Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium

An extreme diversity of substrates and catalytic reactions of cytochrome P450 (P450) enzymes is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands. Here we report the presence of numerous natural variants of P450 BM3 (CYP102A1) within a species of Bacillus megaterium. Extensive amino acid substitutions (up to 5% of the total 1049 amino acid residues) were identified from the variants. Phylogenetic analyses suggest that this P450 gene evolve more rapidly than the rRNA gene locus. It was found that key catalytic residues in the substrate channel and active site are retained. Although there were no apparent variations in hydroxylation activity towards myristic acid (C14) and palmitic acid (C16), the hydroxylation rates of lauric acid (C12) by the variants varied in the range of >25-fold. Interestingly, catalytic activities of the variants are promiscuous towards non-natural substrates including human P450 substrates. It can be suggested that CYP102A1 variants can acquire new catalytic activities through site-specific mutations distal to the active site

A Highly Efficient CRISPR-Cas9-Mediated Large Genomic Deletion in Bacillus subtilis

In Bacillus subtilis, large genomic deletions have been carried out for genome reduction, antibiotic overproduction, and heterologous protein overexpression. In view of the eco-friendliness of B. subtilis, it is critical that engineering preserves its food-grade status and avoids leaving foreign DNA in the genome. Existing methods of generating large genomic deletions leave antibiotic resistance markers or display low mutation efficiency. In this study, we introduced a clustered regularly interspaced short palindromic repeat-derived genome engineering technique to develop a highly efficient method of generating large genomic deletions in B. subtilis without any trace of foreign DNA. Using our system, we produced 38 kb plipastatin-synthesizing pps operon deletion with 80% efficiency. The significant increase in mutation efficiency was due to plasmids-delivered Streptococcus pyogenes-originated SpCas9, target-specific sgRNA and a donor DNA template, which produces SpCas9/sgRNA endonuclease complex continuously for attacking target chromosome until the mutagenic repair occurs. Our system produced single-gene deletion in spo0A (∼100%), point mutation (∼68%) and GFP gene insertion (∼97%) in sigE and demonstrated its broad applicability for various types of site-directed mutagenesis in B. subtilis

The United States COVID-19 Forecast Hub dataset

Academic researchers, government agencies, industry groups, and individuals have produced forecasts at an unprecedented scale during the COVID-19 pandemic. To leverage these forecasts, the United States Centers for Disease Control and Prevention (CDC) partnered with an academic research lab at the University of Massachusetts Amherst to create the US COVID-19 Forecast Hub. Launched in April 2020, the Forecast Hub is a dataset with point and probabilistic forecasts of incident cases, incident hospitalizations, incident deaths, and cumulative deaths due to COVID-19 at county, state, and national, levels in the United States. Included forecasts represent a variety of modeling approaches, data sources, and assumptions regarding the spread of COVID-19. The goal of this dataset is to establish a standardized and comparable set of short-term forecasts from modeling teams. These data can be used to develop ensemble models, communicate forecasts to the public, create visualizations, compare models, and inform policies regarding COVID-19 mitigation. These open-source data are available via download from GitHub, through an online API, and through R packages

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Stabilization of Homoserine-O-Succinyltransferase (MetA) Decreases the Frequency of Persisters in <i>Escherichia coli</i> under Stressful Conditions

<div><p>Bacterial persisters are a small subpopulation of cells that exhibit multi-drug tolerance without genetic changes. Generally, persistence is associated with a dormant state in which the microbial cells are metabolically inactive. The bacterial response to unfavorable environmental conditions (heat, oxidative, acidic stress) induces the accumulation of aggregated proteins and enhances formation of persister cells in <i>Escherichia coli</i> cultures. We have found that methionine supplementation reduced the frequency of persisters at mild (37°C) and elevated (42°C) temperatures, as well as in the presence of acetate. Homoserine-<i>o</i>-succinyltransferase (MetA), the first enzyme in the methionine biosynthetic pathway, is prone to aggregation under many stress conditions, resulting in a methionine limitation in <i>E. coli</i> growth. Overexpression of MetA induced the greatest number of persisters at 42°C, which is correlated to an increased level of aggregated MetA. Substitution of the native <i>metA</i> gene on the <i>E. coli</i> K-12 WE chromosome by a mutant gene encoding the stabilized MetA led to reduction in persisters at the elevated temperature and in the presence of acetate, as well as lower aggregation of the mutated MetA. Decreased persister formation at 42°C was confirmed also in <i>E. coli</i> K-12 W3110 and a fast-growing WErph+ mutant harboring the stabilized MetA. Thus, this is the first study to demonstrate manipulation of persister frequency under stressful conditions by stabilization of a single aggregation-prone protein, MetA.</p></div

Study on Noise Reduction and Data Generation for sEMG Spectrogram Based User Recognition

With the spread of the modern media industry, harmful genre contents are indiscriminately disseminated to teenagers. The password identification method used to block sensational and violent genre content has become a problem that teenagers can easily steal. Therefore, a user identification method with less risk of theft and hacking is required. The surface EMG (sEMG) signal, which is an electrical signal generated inside the body and has individual features, is being studied as a next-generation user identification method. sEMG involves measuring an individual&rsquo;s unique muscular strength activated over time as digital signals, thus giving it the advantage of generating different signal patterns. However, it is difficult to constantly and repeatedly acquire each motion signal and the number of repetitions for each motion is insufficient, thus there is a limit to improving user identification accuracy. In this paper, we propose a user identification system that solves the problem of insufficient data by applying the matching pursuit that enables signal generation to the sEMG signal from which the resting signal has been removed and improves classification accuracy by extracting STFT-based time&ndash;frequency features. As a result of the experiment, the user identification accuracy of the sEMG spectrogram with the resting state signal removed was 85.4%. In addition, when the training data were increased through data generation, the accuracy was improved, showing a user identification accuracy of 96.1%. Improved user recognition accuracy was confirmed when the training data of the sEMG signal from which the resting signal was removed were increased and multidimensional features including time&ndash;frequency were used