replace 100% (TM 100) of FM, whereas the other diet (TM 0) was without TM. To analyse the microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and Qiime pipeline were used to identify bacteria in the gut and skin mucosa, and in the diets. Results The data showed no major effects of full FM substitution with TM meal on bacterial species richness and diversity in both, gut mucosa- and skin mucus-associated microbiome. Skin microbiome was dominated by phylum Proteobacteria and especially by Gammaproteobacteria class that constituted approximately half of the bacterial taxa found. The two dietary fish groups did not display distinctive features, except for a decrease in the relative abundance of Deefgea genus (family Neisseriaceae) in trout fed with insect meal. The metagenomic analysis of the gut mucosa indicated that Tenericutes was the most abundant phylum, regardless of the diet. Specifically, within this phylum, the Mollicutes, mainly represented by Mycoplasmataceae family, were the dominant class. However, we observed only a weak dietary modulation of intestinal bacterial communities. The only changes due to full FM replacement with TM meal were a decreased number of Proteobacteria and a reduced number of taxa assigned to Ruminococcaceae and Neisseriaceae families. Conclusions The data demonstrated that TM larvae meal is a valid alternative animal protein to replace FM in the aquafeeds. Only slight gut and skin microbiota changes occurred in rainbow trout after total FM replacement with insect meal. The mapping of the trout skin microbiota represents a novel contribution of the present study. Indeed, in contrast to the increasing knowledge on gut microbiota, the skin microbiota of major farmed fish species remains largely unmapped but it deserves thorough consideration.

The influence of hydration on the Watson-Crick cytosine-guanine base pair was investigated, testing the ability of the self-consistent field for molecular interaction (SCF-MI) ab initio method to reproduce the hydration pattern present in a real system (the base pair in the DNA framework). The positions of hydration sites around the base pair predicted by a knowledge-based approach employing crystallographic data were compared to the ab initio optimized structures. The SCF-MI method was applied to perform basis set superposition error (BSSE)-free geometry optimization. The hydration shell taken into accountscomprising five water molecules, three on guanine and two on cytosines"saturates" the base pair, engaging all of the available hydrogen bond donors/acceptors. The interaction between water and the base pair was also analyzed from the energetic viewpoint, highlighting the role of water in the pair stabilization.