The ability to read and quantify nucleic acids such as DNA and RNA using sequencing technologies has revolutionized our understanding of life. With the emergence of synthetic biology, these tools are now being put to work in new ways - enabling de novo biological design. Here, we show how sequencing is supporting the creation of a new wave of biological parts and systems, as well as providing the vast data sets needed for the machine learning of design rules for predictive bioengineering. However, we believe this is only the tip of the iceberg and end by providing an outlook on recent advances that will likely broaden the role of sequencing in synthetic biology and its deployment in real-world environments.

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G>T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.

1) Background: One of the most common cancer that affects men worldwide and North American men is prostate cancer. Gleason score is a pathological grading system to examine the potential aggressiveness of the disease in the prostate tissue. The advancement in computing and next-generation sequencing technology now allow us to study the genomic profiles of patients in association with their different Gleason score more accurately and effectively. 2) Methods: In this study, we used a novel machine learning method to analyze gene expression of prostate tumors with different Gleason scores, and identify potential genetic biomarkers for each Gleason group. We obtained a publicly-available RNA-Seq dataset of a cohort of 104 prostate cancer patients from the National Center for Biotechnology Information’s (NCBI) Gene Expression Omnibus (GEO) repository, and categorized patients based on their Gleason scores to create a hierarchy of disease progression. A hierarchical model with standard classifiers in different Gleason groups, also known as nodes, was developed to identify and predict nodes based on their mRNA or gene expression. In each node, patient samples were analyzed via class imbalance and hybrid feature selection techniques to build the prediction model. The outcome from analysis of each node is a set of genes that can differentiate each Gleason group from the remaining groups. To validate the proposed method, the set of identified genes are used to classify a second dataset of 499 prostate cancer patients collected from cBioportal [1]. 3) Results: The overall accuracy of applying this novel method to the first dataset was 93.3%, and further validated to 87% accuracy using the second dataset. This method also identified genes that were not previously reported as potential biomarkers for specific Gleason groups. In particular, PIAS3 was identified as a potential biomarker for Gleason score 4+3=7, and UBE2V2 for Gleason score 6. 4) Insight: Previous reports show that the genes predicted by this newly proposed method strongly correlate with prostate cancer development and progression. Furthermore, pathway analysis shows that both PIAS3 and UBE2V2 share similar protein interaction pathways, the JAK/STAT signaling process.

Mucosal melanomas (MM) are rare aggressive cancers in humans and one of the most common forms of oral cancers in dogs. Similar biological and histological features are shared between MM in both species making dogs a powerful model for comparative oncology studies of melanomas. Although exome sequencing recently identified recurrent coding mutations in canine MM, little is known about changes in non-coding gene expression and more particularly in canine long non-coding RNAs (lncRNAs), which are commonly dysregulated in human cancers. Here, we sampled a large cohort (n= 52) of canine normal/tumor oral MM from three predisposed breeds (poodles, Labrador retrievers and golden retrievers) and used deep transcriptome sequencing to identify more than 400 differentially expressed (DE) lncRNAs. We further prioritized candidate lncRNAs by comparative genomic analysis to pinpoint 26 dog-human conserved DE lncRNAs, including SOX21-AS, ZEB2-AS and CASC15 lncRNAs. Using unsupervised co-expression networks analysis with coding genes, we inferred potential functions of DE lncRNAs suggesting associations with cancer-related genes, cell cycle and carbohydrate metabolism GO terms. Finally, we exploited our multi-breed design to identify DE lncRNAs per breed. This study provides a unique transcriptomic resource for studying oral melanoma in dogs and highlights lncRNAs that may potentially be diagnostic or therapeutic targets for human and veterinary medicine.

In the recent past, there has been a rise in Prostate Cancer (PCa) in Asia, particularly India. Although systematic reviews on PCa have dealt on the genetics, genomics and the environmental influence in causal of PCa, no predictive analytics in comparing the PCa from Caucasian, American to Asian population was attempted. In this review article, we have attempted to elaborate this aspect of PCa and deliberated on challenges related to next generation sequencing methods of PCa’s manifestation when compared to the west.

B chromosomes (Bs) were discovered a century ago, and since then most studies have focused on describing their distribution and abundance using traditional cytogenetics. Only recently have attempts been made to understand their structure and evolution at the level of DNA sequence. Many questions regarding the origin, structure, function and evolution of B chromosomes remain unanswered. Here we identify B chromosome sequences from several species of cichlid fish from Lake Malawi by examining the ratios of DNA sequence coverage in individuals with and without B chromosomes. We examine the efficiency of this method, and compare results using both Illumina and PacBio sequence data. The B chromosome sequences detected in 13 individuals from 7 species were compared to assess the rates of sequence replacement. B-specific sequence common to at least 12 of the 13 datasets are identified as the “Core” B chromosome. The location of B sequence homologs throughout the genome provides further support for theories of B chromosome evolution. Finally, we identified candidate genes located on the B chromosome which may regulate the segregation and maintenance of the B chromosome.

Beta-lactam resistant bacteria, commonly resident in tertiary hospitals, have emerged as a worldwide health problem because of ready-to-eat vegetable intake. We aimed to characterize the genes providing resistance to beta-lactam antibiotics in Enterobacteriaceae, isolated from five commercial salad brands for human consumption in Mexico City. 25 samples were collected, grow in blood agar plates, the bacteria were biochemistry identified and antimicrobial susceptibility testing was done, the carried family genes were identified by endpoint PCR and the specific genes were confirmed with WGS by NGS. 12 positive cultures were identified and their microbiological distribution was as follows, 8.3% for Enterobacter aerogene (n=1), 8.3% for Serratia fonricola (n=1), 16.7% for Serratia marcesens (n=2), 16.7% for Klebsiella pneumoniae (n=2), and 50% (n=6) for Enterobacter cloacae. The endpoint PCR results showed 11 colonies positive for blaBIL (91.7%), 11 for blaSHV (91.7%), 11 for blaCTX (97.7%), 12 for blaDHA (100%),4 for blaVIM (33.3%), 2 for blaOXA (16.7%), 2 for blaIMP (16.7%), 1 for blaKPC (8.3%) and 1 for blaTEM (8.3%) gene, all samples were negative blaROB, blaCMY, blaP, blaCFX and blaLAP gene. The sequencing analysis revels a specific genotypes for Enterobacter cloacae (blaSHV-12, blaCTX-M-15, blaDHA-1, blaKPC-2); Serratia marcescens (blaSHV-1, blaCTX-M-3, blaDHA-1, blaVIM-2); Klebsiella pneumoniae (blaSHV-12, blaCTX-M-15, blaDHA-1); Serratia fonticola (blaSHV-12, blaVIM-1, blaDHA-1) and Enterobacter aerogene (blaSHV-1, blaCTX-M-1, blaDHA-1, blaVIM-2, blaOXA-9). Our results indicate that beta-lactam resistant bacteria have acquired integrons with a different number of genes that providing panresistance to beta-lactam antibiotics, including penicillins, oxacillins, cefalosporins, monobactams, carbapenems and imipenems.

Retinoic acid (RA) is the biologically active metabolite of vitamin A and has become a well-established factor that induces neurite outgrowth and regeneration in both vertebrates and invertebrates. However, the underlying regulatory mechanisms that may mediate RA-induced neurite sprouting remain unclear. In the past decade, microRNAs have emerged as important regulators of nervous system development and regeneration, and have been shown to contribute to processes such as neurite sprouting. However, few studies have demonstrated the role of miRNAs in RA-induced neurite sprouting. By miRNA-Sequencing analysis, we identify 482 miRNAs in the regenerating CNS of the mollusc Lymnaea stagnalis, 219 of which represent potentially novel miRNAs. Of the remaining conserved miRNAs, 38 show a statistically significant up or downregulation in regenerating CNS as a result of RA treatment. We further characterized the expression of one neuronally-enriched miRNA upregulated by RA, miR-124. We demonstrate for the first time that miR-124 is expressed within the cell bodies and neurites of regenerating motorneurons. Moreover, we identify miR-124 expression within the growth cones of cultured ciliary motorneurons (Pedal A), whereas expression in the growth cones of another class of respiratory motorneurons (RPA) was absent in vitro. These findings support our hypothesis that miRNAs are important regulators of retinoic acid-induced neuronal outgrowth and regeneration in regeneration-competent species.

Retinoic acid (RA) is the biologically active metabolite of vitamin A,and has become a well-established factor that induces neurite outgrowth and regeneration in both vertebrates and invertebrates. However, the underlying regulatory mechanisms that may mediate RA-induced neurite sprouting remain unclear. In the past decade, microRNAs have emerged as important regulators of nervous system development and regeneration, and have been shown to contribute to processes such as neurite sprouting. However, few studies have demonstrated the role of miRNAs in RA-induced neurite sprouting. By R-Seq analysis, we identify 482 miRNAs in the regenerating CNS of the mollusc Lymnaea stagnalis, 219 of which represent potentially novel miRNAs. Of the remaining conserved miRNAs, 38 show a statistically significant up or downregulation in regenerating CNS as a result of RA treatment. We further characterized the expression of one neuronally-enriched miRNA upregulated by RA, miR-124. We demonstrate for the first time that miR-124 is expressed within the cell bodies and neurites of regenerating motorneurons. Moreover, we identify miR-124 expression within the growth cones of cultured ciliary motorneurons (Pedal A), whereas expression from the growth cones of another class of respiratory motorneurons (RPA) was absent in vitro. These findings support our hypothesis miRNAs are important regulators of retinoic acid induced neuronal outgrowth and regeneration in regeneration-competent species.

