Comparative assessment of mortality risk factors between admission and follow-up models among patients hospitalized with COVID-19

Objectives: This study aimed to compare differences in mortality risk factors between admission andfollow-up incorporated models.Methods: A retrospective cohort study of 524 patients with confirmed COVID-19 infection admitted to atertiary medical center in São Paulo, Brazil from 13 March to 30 April 2020. Data were collected onadmission, and the third, eighth and fourteenth days of hospitalization. The hazard ratio (HR) wascalculated and 28-day in-hospital mortality risk factors were compared between admission and follow-up models using a time-dependent Cox regression model.Results: Of 524 patients, 50.4% needed mechanical ventilation. The 28-day mortality rate was 32.8%.Compared with follow-up, admission models under-estimated the mortality HR for peripheral oxygensaturation 100 bpm (1.19 versus 2.04), respiratory rate >24/min (1.01versus 1.82) and mechanical ventilation (1.92 versus 12.93). Low oxygen saturation, higher oxygensupport and more biomarkers–including lactate dehydrogenase, C-reactive protein, neutrophil-lymphocyte ratio, and urea remained associated with mortality after adjustment for clinical factorsat follow-up compared with only urea and oxygen support at admission.Conclusions: The inclusion of follow-up measurements changed mortality hazards of clinical signs andbiomarkers. Low oxygen saturation, higher oxygen support, lactate dehydrogenase, C-reactive protein,neutrophil-lymphocyte ratio, and urea could help with prognosis of patients during follow-up

Genome-wide characterization of genetic variants and putative regions under selection in meat and egg-type chicken lines

Abstract\ud \ud Background\ud Meat and egg-type chickens have been selected for several generations for different traits. Artificial and natural selection for different phenotypes can change frequency of genetic variants, leaving particular genomic footprints throghtout the genome. Thus, the aims of this study were to sequence 28 chickens from two Brazilian lines (meat and white egg-type) and use this information to characterize genome-wide genetic variations, identify putative regions under selection using Fst method, and find putative pathways under selection.\ud \ud \ud Results\ud A total of 13.93 million SNPs and 1.36 million INDELs were identified, with more variants detected from the broiler (meat-type) line. Although most were located in non-coding regions, we identified 7255 intolerant non-synonymous SNPs, 512 stopgain/loss SNPs, 1381 frameshift and 1094 non-frameshift INDELs that may alter protein functions. Genes harboring intolerant non-synonymous SNPs affected metabolic pathways related mainly to reproduction and endocrine systems in the white-egg layer line, and lipid metabolism and metabolic diseases in the broiler line. Fst analysis in sliding windows, using SNPs and INDELs separately, identified over 300 putative regions of selection overlapping with more than 250 genes. For the first time in chicken, INDEL variants were considered for selection signature analysis, showing high level of correlation in results between SNP and INDEL data. The putative regions of selection signatures revealed interesting candidate genes and pathways related to important phenotypic traits in chicken, such as lipid metabolism, growth, reproduction, and cardiac development.\ud \ud \ud Conclusions\ud In this study, Fst method was applied to identify high confidence putative regions under selection, providing novel insights into selection footprints that can help elucidate the functional mechanisms underlying different phenotypic traits relevant to meat and egg-type chicken lines. In addition, we generated a large catalog of line-specific and common genetic variants from a Brazilian broiler and a white egg layer line that can be used for genomic studies involving association analysis with phenotypes of economic interest to the poultry industry.CB received a fellowship from the program Science Without Borders - National Council for Scientific and Technological Development (CNPq, grant 370620/2013–5). GCMM and TFG received fellowships from São Paulo Research Foundation (FAPESP, grants 14/21380–9 and 15/00616–7). LLC is recipient of productivity fellowship from CNPq. This project was funded by São Paulo Research Foundation (FAPESP) - thematic project (2014/08704–0)

Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences

The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & Nemésio 2007; Donegan 2008, 2009; Nemésio 2009a–b; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on 18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based researchers who signed it in the short time span from 20 September to 6 October 2016

Catálogo Taxonômico da Fauna do Brasil: setting the baseline knowledge on the animal diversity in Brazil

