The estimation of the inbreeding coefficient (F) is essential for the study of inbreeding depression (ID) or for the management of populations under conservation. Several methods have been proposed to estimate the realized F using genetic markers, but it remains unclear which one should be used. Here we used whole-genome sequence data for 245 individuals from a Holstein cattle pedigree to empirically evaluate which estimators best capture homozygosity at variants causing ID, such as rare deleterious alleles or loci presenting heterozygote advantage and segregating at intermediate frequency. Estimators relying on the correlation between uniting gametes (FUNI) or on the genomic relationships (FGRM) presented the highest correlations with these variants. However, homozygosity at rare alleles remained poorly captured. A second group of estimators relying on excess homozygosity (FHOM), homozygous-by-descent segments (FHBD), runs-of-homozygosity (FROH) or on the known genealogy (FPED) was better at capturing whole-genome homozygosity, reflecting the consequences of inbreeding on all variants, and for young alleles with low to moderate frequencies (0.10 < . < 0.25). The results indicate that FUNI and FGRM might present a stronger association with ID. However, the situation might be different when recessive deleterious alleles reach higher frequencies, such as in populations with a small effective population size. For locus-specific inbreeding measures or at low marker density, the ranking of the methods can also change as FHBD makes better use of the information from neighboring markers. Finally, we confirmed that genomic measures are in general superior to pedigree-based estimates. In particular, FPED was uncorrelated with locus-specific homozygosity.

The purpose of this study was to assess Analgesia/Nociception Index (ANI) and bispectral index (BIS) variations in supine and prone position during closed-tracheal suction in intensive care unit (ICU) patients with severe COVID-19 pneumonia requiring myorelaxation and prone positioning. We retrospectively reviewed the data of 15 patients hospitalized in ICU for severe COVID-19 pneumonia requiring sedation, myorelaxation and prone positioning. The BIS, instant ANI (ANIi), mean ANI (ANIm), heart rate (HR), systolic blood pressure (SBP) and SpO2 were retrieved in supine and prone position 1 min before tracheal suction then every minute from the beginning of tracheal suction during 4 min and compared using ANOVA for repeated measures (p < 0.05 considered as statistically significant). Both ANIm and ANIi decreased significantly during tracheal suction with no difference between positions, whereas BIS showed no significant variation within time and between groups. The median [Q1-Q3] ANIm value decreased from 87 [68-98] to 79 [63-09] in supine position and from 79 [63-95] to 78 [66-98] in prone position 2 min after the beginning of tracheal suction. The median [Q1-Q3] ANIi value decreased earlier 1 min after the beginning of tracheal suction from 84 [69-98] to 73 [60-90] in supine position and from 84 [60-99] to 71 [51-88] in prone position. Both HR, SBP and SpO2 varied modestly but significantly during tracheal suction with no difference between positions. Monitoring ANI, but not BIS, may be of interest to detect noxious stimuli such as tracheal suction in ICU myorelaxed patients with severe COVID-19 pneumonia requiring prone positioning.

A systematic review and meta-analysis basing on the prospective cohort studies were conducted to explore the risk of all-cause mortality and dementia in cognitively frail older adults compared to robust older adults and to determine whether it was a predictor of adverse outcomes.