Rhizosphere microbiomes which have been implicated to enhance plant growth and yield are modulated or influenced by a couple of environmental factors such as soil type, plant cultivar, climate change and anthropogenic activities. In particular, anthropogenic activity such as the use of nitrogen-based chemical fertilizers is associated with environmental destruction and this call for a more ecofriendly strategy to increase nitrogen level of agricultural land. This feat is attainable by harnessing nitrogen-fixing endophytic and free-living rhizobacteria. Rhizobium, Pseudomonas, Azospirillum and Bacillus have been found to have positive impacts on crops by enhancing both above and belowground biomass and could therefore play positive roles in achieving sustainable agriculture. Thus, it is needful to study these rhizosphere microbiomes with more sophisticated culture-independent technologies such as next generation sequencing (NGS) with the prospect of discovering novel bacteria with plant growth promoting traits. This review is therefore aimed at discussing factors that can modulate rhizosphere microbiomes with focus on the contributions of nitrogen fixing bacteria towards sustainable agricultural development and the techniques that can be used for their study.

With the rise of long-read sequencers and long-range technologies, delivering high-quality plant genome assemblies is no longer reserved to large consortia. Indeed, sequencing techniques but also computer algorithms have reached a point where the reconstruction of assemblies at the chromosome-scale is now feasible at the laboratory scale. Current technologies, and especially long-range technologies, are numerous and selecting the most promising one for the genome of interest is crucial to obtain optimal results. In this study, we resequenced the genome of the yellow sarson, Brassica rapa cv. Z1, using the Oxford Nanopore PromethION sequencer and assembled the sequenced data using current assemblers. To reconstruct complete chromosomes, we used and compared three long-range techniques, optical mapping, Omni-C and Pore-C sequencing libraries commercialized by Bionano Genomics, Dovetail Genomics and Oxford Nanopore Technologies respectively, or a combination of the three, in order to evaluate the capability of each technology.

A three-arm, randomized, placebo controlled clinical study has been performed to evaluate the effects of a lyophilized pineapple extract titrated in bromelain (Brome-Inf®), and purified bro-melain on pain, swelling, trismus and quality of life (QoL) after the surgical removal of the lower third molars. Moreover, the need for non-steroidal anti-inflammatory drugs (NSAIDs) has also been evaluated and compared with a placebo group. The study included 42 people that required the extraction of a single mandibular third molar under local anesthesia. The patients were ran-domized and distributed to receive Brome-Inf®, purified bromelain or placebo and started the treatment the day of surgery and continued it for the next 7 days. The primary outcome was the requirement of NSAIDs in the three groups. The outcome variables were pain, swelling and trismus, which were measured postoperatively at 1, 3, and 7 days. The differences in the efficacy of the freeze-dried pineapple extract and single-component bromelain were also evaluated. At the end of the study, the assumption of ibuprofen (from days 1-7) was statistically higher in the placebo group (p

Transdermal drug delivery systems (TDDS) are designed to administer a consistent and effective dose of medication through the patient's skin. These pharmaceutical preparations are self-contained, discrete dosage forms designed to be placed topically on intact skin to release the active component at a controlled rate by penetrating the skin barriers. This medication provides a continuous and prolonged administration of a substance at a consistent rate. TDDS, or transdermal drug delivery system, has gained significant attention as a non-invasive method of administering drugs to vulnerable patient populations, such as pediatric and geriatric patients. This approach is considered easy to administer and helps overcome bioavailability issues associated with conventional drug delivery, which can be hindered by poor absorption and metabolism. TDDS has various advantages compared to conventional methods of drug administration. It is less intrusive, more patient-friendly, and can circumvent first-pass metabolism and the corrosive acidic environment of the stomach that happens when medications are taken orally. Multiple methodologies have been devised to augment the transdermal permeability of diverse pharmaceutical substances. Recent advancements in transdermal drug delivery systems have facilitated the precise delivery of medications to the intended site of action by improving the penetration of drugs through the outermost layer of the skin, known as the stratum corneum, resulting in enhanced drug availability throughout the body. This review seeks to provide a concise overview of the several methods employed in the production of TDDS, as well as their evaluation, therapeutic uses, clinical considerations, and current advancements. These advancements have resulted in the development of intelligent, biodegradable, and highly efficient transdermal drug delivery systems.

Tomato pomace (TP), an agricultural industrial waste product from the tomato processing industry, was valorized by being used as a rearing substrates for Tenebrio molitor (L.). This study evaluated bran-based diets with increasing tomato pomace (0%, 27%, 41% and 100%). Protein sources, as brewer’s spent grain and yeast, are used in TP27 and TP41 diets, respectively, to ensure equal protein content to control diet (TP0). Results showed larval survival, larval and pupal weight no different between diets; however, growth time significantly increases in TP100 compared at all diets. The feed conversion rate progressively increases from 2.7 to 4.3, respectively from TP0 to TP100. Conversely, Lycopene and β-Carotene increases in the larvae. Fatty acid composition improves by increasing polyunsaturated fatty acids (mainly -Linoleic acid). Although the best nutritional quality was obtained in T100, the TP41 is the optimal diet for balance between larval performance and qualitative improvement of larvae. Therefore, tomato pomace is suitable for the formulation of mealworm diets, even in high dosages when supplemented with sustainable protein sources.

A balanced diet model, rich in fruits and vegetables and ensuring the intake of natural products, has been shown to reduce or prevent the occurrence of many chronic diseases. However, the choice to consume large quantities of fruits and vegetables leads to an increase in the amount of waste, which can cause the alteration in environmental sustainability. To date, the concept of "by-product" has evolved, understood as a waste product, from which it is still possible obtain useful compounds. Therefore, the by-products in the agricultural sector, are a rich source of bioactive compounds, capable to possess a second life, decreasing the amount of waste products, the disposal costs and the environmental pollution. A promising and well-known citrus of the Mediterranean diet is the bergamot (Citrus bergamia, Risso et Poiteau). The composition of bergamot is known and the rich presence of phenolic compounds and essential oils has justified countless beneficial properties found, including anti-inflammatory, antioxidant, anti-cholesterolemic and protective activity for the immune system, heart failure and coronary heart diseases. The industrial processing of bergamot fruits leads to the formation of bergamot juice and bergamot oil. The solid residues, referred as "pastazzo", are normally used as feed for livestock or pectin production. The fiber of bergamot (BF) can be obtained from pastazzo and could exert an interesting effect thanks to its content of polyphenols. The purpose of this work was to test the effects of BF on an in vitro model of neurotoxicity induced by treatment with amyloid beta protein. In particular, this experimental model included both neurons and oligodendrocytes in order to measure the involvement of the glia and compare it with the neurons one. The results obtained showed a protective activity of BF, although the oligodendrocytes were more sensitive and fragile than neurons. Further experiments are necessary and if the trend was confirmed, solid residues of bergamot could be used in AD, and, at the same time, could help to avoid the accumulation of waste products.

The deep neural network - BERT model (Bidirectional Encoder Representations from Transformers) and the stocks cumulative abnormal return is used in this article to analyze the sentiment of financial texts. The proposed approach, unlike those used so far, does not require the creation of dictionaries, takes into account the broad context of words and their meaning in financial texts, eliminates the problem of ambiguity of words in various contexts, does not require manual labelling of data and is free from the subjective assessment of the researcher. The sentiment of financial texts in the meaning presented in this paper is directly related to the market reaction to the information contained in these texts. For texts belonging to one of the two classes (positive or negative) with the highest probability the BERT model gives the results of predictions with a precision level of 62.38% for the positive class and 55% for the negative class. The results at this level can be used in event study, market efficiency research, investment strategy development or support of investment analysts using fundamental analysis.

Highly-correlated electrons – electrons that engage in strong electron-electron interactions – have been observed in transition metal oxides and quantum dots and can create unusual material behavior that is difficult to model, such as switching between a low resistance metal state and a high resistance Mott insulator state. Tests of devices using a layer-by-layer deposition process for forming multilayer arrays of ferritin (a transition metal (iron) oxide storage protein) have been previously reported that indicate that highly-correlated electron transport is occurring, consistent with models of electron transport in quantum dots. This paper reports the results of the effect of various degrees of structural homogeneity on the electrical characteristics of these ferritin arrays, as well as demonstrating that these structures can provide a switching function associated with the circuit that they are contained within, consistent with the observed behavior of highly-correlated electrons.

The present paper introduces a new class of Stancu-Kantorovich operators constructed in the King sense. For this class of operators we establish some convergence results, error estimations theorems and graphical properties of approximation for the cases considered, namely, operators that preserve the test functions $e_{0}(x)=1$ and $e_{1}(x)=x$, $% e_{0}(x)=1$ and $e_{2}(x)=x^{2},$ as well as $e_{1}(x)=x$ and $e_{2}(x)=x^{2} $. The new class of operators introduced in this paper is a genuine generalization of the class introduced by Indrea et al. (see Indrea A., Indrea A., Pop O. A New Class of Kantorovich-Type Operators, Constructive Mathematical Analysis).

In this paper, we work with a diffusion-perturbed risk model comprising a surplus generating process and an investment return process. The investment return process is of standard Black-Scholes type, that is, it comprises a single risk-free asset that earns interest at a constant rate and a single risky asset whose price process is modelled by a geometric Brownian motion. Additionally, the company is allowed to purchase noncheap proportional reinsurance priced via the expected value principle. Using the Hamilton-Jacobi-Bellman approach, we derive a second-order Volterra integrodifferential equation which we transform into a linear Volterra integral equation of the second kind. We proceed to solve this integral equation numerically using the block-by-block method for the optimal reinsurance retention level that minimizes the ultimate ruin probability. The numerical results based on light- and heavy-tailed distributions show that proportional reinsurance and investments play a vital role in enhancing the survival of insurance companies. But the ruin probability exhibits sensitivity to the volatility of the stock price.

