EGFR Regulation of Epidermal Barrier Function

Keratinocyte terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammals to survive in the ex utero environment. This process is tightly controlled by the expression of many well-characterized genes. Although a few of these genes are known to be regulated by the epidermal growth factor receptor (EGFR), an important regulator of multiple epidermal functions, neither the genome-wide scale of EGFR-mediated regulation nor the mechanisms by which EGFR signaling controls keratinocyte differentiation are well understood. Using microarray analysis we identified 2,676 genes that are regulated by EGF, a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all essential metabolic processes of keratinocyte differentiation by (1) decreasing the expression of lipid matrix biosynthetic enzymes, (2) regulating numerous genes forming the cornified envelope, and (3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, the collective effect of EGF impaired epidermal barrier integrity, evidenced by increased transepidermal water loss. As defective epidermal differentiation and disruption of the epidermal barrier are primary features of many human skin diseases, we used bioinformatics analysis to identify genes that are known to be associated with human skin diseases. In comparison to non-EGF-regulated genes, the EGF-regulated gene list was significantly enriched for disease genes. Further validation of the expression profiles of many of the 114 identified skin disease genes included the transcription factors GATA binding protein 3 (GATA3) and Kruppel-like factor 4 (KLF4), both required for establishing the barrier function of the skin in developing mice. These results provide a new systems level understanding of the actions of EGFR signaling to inhibit keratinocyte differentiation. As the overall effect of this inhibition is to impair epidermal barrier integrity, this study clarifies how dysregulation of the EGFR and its ligands may contribute to diseases of the skin

Perceptions and Experiences of Perinatal Mental Disorders in Rural, Predominantly Ethnic Minority Communities in Northern Vietnam

Background: Preliminary research has suggested that perinatal mental disorders (PMDs), including post-partum depression, are prevalent in Vietnam. However the extent to which these disorders are recognized at the community level remains largely undocumented in the literature. PMDs have also never been investigated within Vietnam’s significant ethnic minority populations, who are known to bear a greater burden of maternal and infant health challenges than the ethnic majority. Objective: To investigate knowledge and perceptions of PMDs and their treatments at the community level in a rural, predominantly ethnic minority region of northern Vietnam

Deformation forecasting of a hydropower dam by hybridizing a long short-term memory deep learning network with the coronavirus optimization algorithm

The safety operation and management of hydropower dam play a critical role in social-economic development and ensure people’s safety in many countries; therefore, modeling and forecasting the hydropower dam’s deformations with high accuracy is crucial. This research aims to propose and validate a new model based on deep learning long short-term memory (LSTM) and the coronavirus optimization algorithm (CVOA), named CVOA-LSTM, for forecasting the defor mations of the hydropower dam. The second-largest hydropower dam of Viet nam, located in the Hoa Binh province, is focused. Herein, we used the LSTM to establish the deformation model, whereas the CVOA was utilized to opti mize the three parameters of the LSTM, the number of hidden layers, the learn ing rate, and the dropout. The efficacy of the proposed CVOA-LSTM model is assessed by comparing its forecasting performance with state-of-the-art bench marks, sequential minimal optimization for support vector regression, Gaussian process, M5’ model tree, multilayer perceptron neural network, reduced error pruning tree, random tree, random forest, and radial basis function neural net work. The result shows that the proposed CVOA-LSTM model has high fore casting capability (R2 = 0.874, root mean square error = 0.34, mean absolute error = 0.23) and outperforms the benchmarks. We conclude that CVOA-LSTM is a new tool that can be considered to forecast the hydropower dam’s deforma tions.Ministerio de Ciencia, Innovación y Universidades PID2020-117954RB-C2

Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii

Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii. IMPORTANCE Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various selective conditions. We applied fluorescence-activated cell sorting (FACS) to physically enrich for phenotypes of interest within a mutant population prior to TraDIS. To our knowledge, this is the first time that a physical selection method has been applied in parallel with TraDIS rather than a fitness-induced selection. The results demonstrate the feasibility of this combined approach to generate significant results and highlight the major multidrug efflux pumps encoded in an important pathogen. This FACS-based approach, TraDISort, could have a range of future applications, including the characterization of efflux pump inhibitors, the identification of regulatory factors controlling gene or protein expression using fluorescent reporters, and the identification of genes involved in cell replication, morphology, and aggregation