Essential oils (EOs) have attracted increased interest for different applications such as food preservatives, feed additives and ingredients in cosmetics. Due to their reported variable composition of components, they might be acutely toxic to humans and animals in small amounts. Despite the necessity, rigorous toxicity testing in terms of safety evaluation has not been reported so far, especially using alternatives to animal models. Here, we provide a strategy by use of alternative in vitro (cell cultures) and in vivo (Caenorhabditis elegans, hen's egg test) approaches for detailed investigation of the impact of commonly used rosemary, citrus and eucalyptus essential oil on acute, developmental and reproductive toxicity as well as on mucous membrane irritation. In general, all EOs under study exhibited a comparable impact on measured parameters, with a slightly increased toxic potential of rosemary oil. In vitro cell culture results indicated a concentration-dependent decrease of cell viability for all EOs, with mean IC50 values ranging from 0.08 to 0.17% [v/v]. Similar results were obtained for the C. elegans model when using a sensitized bus-5 mutant strain, with a mean LC50 value of 0.42% [v/v]. In wild-type nematodes, approximately tenfold higher LC50 values were detected. C. elegans development and reproduction was already significantly inhibited at concentrations of 0.5% (wild-type) and 0.1% (bus-5) [v/v] of EO, respectively. Gene expression analysis revealed a significant upregulation of xenobiotic and oxidative stress genes such as cyp-14a3, gst-4, gpx-6 and sod-3. Furthermore, all three EOs under study showed an increased short-time mucous membrane irritation potential, already at 0.5% [v/v] of EO. Finally, GC-MS analysis was performed to quantitate the relative concentration of the most prominent EO compounds. In conclusion, our results demonstrate that EOs can exhibit severe toxic properties, already at low concentrations. Therefore, a detailed toxicological assessment is highly recommended for each EO and single intended application.

Options for the effective treatment of hereditary optic neuropathies have been a long time coming. The successful launch of the antioxidant idebenone for Leber's Hereditary Optic Neuropathy (LHON), followed by its introduction into clinical practice across Europe, was an important step forward. Nevertheless, other options, especially for a variety of mitochondrial optic neuropathies such as dominant optic atrophy (DOA), are needed, and a number of pharmaceutical agents, acting on different molecular pathways, are currently under development. These include gene therapy, which has reached Phase III development for LHON, but is expected to be  developed also for DOA, whilst most of the other agents (other antioxidants, anti-apoptotic drugs, activators of mitobiogenesis, etc.) are almost all at Phase II or at preclinical stage of research. Here, we review proposed target mechanisms, preclinical evidence, available clinical trials with primary endpoints and results, of a wide range of tested molecules, to give an overview of the field, also providing the landscape of future scenarios, including gene therapy, gene editing, and reproductive options to prevent transmission of mitochondrial DNA mutations.

In the neocortex, each sensory modality engages distinct sensory areas that route information to association areas. Where signal flow converges for maintaining information in short-term memory and how behavior may influence signal routing remain open questions. Using wide-field calcium imaging, we compared cortex-wide neuronal activity in layer 2/3 for mice trained in auditory and tactile tasks with delayed response. In both tasks, mice were either active or passive during stimulus presentation, moving their body or sitting quietly. Irrespective of behavioral strategy, auditory and tactile stimulation activated distinct subdivisions of the posterior parietal cortex, anterior area A and rostrolateral area RL, which held stimulus-related information necessary for the respective tasks. In the delay period, in contrast, behavioral strategy rather than sensory modality determined short-term memory location, with activity converging frontomedially in active trials and posterolaterally in passive trials. Our results suggest behavior-dependent routing of sensory-driven cortical signals flow from modality-specific posterior parietal cortex (PPC) subdivisions to higher association areas.

Metabolomics is a study of the entire repertoire of metabolites in a cell at a particular time point. Here, we investigate the mouse lens at multiple embryonic and postnatal time points to establish the metabolome profile during early lens development. The lenses were isolated at six time points including embryonic day 15 (E15) and E18 and postnatal day 0 (P0), P3, P6, and P9. A total of four biological replicates of each time point, each consisting of 25 mg of lens tissue were preserved. Sample preparation was performed by protein precipitation followed by centrifugation to remove proteins and recover metabolites. The resulting extract was subjected to reverse phase/ultra-performance liquid chromatography-tandem mass spectrometry. Metabolome profiling identified a total of 353 metabolites in mouse lens, marked with an abundance of collagen, antioxidant, glycosaminoglycans, lipid, amino acid, and energy-related metabolites. A comparative metabolome analysis identified >200 metabolites exhibiting increased levels (p < 0.05) at latter time points relative to E15. Principal component analysis revealed distinct metabolomic signatures running from E15 to P9 while random forest analysis categorized lipid-, amino acid-, and nucleotide-related metabolites contributing significantly to the separation of the time points. To the best of our knowledge, this is the first report investigating the mouse lens metabolome at multiple embryonic and postnatal time points.