The Mexican Central Pacific (MCP) has discontinuous coral ecosystems with different protection and anthropogenic disturbance. Characterizing the bacterial assemblage associated with the sea urchin Toxopneustes roseus and its relationship with environmental variables will contribute to understanding the species’ physiology and ecology. We collected sea urchins from coral ecosystems at six sites in the MCP during the summer and winter for two consecutive years. The spatial scale represented the most important variation in the T. roseus bacteriome, particularly because of Isla Isabel National Park (PNII). Likewise, spatial differences correlated with habitat structure variables, mainly the sponge and live coral cover. The PNII exhibited a highly diverse bacterial assemblages compared to other sites, characterized by families associated with diseases and environmental stress (Saprospiraceae, Flammeovirgaceae, and Xanthobacteraceae). The remaining five sites presented a constant spatio-temporal pattern, where the predominance of Campylobacteraceae and Helicobacteraceae families were key to T. roseus’ holobiont. However, the dominance of certain bacterial families in the second analyzed year, such as Enterobacteriaceae, suggests that Punto B, and Islas e islotes de Bahía Chamela Sanctuary, were exposed to sewage contamination. Overall, our results improve the understanding of host-associated bacterial assemblage in specific time and space and its relationship with the environmental condition.

This study aimed to determine shifts in microbial populations regarding richness and diversity from the daily use of a popular over-the-counter nasal spray. In addition, the finding of nasal commensal bacterial species that overlap with the oral microbiome may prove to be potential probiotics for the “gateway microbiomes.” Nasal swab samples were obtained before and after using the most popular over-the-counter (OTC) nasal spray in 10 participants aged 18–48. All participants were healthy volunteers with no significant medical histories. The participants were randomly assigned a number by randomizing software and consisted of five men and five women. The sampling consisted of placing a nasal swab atraumatically into the nasal cavity. The samples were preserved and sent to Northwestern University Sequencing Center for whole-genome deep sequencing. After 21 days of OTC nasal spray use twice daily, the participants returned for further nasal microbiome sampling. The microbial analysis included all bacteria, archaea, viruses, molds, and yeasts via deep sequencing for species analysis. The Northwestern University Sequencing Center utilized artificial intelligence analysis to determine shifts in species and strains following nasal spray use that resulted in changes in diversity and richness.

[Objective] To investigate the expression of ALDH2 in hepatocellular carcinoma and its correlation with macrophage infiltration and with the activation of specific subtypes of macrophages. [Methods] Transcriptome and clinical data from patients with hepatocellular carcinoma were downloaded from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases and analyzed and collated using R and Perl software. In the TCGA hepatocellular carcinoma dataset, the Wilcoxon test was used to identify macrophage activation-related gene sets that show differential expression between hepatocellular carcinoma and normal tissues. Single-cell sequencing combined with bulk data was used to analyze macrophage infiltration of hepatocellular carcinoma immune cells and the activation and recruitment of different subtypes of macrophages. Single-factor Cox regression and minimum absolute convergence and selection operator (Lasso) were used to identify survival-related genes and construct prognostic models of HCC. The patients were divided into two groups based on their model scores. The Wilcoxon test was used to identify differentially expressed genes (DEGs) between the two groups, and gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses of the DEGs were performed. Single-sample gene set enrichment analysis (ssGSEA) was performed to explore the possible mechanism underlying the differences in prognosis. The reliability of the model was verified using the ICGC hepatocellular carcinoma dataset. A Kaplan‒Meier curve was constructed and used to analyze the influence of macrophage activation-related gene expression on survival and prognosis in patients with hepatocellular carcinoma. The log-rank test and the independent t test were used to analyze survival time data and to compare immune cell infiltration in the two groups. [Results] In a comparison of hepatocellular carcinoma and paracancerous tissues, the expression of six of 137 macrophage activation-related genes (ALDH2, CCNB2, CYP2C9, CYP3A4, F9 and KLKB1) was found to be significantly correlated with overall survival time (OS) in patients with hepatocellular carcinoma. Based on this finding, a prognosis assessment model for hepatocellular carcinoma was constructed. The OS of the patients in the high-scoring group was significantly shorter than the OS of the patients in the low-scoring group (P<0.05), and the model predicted the prognosis of patients with hepatocellular carcinoma independently of sex, age, tumor grade and stage. The area under the receiver operating characteristic (ROC) curve of the model used to predict 1-, 2-, and 3-year survival was greater than 0.7 in both the test set and the validation set. GO enrichment of DEGs between the high- and low-scoring groups of the model suggested that the DEGs are mainly related to immune function. GSEA suggested that the tumor-infiltrating immune cells in the high-scoring group were macrophages, Th2 cells and Treg cells and showed that the number of tumor-infiltrating immune cells, including NK cells, was lower in the high-scoring group than in the low-scoring group. With respect to immune function, the immune checkpoint of the high-scoring group was higher than that of the low-scoring group. Single-cell sequencing data indicated the recruitment of B cells, M0 cells, M1 cells and M2 cells to the hepatocellular carcinoma microenvironment and suggested that ALDH2 regulates related signaling pathways in a way that promotes the differentiation of macrophages into M2-type macrophages. [Conclusion] M2-type macrophages are the dominant type of macrophages associated with immune infiltration in hepatocellular carcinoma. The macrophage-associated activation gene ALDH2 activates the macrophage polarization signaling pathway and negatively regulates the differentiation of macrophages into M2-type macrophages.

Chimeric antigen receptor (CAR) T-cell therapy has greatly improved outcomes for patients with relapsed or refractory hematological malignancies. However, challenges such as treatment resistance, relapse, and severe toxicity still hinder its widespread clinical application. Traditional transcriptome analysis has provided limited insights into the complex transcriptional landscape of both leukemia cells and engineered CAR-T cells, as well as their interactions within the tumor microenvironment. However, with the advent of single-cell sequencing techniques, a paradigm shift has occurred, providing robust tools to unravel the complexities of these factors. These techniques enable unbiased analysis of cellular heterogeneity and molecular patterns. These insights are invaluable for precise receptor design, guiding gene-based T cell modification, and optimizing manufacturing conditions. Consequently, this review utilizes modern single-cell sequencing techniques to clarify the transcriptional intricacies of leukemia cells and CAR-Ts. The aim of this manuscript is to discuss the potential mechanisms that contribute to the clinical failures of CAR-T immunotherapy. We will examine the biological characteristics of CAR-Ts, the mechanisms that govern clinical responses, and the intricacies of adverse events. By exploring these aspects, we hope to gain a deeper understanding of CAR-T therapy, which will ultimately lead to improved clinical outcomes and broader therapeutic applications.

Transcription of glycine-rich RNA-binding protein 2 (PeGRP2) transiently increased in root and shoot of Populus euphratica (salt-resistant poplar) upon initial salt exposure and tended to decrease after long-term NaCl stress (100 mM, 12 days). PeGRP2 overexpression in the hybrid Populus tremula × P. alba ‘717-1B4’ (P. × canescens) increased salt sensitivity, which was reflected in plant growth and photosynthesis. PeGRP2 contains a conserved RNA recognition motif domain at the N-terminus, and RNA affinity purification (RAP) sequencing was developed to enrich target mRNAs that physically interact with PeGRP2 in P. × canescens. RAP sequencing combined with RT-qPCR revealed that NaCl decreased the transcripts of PeGRP2-interacting mRNAs encoding photosynthetic proteins, antioxidative enzymes, ATPases, and Na+/H+ antiporters in transgenic poplar. Particularly, PeGRP2 negatively affected the stability of target mRNAs encoding photosynthetic proteins (PETC and RBCMT), antioxidant enzymes (SOD[Mn], CDSP32 and CYB1-2), ATPases (AHA11, ACA8 and ACA9), and Na+/H+ antiporter (NHA1). This resulted in (i) a greater reduction in Fv/Fm, YII, ETR and Pn, (ii) a less pronounced salt activation of antioxidative enzymes, and (iii) a reduced ability to maintain Na+ homeostasis in transgenic poplars during long-term salt stress, leading to a lowered ability to tolerate salinity stress.

Flax seed is one of the richest plant sources of linolenic acid (LIN) and also contains unsaturated linoleic acid (LIO) and oleic acid (OLE). Stearoyl-ACP desaturases (SAD) and fatty acid desaturases (FAD) play a key role in the synthesis of flax fatty acids (FA). We sequenced flax seed transcriptomes at the 3, 7, 14, 21, and 28 day after flowering (DAF) for ten flax varieties with different oil FA compositions grown under three temperature/watering conditions. Expression levels of 25 genes of SAD, FAD2, and FAD3 families were evaluated. FAD3b, FAD3a, FAD2b-2, SAD3-1, SAD2-1, SAD2-2, SAD3-2, FAD2a-1, and FAD2a-2 had the highest expression levels, which changed significantly during seed development. These genes probably play a key role in the FA synthesis in flax seeds. High temperature and insufficient watering shifted the maximum expression levels of the FAD and SAD genes to earlier development stages, while the opposite trend was observed for low temperature and excessive watering. Differences in FAD and SAD expression profiles under different growth conditions could contribute to the FA composition of linseed oil. Stop codons in the FAD3a gene, resulting in reduced LIN content, decreased the level of the FAD3a transcript. Some difference in reaching the maximum expression level during seed development was observed between 1) SAD3-1, SAD3-2, and FAD2b-2 and 2) SAD2-1, SAD2-2, FAD2a-1, FAD2a-2, FAD3a, and FAD3b. These groups possibly contribute somewhat differently to the synthesis of FA at different development stages.

Wool quality and yield are important economic traits of livestock. In this study, the skin tissues of the upper scapula of Super Merino sheep (SM) and Small-Tailed Han sheep (STH) during the growing period were studied. Differentially expressed (DE) long non-coding RNAs (lncRNAs) and messenger RNAs (mRNA) were identified via histopathological observation and high-throughput RNA sequencing. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze differentially expressed genes. Reverse transcription quantitative Polymerase Chain Reaction (RT-qPCR) was used to verify nine selected DE lncRNAs and five DE mRNAs. Significant differences were found in the expression levels of LOC114113396 (KRTAP15-1), CRABP1, KRT10, KRTAP1-3, KRTAP4.3, KRT2, FGF1, IGF-1, and KIT in SM and STH skin tissues. These results indicate that these mRNAs may play an important role in the growth and development of hair follicles and the regulation of hair fiber traits. Differentially expressed lncRNA target genes and mRNAs were mainly enriched in biological processes and pathways related to skin development and fiber traits, which is consistent with previous studies. LncRNA MSTRG.9225.1, MSTRG.124320.1, MSTRG.96951.1, MSTRG.91685.1, MSTRG.122229.1, MSTRG.89255.1, MSTRG.98769.1, and MSTRG.84658.1 may indirectly participate in the growth and development of sheep hair follicles and the regulation of hair fiber diameter by regulating the expression of their respective target genes. These candidate lncRNAs and mRNAs may provide a basis for the study of sheep hair follicle growth and development and hair fiber traits.