Altering, Improving, And Defining The Specificities Of Crispr-Cas Nucleases

CRISPR-Cas9 nucleases have been widely adopted for genome editing applications to knockout genes or to introduce desired changes. While these nucleases have shown immense promise, two notable limitations of the wild-type form of the broadly used Streptococcus pyogenes Cas9 (SpCas9) are the restriction of targeting range to sites that contain an NGG protospacer adjacent motif (PAM), and the undesirable ability of the enzyme to cleave off-target sites that resemble the on-target site. Scarcity of PAM motifs can limit implementations that require precise targeting, whereas off-target effects can confound research applications and are important considerations for therapeutics. To improve the targeting range of SpCas9 and an orthogonal Cas9 from Staphylococcus aureus (called SaCas9), we optimized a heterologous genetic selection system that enabled us to perform directed evolution of PAM specificity. With SpCas9, we evolved two separate variants that can target NGA and NGCG PAMs1, and with SaCas9 relaxed the PAM from NNGRRT to NNNRRT2, increasing the targetability of these enzyme 2- to 4-fold. The genome-wide specificity profiles of SpCas9 and SaCas9 variants, determine by GUIDE-seq3, indicate that they are at least as, if not more, specific than the wild-type enzyme1,2. Together, these results demonstrate that the inherent PAM specificity of multiple different Cas9 orthologues can be purposefully modified to improve the accuracy of targeting. Existing strategies for improving the genome-wide specificity of SpCas9 have thus far proven to be incompletely effective and/or have other limitations that constrain their use. To address the off-target potential of SpCas9, we engineered a high-fidelity variant of SpCas9 (called SpCas9-HF1), that contains alterations designed to reduce non-specific contacts to the target strand DNA backbone. In comparison to wild-type SpCas9, SpCas9-HF1 rendered all or nearly all off-target events imperceptible by GUIDE-seq and targeted deep-sequencing methods with standard non-repetitive target sites in human cells4. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 and optimized derivatives were not detected4. With its exceptional precision, SpCas9-HF1 provides an important and easily employed alternative to wild-type SpCas9 that can eliminate off-target effects when using CRISPR-Cas9 for research and therapeutic applications. Finally, on-target activity and genome-wide specificity are two important properties of engineered nucleases that should be characterized prior to adoption of such technologies for research or therapeutic applications. CRISPR-Cas Cpf1 nucleases have recently been described as an alternative genome-editing platform5, yet their activities and genome-wide specificities remain largely undefined. Based on assessment of on-target activity across more than 40 target sites, we demonstrate that two Cpf1 orthologues function robustly in human cells with efficiencies comparable to those of the widely used Streptococcus pyogenes Cas9. We also demonstrate that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 are insensitive to single base mismatches, but that many of the other bases within the crRNA targeting region are highly sensitive to single or double substitutions6. Consistent with these results, GUIDE-seq performed in multiple cell types and targeted deep sequencing analyses of two Cpf1 nucleases revealed no detectable off-target cleavage for over half of 20 different crRNAs we examined. Our results suggest that the two Cpf1 nucleases we characterized generally possess robust on-target activity and high specificities in human cells, findings that should encourage broader use of these genome editing enzymes. 1. Kleinstiver, BP, et al. (2015) Nature, 523(7561):481-5 2. Kleinstiver, BP, et al. (2015) Nature Biotechnology, 33(12):1293-98 3. Tsai, SQ et al. (2015) Nature Biotechnology, 33(2):187-97 4. Kleinstiver, BP and Pattanayak, V, et al. (2016), Nature, 529(7587):490-5 5. Zetsche, B, et al. (2015) Cell, 163(3):759-71 6. Kleinstiver, BP and Tsai, SQ, et al. (2016), Nature Biotechnology, 34(8):869-7