Molting in decapod crustaceans is controlled by ecdysteroid hormones synthesized and secreted by the molting gland, or Y-organ (YO). Halloween genes encode cytochrome P450 enzymes in the ecdysteroid synthetic pathway. The current paradigm is that YOs secrete an inactive precursor (e.g., ecdysone or E), which is hydroxylated at the #20 carbon to form an active hormone (20-hydroxyecdysone or 20E) by a mitochonrial 20-monooxygenase (CYP314A1) in peripheral tissues. 20-Monooxygenase is encoded by Shed in decapods and Shade in insects. We used eastern spiny lobster Shed sequences to extract six orthologs in the G. lateralis YO transcriptome. Phylogenetic analysis of the deduced amino acid sequences from six decapod species organized the Sheds into seven classes (Sheds 1-7), resulting in the assignment of the G. lateralis Sheds to Gl-Shed1, 2, 4A, 4B, 5A, and 5B. The mRNA levels of the six Gl-Sheds in the YO of intermolt animals were comparable to those in nine other tissues that included hepatopancreas and muscle. qPCR was used to compare the effects of molt induction by multiple leg autotomy (MLA) and eyestalk ablation (ESA) on Gl-Shed mRNA levels in the YO. Molt stage had little effect on Gl-Shed1 and Gl-Shed5B expression in the YO of MLA animals. Gl-Shed5A was expressed at the highest mRNA levels in the YO and was significantly increased during early and mid premolt stages. By contrast, ESA ± SB431542 had no effect on Gl-Shed expression at 1, 3, 5, and 7 days post-ESA. SB431542, which inhibits Transforming Growth Factor-β/activin signaling and blocks YO commitment, decreased Gl-Shed2 and Gl-Shed4A mRNA levels at 14 days post-ESA. A targeted metabolomic analysis showed that YOs cultured in vitro secreted E and 20E to the medium. These data suggest that the YO expresses 20-monooygenases that can convert E to 20E, which may contribute to the increase in active hormone in the hemolymph during premolt.

The methods of solution NMR, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study two zinc-containing L-alanyl-D-glutamate peptidases - endolysins of the pseudo T-even myoviruses RB43 and RB49 (EndoRB43 and EndoRB49, respectively), which are orthologous to the EndoT5, which is a zinc-containing L-alanyl-D-glutamate peptidase of the T5 siphovirus. The spatial conservation of the Zn2+-binding sites for the enzymes EndoT5, EndoRB43, and EndoRB49 was established, and the key role of Zn2+ ions in the stabilization of the spatial structures of these three peptidases was confirmed. We are showing here that the binding of the Zn2+ ion in the active center of EndoRB49 peptidase causes conformational rearrangements similar to those observed in the EndoT5 peptidase upon binding of Zn2+ and Ca2+ ions and lead to the formation of a catalytically active form of the enzyme. Therefore, the binding of the Zn2+ ion to the active site of EndoRB49 peptidase is a necessary and sufficient condition for functioning of this protein.