(1) Background: Breast cancer (BC) is the second largest cause of mortality in female worldwide. Besides from basic risk factors, there are several hereditary variables that contribute to BC. Breast cancer has been linked to a variety of nuclear DNA changes as well as mitochondrial DNA alterations. The aim of this study was to analysis the association of mitochondrial tRNA leucine2 gene with BC. (2) Material and methods: In the current study, 24 samples have been collected from various families in Peshawar. DNA was extracted from Blood. PCR was used to amplify the mitochondrial tRNA MT-TL2 gene, and 22 samples were sequenced. The sequence was compared with accession #NC-012920.1 of the revised Cambridge Reference Sequence (rCRS). (3) Results: The results (Chromatograph, nucleotide sequence and rCRS alignment) shows mutations in mitochondrial tRNA MT-TL2 gene in our participants is not the cause of Breast cancer. (4) Conclusion: Yet, a significant number of BC patients must be studied, and their full mitochondrial DNA must be analyzed. This will provide an indication of the potential DNA marker that might be used to prevent BC deaths at the earliest stages.

Aluminum (Al) toxicity is the most common factor limiting the growth of alfalfa in acidic soil conditions. Melatonin (MT), a significant pleiotropic molecule present in both plants and animals, has shown promise in mitigating Al toxicity in various plant species. This study aims to elucidate the underlying mechanism by which melatonin alleviates Al toxicity in alfalfa through a combined physiological and transcriptomic analysis. The results reveal that 5 μM MT significantly alleviates Al-induced growth inhibition and preserves the morphology of root tip cells. Moreover, MT application reduces Al accumulation in alfalfa roots and shoots by 28.6% and 27.6%, respectively. Additionally, MT plays a crucial role in scavenging Al-induced excess H2O2 by enhancing the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), consequently reducing malondialdehyde (MDA) levels. The RNA-seq results reveal that MT application significantly upregulates the expression of xyloglucan endotransglucosylase/hydrolase (XTH) and carbon metabolism-related genes, including those involved in the glycolysis process, as well as sucrose and starch metabolism, suggesting that MT application may mitigate Al toxicity by facilitating the binding of Al to the cell walls, thereby reducing intracellular Al accumulation, and by improving respiration and the content of sucrose and trehalose. Taken together, our study demonstrates that MT alleviates Al toxicity in alfalfa by restoring redox homeostasis, enhancing Al binding to the cell wall, and modulating carbon metabolism. This research advances our understanding of the mechanisms underlying MT's effectiveness in mitigating Al toxicity, offering potential strategies for reducing Al accumulation in alfalfa.

The genome sequencing technologies reveal the molecular mechanisms of differentiated thyroid cancer (DTC). Unlike somatic mutation analysis from thyroidectomy samples, germline mutations showing genetic susceptibility to DTC are less understood. The study aimed to assess the prevalence of germline mutations predisposing to DTC in a cohort of Polish individuals based on their whole genome sequencing (WGS) data. We analyzed sequencing data from 1076 unrelated individuals, released openly for academic and clinical research as The Thousand Polish Genomes database (https://1000polishgenomes.com). The list of genes chosen for further analysis was based on the review of previous studies. The cohort contained 104 variants located within the coding and noncoding DNA sequences of 90 genes selected by ClinVar classification as pathogenic and potentially pathogenic. The frequency of variants in the Polish cohort (our study) was compared to the frequency estimated for the non-Finnish European population, obtained from the gnomAD database (gnomad.broadinstitute.org). Statistically significant variants included 23 genes. Even though the Polish population is genetically similar to other European populations, there are significant differences in variant frequencies contributing to the disease development and progression, such as RET, CHECK2, BRCA1, SLC26A4 or TERT. Further studies are needed to identify genomic variants associated directly with DTC.

Abstract: (1) Background: Cancer immune escape is associated with the metabolic reprogramming of TME, and combining metabolic targets with immunotherapy has great potential to improve clinical outcomes. Among all metabolic processes, lipid metabolism, especially fatty acid (FA) metabolism, has a huge role in cancer cell survival, migration, and proliferation, but its mechanism and role in the tumor immune microenvironment remain to be investigated. (2) Methods: We comprehensively analyzed 309 fatty acid-related genes, screened 121 different genes, and used one-way COX regression to select 15 genes with prognostic impact. Systematically evaluated the correlation between FMGs modification patterns and Tumor Microenvironment Infiltration, prognosis, and Immunotherapy. The FMGs-Score was constructed to quantify the FMGs modification patterns using principal component analysis. (3) Results: Three clusters based on FMGs-related genes were demonstrated in breast cancer, with three patterns of distinct immune cell infiltration and biological behavior. An FMGsScore signature was constructed to reveal that patients with a low FMGsScore had higher immune checkpoint expression, higher immune checkpoint inhibitor (ICI) scores, increased immune microenvironment infiltration, better survival advantage, and were more sensitive to immunotherapy than those with a high FMGsScore. Finally, the expression and function of the signature key gene NDUFAB1 were examined by in vitro experiments. (4) Conclusions: This study significantly demonstrates the non-negligible role of FMGs in the immune microenvironment of breast cancer, and that FMGsScores can be used to guide the prediction of immunotherapy in breast cancer patients. In in vitro experiments, knockdown of the NDUFAB1 gene resulted in reduced proliferation and migration of MCF-7 cell lines.

The snATAC + snRNA platform allows epigenomic profiling of open chromatin and gene expression with single-cell resolution. The most critical assay step is to isolate high-quality nuclei to proceed with droplet-base single nuclei isolation and barcoding. With the increasing popularity of multiomic profiling in various fields, there is a need for optimized and reliable nuclei isolation methods, mainly for human tissue samples. Herein we compared different nuclei isolation methods for cell suspensions, such as peripheral blood mononuclear cells (PBMC, n=18) and a solid tumor type, ovarian cancer (OC) (n=18), derived from debulking surgery. Nuclei morphology and sequencing output parameters were used to evaluate the quality of preparation. Our results show that NP-40 detergent-based nuclei isolation yields better sequencing results than collagenase tissue dissociation for OC, significantly impacting cell identification and analysis. Given the utility of applying such techniques to frozen samples, we also tested frozen preparation and digestion (n=6). A paired comparison between frozen and fresh samples validated the quality of both specimens. Finally, we demonstrate the reproducibility of scRNA and snATAC + snRNA platform, by comparing the gene expression profiling of PBMC. Our results highlight how the choice of nuclei isolation methods is critical for obtaining quality data in multiomic assays. It also shows that the measurement of expression between scRNA and snRNA is comparable and effective for cell type identification.

Black barley seeds are a health-beneficial diet resource because of their special chemical composition and antioxidant properties. The black lemma and pericarp (BLP) locus was mapped in a genetic interval of 0.807 Mb on chromosome 1H, but its genetic basis remains unknown. In this study, targeted metabolomics and conjunctive analyses of BSA-seq and BSR-seq were used to identify candidate genes of BLP and the precursors of black pigments. The results revealed that five differentially expressed genes identified on the 1H chromosome were candidate genes of the BLP locus, and 17 differential metabolites, including the precursor and repeating unit of allomelanin, were accumulated in the grain-filling stage of black barley. Phenol nitrogen-free precursors such as catechol (protocatechuic aldehyde) or catecholic acids (caffeic, protocatechuic, and gallic acids) may promote black pigmentation. BLP can manipulate the accumulation of benzoic acid derivatives (salicylic acid, 2,4-dihydroxybenzoic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid, protocatechuic aldehyde, and syringaldehyde) through the shikimate/chorismite pathway other than the phenylalanine pathway and alter the metabolism of the phenylpropanoid-monolignol branch. Collectively, it is reasonable to infer that black pigmentation in barley is due to allomelanin biosynthesis in lemma and pericarp, and BLP regulates melanogenesis by manipulating the biosynthesis of its precursors.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, which function as regulators of gene expression and contribute in numerous physiological processes. However, little is known referring to miRNAs function in insect chemosensation. In the current study, nine small RNA libraries were constructed and sequenced from the antennae of nymphs, adult males and females of Apolygus lucorum. In total, 399 miRNAs were identified including 275 known and 124 novel miRNAs. Known miRNAs were classified into 71 families, amongst which, 23 families were insect-specific. Expression profile analysis showed that miR-7-5p_1 was the most abundant miRNAs in the antennae of A. lucorum. Altogether, 69708 target genes related to biogenesis, membrane and binding activities were predicted for 399 miRNAs. Particularly, 15 miRNAs were found to target 16 olfactory genes. These miRNAs could be involved in regulation of olfactory-associated genes ex-pression. Comparing the antennae of nymphs, adult males and females, 94 miRNAs were found to be differentially expressed. The expression levels of some differentially expressed miRNAs measured by qPCR were consistent with sequencing results. This study provides a global miRNAs transcriptome in the antennae of A. lucorum and valuable information for further investigation on miRNA-mRNA interactions, especially the functions of miRNAs in regulating chemosensation.