The limited temporal completeness and taxonomic accuracy of species lists, made available in a traditional manner in scientific publications, has always represented a problem. These lists are invariably limited to a few taxonomic groups and do not represent up-to-date knowledge of all species and classifications. In this context, the Brazilian megadiverse fauna is no exception, and the Catálogo Taxonômico da Fauna do Brasil (CTFB) (http://fauna.jbrj.gov.br/), made public in 2015, represents a database on biodiversity anchored on a list of valid and expertly recognized scientific names of animals in Brazil. The CTFB is updated in near real time by a team of more than 800 specialists. By January 1, 2024, the CTFB compiled 133,691 nominal species, with 125,138 that were considered valid. Most of the valid species were arthropods (82.3%, with more than 102,000 species) and chordates (7.69%, with over 11,000 species). These taxa were followed by a cluster composed of Mollusca (3,567 species), Platyhelminthes (2,292 species), Annelida (1,833 species), and Nematoda (1,447 species). All remaining groups had less than 1,000 species reported in Brazil, with Cnidaria (831 species), Porifera (628 species), Rotifera (606 species), and Bryozoa (520 species) representing those with more than 500 species. Analysis of the CTFB database can facilitate and direct efforts towards the discovery of new species in Brazil, but it is also fundamental in providing the best available list of valid nominal species to users, including those in science, health, conservation efforts, and any initiative involving animals. The importance of the CTFB is evidenced by the elevated number of citations in the scientific literature in diverse areas of biology, law, anthropology, education, forensic science, and veterinary science, among others

A defective TLR4 signaling for IFN-β expression is responsible for the innately lower ability of BALB/c macrophages to produce NO in response to LPS as compared to C57BL/6.

C57BL/6 mice macrophages innately produce higher levels of NO than BALB/c cells when stimulated with LPS. Here, we investigated the molecular events that account for this intrinsic differential production of NO. We found that the lower production of NO in BALB/c is not due to a subtraction of L-arginine by arginase, and correlates with a lower iNOS accumulation, which is independent of its degradation rate. Instead, the lower accumulation of iNOS is due to the lower levels of iNOS mRNA, previously shown to be also independent of its stability, suggesting that iNOS transcription is less efficient in BALB/c than in C57BL/6 macrophages. Activation of NFκB is more efficient in BALB/c, thus not correlating with iNOS expression. Conversely, activation of STAT-1 does correlate with iNOS expression, being more prominent in C57BL/6 than in BALB/c macrophages. IFN-β and IL-10 are more highly expressed in C57BL/6 than in BALB/c macrophages, and the opposite is true for TNF-α. Whereas IL-10 and TNF-α do not seem to participate in their differential production of NO, IFN-β has a determinant role since 1) anti-IFN-β neutralizing antibodies abolish STAT-1 activation reducing NO production in C57BL/6 macrophages to levels as low as in BALB/c cells and 2) exogenous rIFN-β confers to LPS-stimulated BALB/c macrophages the ability to phosphorylate STAT-1 and to produce NO as efficiently as C57BL/6 cells. We demonstrate, for the first time, that BALB/c macrophages are innately lower NO producers than C57BL/6 cells because they are defective in the TLR-4-induced IFN-β-mediated STAT-1 activation pathway

IFN-β mRNA expression and effect of IFN-β neutralization on NO production and STAT-1 activation in BALB/c and C57BL/6 macrophages.

<p>Peritoneal macrophages (5×10⁶) were stimulated with 1 µg/mL LPS for the indicated periods of time. Total RNA was extracted and IFN-β mRNA levels were determined by Real Time RT-PCR. The relative levels of mRNA expression were calculated by reference to the β-actin expression in each sample, using the 2^−ΔΔCt method (A). Alternatively, cells (1×10⁵) were stimulated for 24 h (B) or for the indicated periods of time (C–E) with 1 µg/mL LPS in the presence or absence of the indicated concentrations (B) or with 200 U/mL of polyclonal anti-IFN-β (C–E). Culture supernatants were analyzed for NO⁻₂ levels using the Griess reaction, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#s2" target="_blank">Materials and Methods</a>. Purified normal rabbit IgG was used as an isotype control. (B) represents the dose-response of one experiment and (C) represents the time course of a second one, out of three independent experiments. Values represent the mean of samples assayed in triplicate. (D and E) represents STAT-1 activation as analyzed by Western blot of total protein extracts of C57BL/6 peritoneal macrophages (3×10⁵) using antibodies specific for pSTAT-1 (Y701) and β-actin. The levels of pSTAT-1 (E) were expressed as ratios of the signal intensity of the bands normalized to that of β-actin. Data are representative of three independent and reproducible experiments. Additional experiments to illustrate the variability in the results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#pone.0098913.s004" target="_blank">Fig. S4</a>.</p

Effect of exogenous IFN-β on NO production and STAT-1 activation in BALB/c and C57BL/6 macrophages.