Acute ischemic stroke (AIS) causes both central and peripheral inflammation, while activation of α7 nicotinic acetylcholine receptors (nAChRs) provides both central and peripheral anti-inflammatory and anti-apoptotic effects. Here, we provide evidence that 4OH-GTS-21, a selective α7 agonist, produces its therapeutic effects via primarily central sites of action because 4OH-GTS-21 was found equally effective in splenectomized and non-spenectomized rats in the sub-acute phase of ischemic stroke (≤1 week). However, the spleen may boost the therapeutic efficacy of 4OH-GTS-21 in certain behavioral tasks as our data also indicated. In our tests, AIS was modeled by transient middle cerebral artery occlusion (tMCAO). Splenectomy was done 2 weeks before tMCAO. We determined that: 1) Daily 4OH-GTS-21 treatments for 7 days after tMCAO significantly reduced neurological deficits and brain injury in both splenectomized and non-spelenectomized rats demonstrating that the spleen is not required for therapeutic benefits of 4OH-GTS-21; 2) The effects of 4OH-GTS-21 in the adhesive sticker removal test were significantly weaker in splenectomized animals suggesting that the spleen boosts the efficacy of 4OH-GTS-21 in the first week after tMCAO; and 3) Ischemic brain injury was not significantly affected by splenectomy in both vehicle-treated and 4OH-GTS-21-treated animals. These data support the hypothesis that the therapeutic efficacy of sub-chronic (≤1 week) 4OH-GTS-21 primarily originates from central sites of action. These results validate brain availability as a critical factor for developing novel α7 ligands for AIS.

Calcium/calmodulin-dependent serine protein kinase (CASK) knockdown reduces insulin vesicle docking to cell membranes. Here, we explored CASK interactions with other proteins during insulin secretion. Using co-immunoprecipitation, liquid chromatography-mass spectrometry and bioinformatic analysis, we identified that CASK, Adapter protein X11 alpha (APBA1), and Syntaxin binding protein 1 (STXBP1) formed tripartite complex during insulin secretion. CASK enhanced APBA1-STXBP1 interaction and mediated their traffic from cytoplasm to plasma membrane during insulin release. High fatty acid stimulation decreased insulin secretion along with CASK, APBA1, and STXBP1 expression; Cask overexpression enhanced CASK/APBA1/STXBP1 tripartite complex function, and may thereby rescue lipotoxicity-induced insulin-release defects. Collectively, our results illustrated the function of CASK in insulin granules exocytosis, which broadens the underlying mechanism of insulin secretion and highlights the clinical potential of CASK as a drug target of type 2 Diabetes Mellitus (T2DM).

Advances in gene editing and cell therapies have recently led to outstanding clinical successes. However, the lack of a cost-effective manufacturing process prevents the democratization of these innovative medical tools. Due to the common use of viral vectors, the step of transfection in which cells are engineered to gain new functions, is a major bottleneck in making safe and affordable cell products. A promising opportunity lies in Single-Cell Transfection Technologies (SCTTs). SCTTs have demonstrated higher efficiency, safety and scalability than conventional transfection methods. They can also feature unique abilities such as substantial dosage control over the cargo delivery, single-cell addressability and integration in microdevices comprising multiple monitoring modalities. Unfortunately, the potential of SCTTs is not fully appreciated: they are most often restricted to research settings with little adoption in clinical settings. To encourage their adoption, we review and compare recent developments in SCTTs, and how they can enable selected clinical applications. To help bridge the gap between fundamental research and its translation to the clinic, we also describe how Good Manufacturing Practices (GMP) can be integrated in the design of SCTTs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

In the present study, mitogenomes of the species Trachypenaeus curvirostris and Parapenaeus fissuroides (Decapoda: Dendrobranchiata: Penaeidae) were sequenced. The total lengths of the two species were 15,956 bp and 15,937 bp in length with A + T biases of 67.08% and 67.69%, respectively. Both two species showed positive AT skews (0.016, 0.058) and negative GC skews (-0.254, -0.310). Both mitogenomes contained 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Results of phylogenetic analyses support close relationships among Aristeidae, Benthesicymidae and Solenoceridae. The family Sicyoniidae was observed to be deeply nested within Penaeidae. Within Penaeidae, T. curvirostris and P. fissuroides were most closely related to the genus Parapenaeopsis and Metapenaeopsis, respectively, indicated that these two species belong to Penaeidae. These results will help to better understand the evolutionary position of Penaeidae and provide reference for further phylogenetic research on Penaeoidea species.