The World Anti-Doping Agency (WADA) has prohibited gene doping in the context of progress in gene therapy. In addition, there is a risk of the EPO gene being applied in gene doping among athletes. Along with this, development of a gene-doping test has been underway in worldwide. Here, we had two purposes: to develop a robust gene doping mouse model using the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to prove gene doping using this model. The rAdV including the hEPO gene were injected intravenously to transfer the gene to the liver. After injection, the mice developed significantly increased red blood cell counts in whole blood and increased gene expressions of hematopoietic markers in the spleen, indicating successful development of the gene doping model. Next, we detected direct and indirect proof of gene doping in whole blood DNA and RNA using qPCR assay and RNA sequencing. Proof was detected in one drop of whole blood DNA and RNA over a long period; furthermore, the overall RNA expression profiles significantly changed. Therefore, we have advanced detection of hEPO gene doping in humans.

(1) Background: the NGS based mutational study of hereditary cancer genes is crucial to design tailored prevention strategies in subjects with different hereditary cancer risk. The ease of amplicon-based NGS library construction protocols contrasts with the greater uniformity of enrichment provided by capture-based protocols and so with greater chances for detecting larger genomic rearrangements and copy-number variations. Capture-based protocols, however, are characterized by a higher level of complexity of sample handling, extremely susceptible to human bias. Robotics platforms may definitely help dealing with these limits, reducing hands-on time, limiting random errors and guaranteeing process standardization. (2) Methods: We implemented and validated the complete automation of the SOPHiA GENETICS’ CE-IVD Hereditary Cancer Solution™ (HCS) libraries preparation workflow on the Hamilton’s STARlet platform. (3) Results: We demonstrate that this automated workflow, used for more than 1000 samples achieved the same performances of manual setup in terms of coverages and reads uniformity, with extremely lower variability of reads mapping rate onto the regions of interest. (4) Conclusions: This automated solution offers same reliable and affordable NGS data, but with the essential advantages of a flexible, automated and integrated framework, minimizing possible human errors and depicting a laboratory’s walk-away scenario.

Genetic disorders are preeminent determinants of infant mortality. The inherited pediatric-onset genetic disorders have consequential stress on child growth and development: several congenital, complex and rare disorders with indistinguishable clinical symptoms where diagnosis always remains a challenging task. Traditional diagnosis methods include biochemical tests followed by chromosomal microarray and sequencing of a single gene or panel of genes. These methods had several limitations, but with the advent of whole-exome sequencing (WES), genetic testing has become cost-effective and transformative. Exome sequencing has been known for its effectiveness, which appropriately elucidates and distinguishes the heterogeneous disorders to avoid misdiagnosis and decode the underlying genetic alterations. WES has led to discovering genes and genomic variants in a broad spectrum of diseases, including autism, epilepsy, congenital heart diseases, neurodevelopmental diseases, cancer, nephrotic disorders, neural tube defects and fetal structural anomalies. WES is significant in producing immense genomic biomarkers that can be made as appropriate pharmacogenomic targets for drug therapy. In this article, we analyze the recent exploration of WES technology to revolutionize not only the process of genetic variation and disease detection but also the convention of preventative and targeted drug discovery.

Cancer is the second leading cause of death worldwide. It is theorized that underlying genetic and epigenetic changes enable cells to proliferate out of control by escaping regulatory mechanisms. Although traditional molecular profiling techniques, i.e., bulk sequencing, can identify common mutations and gene expression patterns in cancer cells, they cannot detect tumour heterogeneity. However, single-cell technology has provided an ample opportunity to overcome this difficulty. Since this technology allows us to detect the heterogeneous properties of all cancer cells, this can further our knowledge of the signaling pathways in cancer cells. Indeed, single-cell transcriptomics technology has paved the road for identifying novel biomarkers and signaling pathways, which can serve as targets. This study aims to review the current knowledge about pathways involved in developing cancer cells and shed light on single-cell studies as promising therapeutic approaches.

Cystoseira is a relevant alga, which forms dense meadows on rocky substrates up to 100 meters deep. They are widely recognized as bioindicators for their restricted habitat (sub-coastal zone) and low tolerance to pollution. We have monitored Cystoseira sensu lato (sl) spp. in abrasion platforms in Cabo de Las Huertas (Alicante, SE Spain), a total of seven species. A cladogram has been constructed using fifteen phenotypic taxonomic relevant characters. We have also optimized Cystoseira DNA extraction and sequenced amplicons from mt23S, tRNA-Lys, and psbA genes of five Cystoseira sl species from Cabo de Las Huertas. Our phylogenetic supertree, built with these sequences, backs three clades in the genus. Rare species Treptacantha sauvageauana and Treptacantha algeriensis, sequenced for the first time, are included with T. elegans and the Atlantic species T. baccata and T. abies-marina in Cystoseira-VI or Cystoseira-II clades. Our data supports a complex colonization of Cystoseira (sl) spp. in the Mediterranean, perhaps involving Atlantic clades.

Young women with breast cancer represent 15% of cancer cases in Latin America. Genomic studies have found that early-onset breast-cancer cases exhibit a higher genetic susceptibility and a specific genomic signature as compared to their older counterparts. The aim of this study was to describe clinically relevant germline mutations in a cohort of young women with breast cancer. To achieve this, we analyzed hereditary-cancer genes from whole-exome sequencing data in 108 unrelated women with an extreme phenotype of breast cancer (≤40 years of age), diagnosed and treated at the National Cancer Institute of Mexico; 11% of the patients carried a pathogenic variant. BRCA2 comprised 46% of the mutations, followed by BRCA1 with 23%; PALB2 with 15%; and TP53 and RAD51C with 8 % each. This article describes a novel pathogenic mutation in RAD51C c.519dupT. The median age at diagnosis was 35 years overall; however, it was six years younger in patients with mutations. Age at diagnosis (OR=0.82, CI 95% 0.71-0.94; P= 0.008) and first-degree family history of cancer (OR=8.26, CI95% 1.35-50; P= 0.022) were the only epidemiological variables associated with mutational status. We found no differences in disease-free survival (p=0.403) or overall survival (p=0.735) among mutational status subgroups.

Single molecule de novo protein sequencing based on the 'superspecificity' of amino-acyl tRNA synthetases (aaRS) is proposed. An unfolded protein molecule is threaded through a nanopore in an electrolytic cell (e-cell) to expose the terminal residue in the e-cell's trans chamber. After the residue is cleaved with an exopeptidase, a set of tRNAs, their aaRSs, and ATP are added to trans. An aaRS charges a cognate tRNA molecule with the residue. The charged tRNA (along with the other reactants) is transferred to an extended e-cell with N (20 ≤ N ≤ 61) pores in N individual cis chambers and a single trans chamber. Each pore holds an RNA molecule ending in a unique codon that is exposed in trans. The charged tRNA's anticodon base-pairs with the terminal codon of an RNA. If tRNAs and residues are fluorescently tagged with two different colors, the residue can be identified from the observed position of the resulting color pair. As charging is 'superspecific' identification is unambiguous. The protein molecule in the first e-cell is advanced by one residue and the process repeated. In this approach there is no need for precise pore current or optical intensity measurements. Potential implementation issues are discussed. Other possibilities, including one in which the terminal residue is cleaved after charging, are also examined.

Here we present 1. a model for amplicon sequencing 2. a definition of the best assignment of a read to a set of reference sequences 3. strategies and structures for indexing reference sequences and computing the best assign- ments of a set of reads efficiently, based on (ultra)metric spaces and their geometry The models, techniques, and ideas are particularly relevant to scenarios akin to 16S taxonomic profiling, where both the number of reference sequences and the read diversity is considerable.

Precision medicine seeks to use genomic data to help provide the right treatment to the right patient at the right time. Next-generation sequencing technology allows for the rapid and accurate sequencing of many genes at once. This technology is becoming more common in oncology, though the clinical benefit of incorporating it into precision medicine strategies remains under significant debate. In this manuscript, we discuss the early findings of the impact of next-generation sequencing on cancer patient outcomes. We investigate why not all patients with genomic variants linked to a specific therapy receive that therapy and describe current barriers. Finally, we explore the current state of health insurance coverage for individual genome sequencing and targeted therapies for cancer. Based on our analysis, we recommend increased transparency around the determination of “actionable mutations” and a heightened focus on investigating the variations in health insurance coverage across patients receiving sequencing-matched therapies.

Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and precursor lesions at young ages, mainly at the oral cavity. As patients have defects in DNA repair mechanisms, standard-of-care treatments to OSCC such as radiotherapy and chemotherapy give rise to severe toxicities. New methods for early diagnosis are urgently necessary to allow treatments in early disease stages and achieve better clinical outcomes. We have conducted a prospective, longitudinal study whereby liquid biopsies from sixteen lesion/tumor-free patients were analyzed for the presence of mutations in cancer genes. DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clinical evolution followed during a median time of around 2 years. In 9/16 FA patients we detected mutations in cancer genes (mainly TP53) with molecular allele frequencies (MAF) down to 0.07 %. Importantly, all patients having mutations and clinical follow-up data after mutation detection (n=6) developed oral precursor lesions or OSCC. Lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations display significantly lower risk of developing precursor lesions or OSCC. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow closer surveillance for OSCC or precursor lesions.

Matrices and their inverses allow accurate calculations to be produced quickly and compactly and can be used to control any process represented by a system of equations. Singular matrices require extensive workarounds (costing time and money) to solve these systems of equations because they have no inverse. Finding their inverse involves division by zero. Nevertheless, recently a new number set called semi-structured complex numbers was developed to enable division by zero in algebraic equations. The aim of this research was to demonstrate that the inverse of a singular matrix can be found using semi-structured complex numbers. This research reveals (1) Singular matrices and their inverses can be used to find a unique solution to a pair of simultaneous equations that appear to have infinitely many solutions or appear to have no solution; (2) Singular matrices map coordinates in projective space to coordinates in Euclidean space and their inverse maps coordinates in Euclidean space to coordinates in projective space; (3) the inverse of a singular matrix proves that parallel lines in Euclidean space do not intersect but in projective space intersect at a “point at infinity” and (4) collinear lines that intersect at infinitely many points in Euclidean space only intersect at one point in projective space.