<p>Peritoneal macrophages (1×10⁶) were stimulated with 1 µg/mL LPS for the indicated periods of time. After 8 hs, supernatants of C57BL/6 and BALB/c cells, still devoid of NO, were swapped and, cells were cultured in the presence (dotted lines) or absence (dashed lines) of anti- IFN-β for the indicated periods of time, when supernatants were analyzed for NO⁻₂ levels using the Griess reaction, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#s2" target="_blank">Materials and Methods</a> (A). Alternatively, 1×10⁶ (B and C) or 3×10⁶ (D and E) were stimulated for 48 h (B) or for the indicated periods of time (C–E) with 1 µg/mL LPS in the presence or absence of the indicated concentrations (B) or with 10 U/mL rIFN-β (C–E). Culture supernatants were analyzed for NO⁻₂ levels as above (B and C). (B) represents the dose-response of one experiment and (C) represents the time course of a second one, out of three independent experiments. Values represent the mean of samples assayed in triplicate. D and E represent STAT-1 activation as analyzed by Western blot of total protein extracts of C57BL/6 peritoneal macrophages (3×10⁵) using antibodies specific for pSTAT-1 (Y701) and β-actin. The levels of pSTAT-1 (F) were expressed as ratios of the signal intensity of the bands normalized to that of β-actin. Data are representative of three independent and reproducible experiments. Additional experiments to illustrate the variability in the results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#pone.0098913.s005" target="_blank">Fig. S5</a>.</p

STAT-1 and NF-κB expression and activation in BALB/c and C57BL/6 macrophages.

<p>Total protein extracts of C57BL/6 or BALB/c peritoneal macrophages (1×10⁶) stimulated with 1 µg/mL LPS for the indicated periods of time were analyzed by Western blot using specific anti-STAT-1, anti-pSTAT-1 (Y701) (A) or anti-pp65 (Ser536), anti-IκB-α (B) and anti-β-actin antibodies (A and B). The levels of STAT-1 (C), p-STAT-1 (E), p-p65 (D) or IκB-α (F) were expressed as ratios of the signal intensity of the bands normalized to that of β-actin. Data are representative of three independent and reproducible experiments.</p

IL-10 and TNF-α expression and effect of IL-10 or TNF-α neutralization on NO production in BALB/c and C57BL/6 macrophages.

<p>Cells (1×10⁶ or 5×10⁶) were stimulated with 1 µg/mL LPS for the indicated periods of time. Total RNA was extracted and IL-10 (A) and TNF-α (B) mRNA levels were determined by Real Time RT-PCR. The relative levels of mRNA expression were calculated by reference to the β-actin expression in each sample, using the 2^−ΔΔCt method. Protein levels of IL-10 (C) or TNF-α (D) were measured in culture supernatants by ELISA. Values represent the mean ±SD of samples assayed in triplicate. Alternatively, cells (1×10⁵) were stimulated for indicated periods of time (E and DR) with 1 µg/mL LPS in the presence or absence of 10 µg/mL of either a rat monoclonal IgG antibody specific for IL-10 receptor (E) or a mouse-human chimeric anti-human TNF-α antibody (Infliximab) (F). Culture supernatants were analyzed for NO⁻₂ levels using the Griess reaction, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#s2" target="_blank">Materials and Methods</a>. Values represent the mean of samples assayed in triplicate. Data are representative of three independent and reproducible experiments. Additional experiments to illustrate the variability in the results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#pone.0098913.s006" target="_blank">Fig. S6</a>.</p

Arginase expression and activity in BALB/c and C57BL/6 macrophages.

<p>Cells (5×10⁶) were stimulated with 1 µg/mL LPS for the indicated periods of time. Total RNA was extracted, and mRNA levels of arginase I (A) and arginase II (B) were determined by Real Time RT-PCR. The relative levels of gene expression were calculated by reference to the β-actin expression in each sample, using the 2^−ΔΔCt method. Values represent the mean of samples assayed in triplicate. After stimulation with 1 µg/mL LPS for the indicated time, peritoneal macrophages (5×10⁵) were washed, lysed and assayed for arginase activity by urea quantification (C). Values correspond to the difference between stimulated and non-stimulated cells, and the data are representative of three independent and reproducible experiments. Additional experiments to illustrate the variability in the results are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098913#pone.0098913.s002" target="_blank">Fig. S2</a>.</p