Hepatic steatosis is a common chronic liver disease that can progress into more severe stages of NAFLD or promote the development of life-threatening secondary diseases for some of those affected. These include the liver itself (nonalcoholic steatohepatitis or NASH; fibrosis and cirrhosis, and hepatocellular carcinoma) or other organs such as the vessels and the heart (cardiovascular disease) or the islets of Langerhans (type 2 diabetes). In addition to elevated caloric intake and a sedentary lifestyle, genetic and epigenetic predisposition contribute to the development of NAFLD and the secondary diseases.

Among diarrheagenic E. coli (DEC), enterohaemorrhagic E. coli (EHEC) are the most virulent anthropozoonotic agents. The ability of bacterial cells to functionally interact with their surrounding essentially relies on the secretion of different protein effectors. To experimentally determine the repertoire of extracytoproteins in E. coli O157:H7, a subproteomic analysis was performed not only considering the extracellular milieu but the cell surface and outer membrane vesicles. Following a secretome-based approach, the proteins trafficking from the interior to the exterior of the cell were depicted considering cognate protein transport systems and subcellular localisation. Label-free quantitative analysis of the proteosurfaceome, proteovesiculome and exoproteome from E. coli O157:H7 grown in three different nutrient media revealed differential protein expression profiles and allowed defining the core and variant subproteomes. Network analysis further revealed the higher abundance of some protein clusters in chemically defined medium over rich complex medium, especially related to some outer membrane proteins, ABC transport and Type III secretion systems. This first comprehensive study of the EHEC secretome unravels the profound influence of environmental conditions on the extracytoplasmic proteome, provides new insight in the physiology of E. coli O157:H7 and identifies potentially important molecular targets for the development of preventive strategies against EHEC/STEC. SIGNIFICANCE: Escherichia coli O157:H7 is responsible for severe diarrhoea especially in young children. Despite years of investigations, the global view of the extracytoplasmic proteins expressed in this microorganism was eluded. To provide the first comprehensive view of the secretome landscape of E. coli O157:H7, the exoproteome, proteosurfaceome and proteovesiculome were profiled using growth conditions most likely to induce changes in bacterial protein secretion. The profound influence of growth conditions on the extracytoplasmic proteome was unravelled and allowed identifying the core and variant subproteomes. Besides new insight in the physiology of enterohaemorrhagic E. coli, these proteins potentially constitute important molecular targets for the development of preventive strategies.

The time-course of pulmonary arterial hypertension in the monocrotaline (MCT) model was investigated. Male rats were divided into two groups: MCT (received a 60 mg/kg i.p. injection) and control (received saline). The MCT and control groups were further divided into three cohorts, based on the follow-up interval: 1, 2, and 3 weeks. Right ventricle (RV) catheterization was performed and RV hypertrophy (RVH) was estimated. The lungs were used for biochemical, histological, molecular, and immunohistochemical analysis, while pulmonary artery rings were used for vascular reactivity. MCT promoted lung perivascular edema, inflammatory cells exudation, greater neutrophils and lymphocytes profile, and arteriolar wall thickness, compared to CTR group. Increases in pulmonary artery pressure and in RVH were observed in the MCT 2- and 3-week groups. The first week was marked by the presence of nitrosative stress (50% moderate and 33% accentuated staining by nitrotyrosine). These alterations lead to an adaptation of NO production by NO synthase activity after 2 weeks. Oxidative stress was evident in the third week, probably by an imbalance between endothelin-1 receptors, resulting in extracellular matrix remodeling, endothelial dysfunction, and RVH. Also, it was found a reduced pulmonary arterial vasodilatory response to acetylcholine after 2 (55%) and 3 (45%) weeks in MCT groups. The relevance of this study is precisely to show that nitrosative and oxidative stress predominate in distinct time windows of the disease progression.