Global food waste has a huge impact on the environment, as it is a source of greenhouse gas (GHG) emissions and wasted natural resources. Across the world, over 30% of food is lost or wasted each year. Aside from this, food industry, as well, is one of the biggest sources of agro-industrial waste and by-products, which can be valorized and used for different purposes. Such waste is a good source of bioactive organic compounds which can be extracted without al-tering their properties, where deep eutectic solvents (DESs) can serve as green solvents and excel-lent replacement for volatile organic solvents. Isolated compounds can be used in innovative food production, chemical production, cosmetics and other industries. DESs have attracted extraordi-nary attention due to their advantages such as environmental friendliness, availability and easy preparation, easy handling and utilization of non-toxic components for their formation. Due to those properties, they are a greener alternative to classic organic solvents for many processes, in-cluding extractions. In this paper, we review the utilization of deep eutectic solvents as potential green media for the extraction of organic compounds such as polyphenols, carbohydrates, pro-teins, alkaloids from by-products of the food industry and agro-industrial-waste.

The increasing demand by consumers for popular healthy fruits, such as the watermelon (Citrullus lanatus), generate substantial bio-wastes, making it imperative to harness the value of these by-products. Watermelon has low-calorie content and several other nutritional benefits that make it suitable for weight management and heart-healthy diets. Within this article, the nutritional composition and bioactives of watermelon which are strongly linked to the prevention of cardiovascular disorders (CVD), cancer, and age-related conditions, will be thoroughly reviewed. In addition, the therapeutic potential effects of watermelon consumption, such as anti-inflammatory, cardio-protective and anti-cancer properties, are also examined, with emphasis to the importance of incorporating watermelon into regular diets for overall health improvement. Additionally, the study explores various applications of watermelon by-products in functional foods, contributing to meeting recommended daily nutrient allowances, as well as in other functional products like cosmetics, with health promoting properties. In conclusion, this comprehensive review underscores the role of watermelon and its by-products for the development of products that can contribute to the prevention of chronic diseases and promote overall well-being

The Developing Countries Vaccine Manufacturing Network coordinated a multi-laboratory study funded by the US National Institute for Innovation in Manufacturing Biopharmaceuticals to assess the performance of the Pertussis Serological Potency Test (PSPT) in the laboratories of seven whole-cell pertussis vaccine (wP) manufacturers and three National Control Laboratories based in Southeast Asian countries. To this end, common protocols for the immunization of mice and serological testing by ELISA were shared with all laboratories. Each laboratory used their own wP vaccines and the reference vaccine routinely used in the regulatory required mouse protection test (MPT) and assayed the products both in the MPT and PSPT. Whereas in all laboratories the PSPT showed decreased serum IgG levels after immunization with sub-potent compared to potent final batches, some issues were encountered during execution of the assay, which prevented drawing of definitive conclusions on assay performance. Nevertheless, the study provided the participants with the opportunity to familiarize themselves with the PSPT protocols and revealed the next steps to be addressed for implementation of the method.

Bioactive surface coatings have retained the attention of researchers and physicians due to their versatility and range of applications in orthopedics, particularly in infection prevention. Antibacterial metal nanoparticles (mNPs) are a promising therapeutic, with vast application opportunities on orthopedic implants. The current research aimed to construct a polyelectrolyte multilayer on a highly porous titanium implant using alternating thin film coatings of chitosan and alginate via the layer-by-layer (LbL) self-assembly technique, along with the incorporation of silver nanoparticles (AgNPs) or titanium dioxide nanoparticles (TiO2NPs), for antibacterial and osteoconductive activity. These mNPs were characterized for their physicochemical properties using quartz crystal microgravimetry with dissipation system, nanoparticle tracking analysis, scanning electron microscopy, and atomic force microscopy. Their cytotoxicity and osteogenic differentiation capabilities were assessed by AlamarBlue and alkaline phosphatase (ALP) activity assays, respectively. The antibiofilm efficacy of the mNPs tested against Staphylococcus aureus. The LbL polyelectrolyte coating was successfully applied to the porous titanium substrate. A dose-dependent relationship between nanoparticle concentration and ALP as well as antibacterial effects was observed. TiO2NP samples were also less cytotoxic than AgNP counterparts, although similarly antimicrobial. Together, these data serve as a proof-of-concept for a novel coating approach for orthopedic implants with antimicrobial and osteoconductive properties.

Membrane-based beverage dealcoholization is a successful process for producing low- and non-alcoholic beer and represents a fast-growing industry. Polyamide NF and RO membranes are commonly applied for this process. Polyelectrolyte multilayer (PEM) NF membranes are emerging as industrially relevant species and their unique properties (usually hollow fiber geometry, high and tunable selectivity, low fouling) underlines the importance of testing them in the food industry as well. To test PEM NF membranes for beer dealcoholization, at small pilot scale we dealcoholized filtered and unfiltered lager beer with the tightest available commercial polyelectrolyte multilayer NF membrane (NX Filtration dNF40), which has a MWCO=400Da which is quite high for these purposes. Dealcoholization is possible with a reasonable flux (10 LMH) at low pressures (5-8.6 bar) with a real extract loss of 16-18% and an alcohol passage of ~100%. Inorganic salt passage is high (which is typical for PEM NF membranes), which greatly affected beer flavor. During the dealcoholization process, the membrane underwent changes which substantially increased its salt rejection values (MgSO4 passage decreased 4-fold) while permeance loss was minimal (less than 10%). According to our sensory examination, the process yielded an acceptable flavored beer which could be greatly enhanced by the addition of the lost salts and glycerol.

Marine invertebrates are model organisms in several areas of biological sciences, being a source of massive biological information. Although, the scientific relevance of marine invertebrates, the research with them can be limited for their tissue characteristics and troubles for the replication of physical and chemical properties of seawater. Thence, the main goal of this laboratory workflow is to provide a useful methodological approach to reduce the experimental limitations during the study of marine invertebrates. The present study describes experimental methodologies for the collection, transport, and maintenance of sessile tunicates. Also, an approach to observe and characterize, a diverse population of blood cells in marine invertebrates, by several cytological stains and electron microscopy. Lastly, suggestions and protocols to extract quality RNA from samples with high concentrations of salts, pigments, secondary metabolites, and polysaccharides. This methodological approach can be easily adapted to other marine invertebrates, moreover uses low-cost reagents and widely available laboratory equipment. Making possible the study of different types of marine animals in diverse locations.

The livelihood of most of the fishers in Indian Sundarbans is dependent on Penaeus monodon post larvae fishing. These post larvae collectors are socially backward lacking economic security. The activity of collecting Penaeus monodon post larvae for rearing in aquaculture, destroy other aquatic species. Many other juveniles of shellfish and fin fish were destroyed in the process of collection of Penaeus monodon post larvae. The removal of juveniles before they reach maturity disturbs the ecological chain by hampering breeding processes and may cause extinction of some fish species in the long run. The present study is an attempt to estimate the economic value of juveniles destroyed in the collection of (Penaeus monodon) post larvae. In total 32 species were identified in P.monodon the post larval by-catch. The economic loss is assessed based on estimating biomass by taking a length-weight relationship from published literature. Further, the paper illustrates how does a profit enterprise is linked with natural resource exploitation. The paper explores government policy and nature conservation issues for social justice and effective conservation.In conclusion, suggestions are given to reduce the burden of livelihood on natural resources to the extent of exploitation and to strengthen institution and policy-making considering socio-ecological vulnerabilities of the area.

The efficacy and cost-effectivness of Multigene Panel Next-Generation Sequencing (NGS) in directing patients towards genomically matched therapies remains uncertain. This study investigated metastatic colorectal cancer (mCRC) patients who underwent NGS analysis on formalin-fixed paraffin-embedded tumor samples. Data from 179 patients were analyzed, revealing no mutations in 39 patients (21.8%), one mutation in 83 patients (46.4%), and two or more mutations in 57 patients (31.8%). KRAS mutations were found in 87 patients (48.6%), including KRAS G12C mutations in 5 patients (2.8%), PIK3CA mutations in 40 patients (22.4%), BRAF mutations in 26 patients (14.5%). Less common mutations were identified: ERBB2 in 5 patients (2.8%) and SMO in 4 patients (2.2%). Additionally, MAP2K1, CTNNB1, and MYC were mutated in 3 patients (2.4%). Two mutations (1.1%) were observed in ERBB3, RAF1, MTOR, JAK1, and FGFR2. No significant survival differences were observed based on mutation numbers. 40% of patients had druggable molecular alterations, but only 1.1% received genomically guided treatment, suggesting limited application in standard practice. Despite this, expanded gene panel testing can identify actionable mutations, aiding personalized treatment strategies in metastatic CRC, although current eligibility for biomarker-guided trials remains limited.

Organic fertilizer application in agricultural land is known to improve soil microbial processes, fertility, and yield. In particular, stable changes in soil chemical composition through multi-year fertilization of organic fertilizers are thought to cause changes in the microbial community. Here, the effects of oil cake amendments (OC) on soil bacterial diversity, community profile and enzyme activity were evaluated and compared to those amended with chemical fertilizer (NPK). The comparison of diversity indices between OC and NPK amended soils shows that application of oil cake has increased microbial diversity potentially with an increase in the number of different microbial groups. The ordination plot distinguished and clustered both treatments showing differential effects of soils chemical factors on the microbial communities of each treatment. Proteobacteria, Verrucomicrobia and Bacteriodetes were significantly abundant in OC amended soil than in the NPK received soil, indicating alterations in community structure, composition, and diversity con-current to the changes in pH, Ca and Mg contents of the soil. These shifts in bacterial community structure and composition could also be observed from the phylum to the genus level in both NPK and OC amended soils partially explained by differences in soil chemical factors. The OC soil compared to the NPK amended soil also contain significantly higher abundance of predicted genes corresponding to enzymes related to biogeochemical cycling, decomposition and plant growth promotion. Collectively, these results support the use of an unconventional organic fertilizer positively altering bacterial populations in jujube orchards. The application of oil cake-based organic fertilizer improved microbial diversity and enhanced potential ecosystem functions related to bio-geochemical cycles, mineralization and plant growth promotion.

The genetic bases of Alzheimer's disease (AD) and frontotemporal dementia (FTD) have been comprehensively studied, which is not the case for atypical cases not classified into these diagnoses. In the present study, we aim to contribute to the molecular understanding of the development of non-AD and non-FTD dementia by surveying rare pathogenic variants. The analysis was performed on 91 patients, the DNA samples of which were pooled and then subjected to whole exome sequencing. Among detected variants two are rare pathogenic and show significant difference in frequency between the analyzed pool and gnomAD Bulgarian control exomes. The rs373478202, G&gt;T, B9D1 variant, most likely present in heterozygous state; is causative for the autosomal recessive ciliopathy- Joubert syndrome, which involves specific CNS malformation. The rs148918985, C&gt;T, ASS1 variant leads to arginosuccinate enzyme deficiency and in compound heterozygosity with other rare pathogenic variant detected in the same gene causes the late-onset form of citrullinemia type I. This disorder causes high ammonia levels, which can lead to cerebral dysfunction. In conclusion, the detected rare pathogenic variants suggest that, in certain cases, the development of non-AD and non-FTD dementia can be mediated by structural alterations in neuronal cilia or by hyperammoniemia.

Eggplant or brinjal is one of the most consumed and important nutrient-dense tropical solanaceous vegetable crops grown worldwide. Little leaf is a disease especially widespread in brinjal in India where it is associated with the presence of phytoplasmas. To clarify the epidemiology of this disease and verify its seed transmission potential, seedlings and their progeny derived from symptomatic mother plants field-collected in Dharwad district of Karnataka State, India, were sowed under insect proof conditions in greenhouse. The resulting first- and second-generation seedlings obtained were tested at different times after germination by DNA extraction and amplification of 16S rRNA gene of phytoplasmas. Tissues from field collected symptomatic brinjal plants were also tested following the same procedure. The amplicons obtained were subjected to restriction fragment length polymorphism (RFLP) analysis and sequencing for phytoplasma identification. The detected phytoplasmas belonged to 16SrI, 16SrII, 16SrIII, 16SrV, 16SrVI, and 16SrXII groups. Several fruits produced from the first-generation seedlings showed germination of the seeds inside the fruit and resulted to be phytoplasma positive. This demonstrates the seed transmission of phytoplasmas in eggplants for two subsequent generations. This finding highlights the risk of additional source of infection represented by asymptomatic and infected seedlings under field conditions and contribute to explain the epidemic spread of phytoplasmas in brinjal cultivations in India and in the other eggplant growing areas of the world.

Abstract: The ability to detect several types of cancer using a non-invasive, blood-based test holds the potential to revolutionize oncology screening. We mined tumor methylation array data from the Cancer Genome Atlas (TCGA) covering 14 cancer types and identified two novel, broadly oc-curring methylation markers at TLX1 and GALR1. To evaluate their performance as a general-ized blood-based screening approach, along with our previously reported methylation bi-omarker, ZNF154, we rigorously assessed each marker individually or combined. Utilizing the TCGA methylation data and applying logistic regression models within each individual cancer type, we found that the three-marker combination significantly increased the average area under the ROC curve (AUC) across the 14 tumor types compared to single markers (p= 1.158e-10; Friedman test). Furthermore, we simulated dilutions of tumor DNA into healthy blood cell DNA and demonstrated increased AUC of combined markers across all dilution levels. Finally, we evaluated assay performance in bisulfite sequenced DNA from patient tumors and plasma, in-cluding early-stage samples. When combining all three markers the assay achieved 100% sensi-tivity and specificity, as demonstrated in lung cancer plasma samples. In patient plasma from hepatocellular carcinoma, ZNF154 alone yielded the highest combined sensitivity and specificity values averaging 68% and 72%, whereas multiple markers could achieve higher sensitivity or specificity, but not both. Altogether this study presents a comprehensive pipeline for the identifi-cation, testing and validation of multi-cancer methylation biomarkers with a considerable poten-tial for detecting a broad range of cancer types in patient blood samples.

Chronic heat stress (CHS) has been exerting great pressure on the poultry industry due to the global warming. CHS-induced negative impacts deeply change the physiology, metabolism, egg production and meat quality of ducks. To reduce the increasing economic loss, different environmental strategies have been adopted. Here, we comprehensively compared three rearing modes for Cherry-Valley ducks (CVds) mainly by changing the environmental temperature. Plus, the transcriptome analysis and physiological anatomy were implied to investigate the underlying molecular biology and pneumonocyte dynamics. It turns out that the CVds affected by CHS have poor growth performance and bad carcass traits. In addition, the irreversible damage caused by CHS in the lung tissue of CVds was observed. Furthermore, several potential genes related to CHS have been found, such as HSPA8, IGF1, FGFR1, ACSL3, ACSL6, NR1H3 and GAPDH. This study strongly indicates the severe injury of CHS from the high-temperature environment and points out one of the adopting directions for duck rearing.

The emergence of new SARSCoV-2 variants can affect vaccine efficacy, laboratory diagnosis and therapies already available, triggering interest in the search for antiviral agents for SARSCoV-2 infections. Ribavirin (RBV) is a broad-spectrum antiviral with demonstrated in vitro activity against multiple viruses, including SARSCoV-2. Methods. This retrospective study evaluated the dynamic and viral clearance of SARSCoV-2 in hospitalized adult participants (PTs) with COVID-19 pneumonia who received RBV aerosol within a compassionate use study. Was also assessed the impact of RBV on the clinical outcome and the mutational profile of SARSCoV-2. Results. The median RNA values measured in nine PTs included in the study de-creased from baseline to discharge (at BL, threshold cycle (Ct)=22.4, IQR 19.84-5.07; at discharge, Ct=27.92, IQR 26.43-36.11). Seven/9 PTs experienced a clinical improvement, while two PTs deceased during hos-pitalization. In PTs with a favorable outcome the virus clearance rate at discharge was 28.6%. The cumulative clearance rate was 71.4% within day 14th from discharge. A mutational pattern after RBV was detected in 3/5 PTs in whom was available whole genome sequencing. Conclusions. RBV may limit SARSCOV-2 replication resulting in a favorable clinical outcome. Ribavirin may also contribute to the mutational spectrum of SARSCoV-2.

Rabies is a fatal zoonosis that is considered a re-emerging infectious disease. Although rabies remains endemic in canines throughout much of the world, vaccination programs have essentially eliminated dog rabies in the Americas and much of Europe. However, despite the goal of eradicating dog rabies in the European Union by 2020, sporadic cases of dog rabies still occur in Eastern Europe, including Georgia. To assess the genetic diversity of strains recently circulating in Georgia, we sequenced 78 RABV-positive samples from brain tissues of rabid dogs and jackals using Illumina short-read sequencing of total RNA shotgun libraries. Seventy-seven RABV genomes were successfully assembled and annotated, 74 of them to the coding complete status. Phylogenetic analyses of the nucleoprotein (N) and attachment glycoprotein (G) genes placed all the assembled genomes into the Cosmopolitan clade, consistent with the Georgian origin of the samples. Amino acid alignment of the G glycoprotein ectodomain identified twelve different sequences for this domain among the samples. Only one of the ectodomain groups contained a residue change in an antigenic site, an R264H change in the G5 antigenic site. Three isolates were cultured, and these were found to be efficiently neutralized by human monoclonal antibody A6. Overall, our data show that recently circulating RABV isolates from Georgian canines are predominantly closely related phylogroup I viruses of the Cosmopolitan clade. Current rabies vaccines should offer protection against infection by Georgian canine RABVs. The genomes have been deposited in GenBank (accessions: OQ603609-OQ603685).

Barth Syndrome, a rare X-linked disorder affecting 1:300,000 live births, results from defects in Tafazzin, an acyltransferase that remodels cardiolipin and is essential for mitochondrial respiration. Barth Syndrome patients develop cardiomyopathy, muscular hypotonia and cyclic neutropenia during childhood, rarely surviving to middle age. At present, no effective therapy exists and downstream transcriptional effects of Tafazzin dysfunction are incompletely understood. To identify novel, cell-specific, pathological pathways that mediate heart dysfunction, we performed single-nucleus RNA-sequencing (snRNA-seq) on wild-type (WT) and Tafazzin-knockout (Taz-KO) mouse hearts. We determined differentially expressed genes (DEGs) and inferred predicted cell-cell communication networks from these data. Surprisingly, DEGs were distributed heterogeneously across the cell types, with fibroblasts, cardiomyocytes, endothelial cells, macrophages, adipocytes and pericytes exhibiting the greatest number of DEGs between genotypes. One differentially expressed gene was detected for the lymphatic endothelial and mesothelial cell types, while no significant DEGs were found in the lymphocytes. A Gene Ontology (GO) analysis of these DEGs showed cell-specific effects on biological processes such as fatty acid metabolism in adipocytes and cardiomyocytes, increased translation in cardiomyocytes, endothelial cells and fibroblasts, in addition to other cell-specific processes. Analysis of ligand-receptor pair expression, to infer intercellular communication patterns, revealed the strongest dysregulated communication involved adipocytes and cardiomyocytes. For the knockout hearts, there was a strong loss of ligand-receptor pair expression involving adipocytes, and cardiomyocyte expression of ligand-receptor pairs underwent reorganization. These findings suggest that adipocyte and cardiomyocyte mitochondria may be most sensitive to mitochondrial Tafazzin deficiency and that rescuing adipocyte mitochondrial dysfunction, in addition to cardiomyocyte mitochondrial dysfunction, may provide therapeutic benefit in Barth Syndrome patients.