Rotigotine-loaded microspheres (RoMS) are sustained-release formulations with prolonged anti-Parkinson's effects. Given that pain is a non-motor symptom of Parkinson's disease, this study investigated the antinociceptive effects of RoMS and their synergistic effects with analgesics on inflammatory pain. A model of inflammatory pain was prepared by intraplantarly injecting male Sprague-Dawley rats with carrageenan. The antinociceptive effects of RoMS, acetaminophen, and tramadol, both alone and in combination, were evaluated using the hind paw withdrawal latency in the hot plate test and Randall-Selitto test. The rotigotine concentrations in serum and tissues were assayed using ultra-performance liquid chromatography-tandem mass spectrometry. Isobolographic analysis was performed to evaluate the nature of the interactions of RoMS with acetaminophen or tramadol. The results showed that hind paw withdrawal latency to thermal and mechanical stimuli was significantly increased on day 3 and 7 after administered RoMS. Rotigotine could be detected in serum and tissues 3 and 7 days after an intramuscular injection of RoMS. However, the rotigotine concentration fell the detection limit of the assay on day 14 after administration. RoMS produced synergistic antinociceptive effects in the inflammatory pain model when RoMS is combined with acetaminophen or tramadol. These findings suggest that RoMS can relieve inflammatory pain in rats. Furthermore, the combination of RoMS with acetaminophen or tramadol produces synergistic antinociception, which may be clinically worthy because combination therapies may reduce the drug doses required for antinociception.

COVID-19 has intensified into a global pandemic with over a million deaths worldwide. Experimental research analyses have been implemented and executed with the sole rationale to counteract SARS-CoV-2, which has initiated potent therapeutic strategy development in coherence with computational biology validation focusing on the characterized viral drug targets signified by proteomic and genomic data. Spike glycoprotein is one of such potential drug target that promotes viral attachment to the host cellular membrane by binding to its receptor ACE-2 via its Receptor-Binding Domain (RBD). Multiple Sequence alignment and relative phylogenetic analysis revealed significant sequential disparities of SARS-CoV-2 as compared to previously encountered SARS-CoV and MERS-CoV strains. We implemented a drug re-purposing approach wherein the inhibitory efficacy of a cluster of thirty known drug candidates comprising of antivirals, antibiotics and phytochemicals (selection contingent on their present developmental status in underway clinical trials) was elucidated by subjecting them to molecular docking analyses against the spike protein RBD model (developed using homology modelling and validated using SAVES server 5.0) and the composite trimeric structures of spike glycoprotein of SARS-CoV-2. Our results indicated that Camostat, Favipiravir, Tenofovir, Raltegravir and Stavudine showed significant interactions with spike RBD of SARS-CoV-2. Proficient bioavailability coupled with no predicted in silico toxicity rendered them as prospective alternatives for designing and development of novel combinatorial therapy formulations for improving existing treatment regimes to combat COVID-19.

A pneumonia-like disease of unknown origin caused a catastrophe in Wuhan city, China. This disease spread to 215 countries affecting a wide range of people. World health organization (WHO) called it a pandemic and it was officially named as Severe Acute Respiratory Syndrome Corona virus 2 (SARS CoV-2), also known as Corona virus disease (COVID-19). This pandemic compelled countries to enforce a socio-economic lockdown to prevent its widespread. This paper focuses on how the particulate matter pollution was reduced during the lockdown period (23 March to April 15, 2020) as compared to before lockdown. Both ground-based and satellite observations were used to identify the improvement in air quality of Pakistan with primary focus on four major cities of Lahore, Islamabad, Karachi and Peshawar. Both datasets have shown a substantial reduction in PM2.5 pollution levels (ranging from 13% to 33% in case of satellite observations, while 23%-58% in ground-based observations) across Pakistan. Result shows a higher rate of COVID-19 spread in major cities of Pakistan with poor air quality conditions. Yet more research is needed in order to establish linkage between COVID-19 spread and air pollution. However, it can be partially attributed to both higher rate of population density and frequent exposure of population to enhanced levels of PM2.5 concentrations before lockdown period.