Popped amaranth is a nutritious food consumed since pre-Hispanic times, currently its consumption has been associated with recovery of malnourished children. However, there is no information on the impact that popped amaranth consumption has on gut microbiota composition. A non-randomized pilot trial was conducted to evaluate the changes on composition, structure, and function of gut microbiota of stunted children who received four grams of popped amaranth daily for three months. Stool and serum were collected at the beginning and at the end of the trial. Short-chain-fatty acids were quantified and gut bacterial composition was analyzed by 16S rRNA gene sequencing. Biome-try and hematology results showed that children have no other pathology more than to be low-height-for age. Increase of total SCFAs levels in feces was ob-served as well as a decrease in relative abundance of Alistipes putredinis, Bac-teroides coprocola, and Bacteroides stercoris, bacteria related with inflammation and colitis. On the contrary, an increase in relative abundance of Akkermansia muciniphila and Streptococcus thermophiles, bacteria associated with health and longevity, was observed. Results have proven that popped amaranth, a nutri-tious food, help to combat children malnutrition through gut microbiota modu-lation.

Ultraviolet (UV) radiation is a strong environmental carcinogen responsible for the pathogenesis of most skin cancers, such as malignant melanoma (MM) and non-melanoma (keratinocyte) skin cancers. The carcinogenic role of UV was firmly established based on epidemiological evidence and molecular findings of the characteristic mutation signatures which occur during excision repair of cyclobutane pyrimidine dimers and 6,4-photoproducts. The role of UV in the pathogenesis of mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma, remains controversial. Here, we performed whole exome sequencing of 61 samples of MF cells microdissected from the cutaneous lesions, and compared their mutational signatures to 340 MM. The vast majority of MM mutations had a typical UV mutational signature (SBS 7, SBS 38, DSB 1) underscoring the key role of ultraviolet as a mutagen. In contrast, the SBS 7 signature in MF comprised &lt;5% of all mutations. SBS 7 was higher in the intraepidermal MF cells (when compared to the dermal cells) and in the cells from tumors as compared to early-stage plaques. In conclusion, our data do not support the pathogenic role of UV in the pathogenesis of MF and suggest that the UV mutations are the result of the cumulative, environmental ultraviolet exposure of cutaneous lesions, rather than an early mutagenic event.

Background: It remains unclear whether transfer RNA-derived small RNAs (tsRNAs) play a role in pathological cardiac hypertrophy (PCH). We aimed to clarify the expression profile of tsRNAs and disclose their relationship to the clinical phenotype of PCH and the putative role. Methods: Small RNA sequencing was performed in the plasma of PCH patients and healthy volunteers. In a larger sample size and angiotensin Ⅱ (Ang II)-stimulated H9c2 cells, the data were validated by real-time qPCR. The atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were examined in Ang II-stimulated H9c2 cells. The role of tsRNAs in the pathogenesis of PCH was explored by bioinformatics analysis. Results: A total of 4185 differentially expressed tsRNAs were identified, of which 4 and 5 tsRNAs were observed to be significantly differentially upregulated and downregulated expressed. Of the 5 down-regulated tsRNAs, 4 of them were verified to be significantly down-regulated in the larger sample group, among which tRF-30-3JVIJMRPFQ5D, tRF-16-R29P4PE, tRF-21-NB8PLML3E, and tRF-21-SWRYVMMV0 had areas under the curve to diagnose concentric hypertrophy. The 4 down-regulated tsRNAs were negatively correlated with left ventricular posterior wall dimensions in PCH patients (r=-0.4227; r=-0.4517; r=-0.5567; r=-0.4223). The levels of ANP and BNP as well as cell size were decreased in Ang II-stimulated H9c2 cells with 21-NB8PLML3E mimic transfection. Bioinformatics analysis revealed that the target genes of tRF-21-NB8PLML3E were mainly enriched in the metabolic pathway and involved in the regulation of ribosomes. Conclusion: The plasma tsRNAs tRF-21-NB8PLML3E might be considered biomarkers in patients with PCH with early screening potential.

The molecular mechanisms underlying heat stress tolerance in animals to high temperatures remain unclear. This experiment was performed with Sprague-Dawley rats housed at 22 ℃ (control group; CT), and 42 ℃ for 30 min (H30), 60 min (H60), and 120 min (H120). Transcriptomic analysis using RNA-Sequencing (RNA-Seq) from blood (CT and H120), liver (CT, H30, H60, and H120), and adrenal glands (CT, H30, H60, and H120) was performed. The differentially expressed mRNA isoforms (DEIs) were identified and annotated by the CLC Genomics Workbench. Biological process and metabolic pathway analyses were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A total of 225, 5,764, and 4,988 DEIs in the blood, liver, and adrenal glands were observed. Furthermore, the number of novel differentially expressed transcript lengths with annotated genes and the novel differentially expressed transcript with non-annotated genes were 136 and 8 in blood, 3,549 and 120 in the liver, as well as 3,078 and 220 in adrenal glands, respectively. A total of 35 genes were involved in the heat stress response, out of which Dnaja1, LOC680121, Chordc1, AABR07011951.1, Hsp90aa1, Hspa1b, Cdkn1a, Hmox1, Bag3 and Dnaja4 were commonly identified in the liver and adrenal glands, suggesting that these genes may regulate heat stress response through interactions between liver and adrenal glands. The results suggest that the identified mRNA isoforms could be considered as potential candidates for selecting mammals to improve thermotolerance.

With the rapid development of industry, Cr has become one of the major heavy metal pollutants in soil, which has caused serious effects on the soil's ecological environment. However, the effects of Cr stress on bacterial communities in plant rhizosphere soils remain unclear. In this study, I. tectorum was selected as the research object, and 16S rRNA high-throughput sequencing technology was used to analyze the effects of Cr stress on the structure and diversity of the bacterial community in the rhizosphere soil of I. tectorum. The results showed that I. tectorum had strong tolerance and enrichment to Cr. However, under Cr stress, the diversity and abundance index of rhizosphere bacteria decreased by 8.5% and 6.8% on average, and the Sobs index decreased by 7.6%. Moreover, the bacterial community changed by 20.1% due to the addition of Cr, further leading to a 15.9% decrease in the common species of the bacterial community, among which Proteobacteria, Actinobacteria, Chloroflexi and Acidobacteriota accounted for more than 74.8% of the total sequence. According to the symbiosis network diagram, it was found that under a two-cultivated pattern, the synergizing effect between dominant bacteria was significantly enhanced, and the soil microenvironment was improved. Redundancy analysis showed that C, N, and P nutrient elements and Cr contents in uncontaminated and contaminated soils were the primary driving factors for the succession of I. tectorum rhizosphere bacterial community, and the response was stronger after Cr(Ⅵ) was added. In conclusion, the results of this study will provide insights into the response of rhizosphere bacterial communities to heavy metal Cr and the interactions between wetland plants and rhizosphere bacteria in wetland phytoremediation.Keywords: Cr stress; rhizosphere bacterial community; Iris tectorum; 16S rRNA sequencing technology; Phytoremediation

Chromosomal rearrangements in N. gonorrhoeae and N. meningitidis were studied with the determination of mobile elements and their role in rearrangements. The results of whole-genome sequencing and de novo genome assembly for 50 N. gonorrhoeae isolates collected in Russia were compared with 96 genomes of N. gonorrhoeae and 138 genomes of N. meningitidis from the databases. Rearrangement events with the determination of the coordinates of syntenic blocks were analyzed using the SibeliaZ software, the minimum number of events that allow one genome to pass into another was calculated using the DCJ-indel model using the UniMoG program. Population-level analysis revealed a stronger correlation between changes in the gene order and phylogenetic proximity for N. meningitidis in contrast to N. gonorrhoeae. Mobile elements were identified, including Correa elements, Spencer-Smith elements (in N. gonorrhoeae), Neisserial intergenic mosaic elements, IS elements of IS5, IS30, IS110, IS1595 groups, Nf1-Nf3 prophages, NgoФ1-NgoФ9 prophages, Mu-like prophages Pnm1, Pnm2, MuMenB (in N. meningitidis). More than 44% of the observed rearrangements most likely occurred with the participation of mobile elements, including prophages. No differences were found between the Russian and global N. gonorrhoeae population both in terms of rearrangement events and in the number of transposable elements in genomes.

Abstract: Since ancient times aging has also been regarded as a disease, and humankind has always strived to extend the natural lifespan. Analyzing the genes involved in aging and disease allows for finding important indicators and biological markers for pathologies and possible therapeutic targets. An example of the use of omics technologies is the research regarding aging and the rare and fatal premature aging syndrome progeria (Hutchinson-Gilford progeria syndrome, HGPS). In our study, we focused on the in silico analysis of differentially expressed genes (DEGs) in progeria and aging, using a publicly available RNA Seq dataset (GEO dataset GSE113957) and a variety of bioinformatics tools. We identified several genes that appear to be involved both in natural aging and progeria (KRT8, KRT18, ACKR4, CCL2, UCP2, ADAMTS15, ACTN4P1, WNT16, IGFBP2). Further analyzing these genes and the pathways involved confirmed their possible roles in aging, suggesting the need for further in vitro and in vivo research. The graphical abstract illustrates the analysis workflow we used and will introduce in the following as an example to demonstrate the power of omics and bioinformatics.

Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plant were isolated from river water in Europe but not fully characterized, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum.

Colletotrichum is one of the most important plant pathogenic genera of fungi due to its scientific and economic impact. Colletotrichum spp. can infect a wide range of hosts, causing losses in crops of major importance worldwide, such as soybean. In the past, soybean anthracnose was mainly caused by C. truncatum, but during the last decade, other species have been identified at an increasing rate, becoming one of the most important limiting factors to soybean production in several regions. To gain a better understanding of the evolutionary origin of soybean anthracnose, we compared the repertoire of effector candidates of four Colletotrichum species pathogenic to soybean and eight pathogens of other hosts. Our results show that the four species infecting soybean belong to two lineages and do not share any of the lineage specific effector candidates identified. These results strongly suggest that two Colletotrichum lineages have acquired the capability to infect soybean independently. This study also provides, for each lineage, a set of candidate effectors encoding genes that may have important roles in pathogenicity towards soybean offering a new resource useful for further research on soybean anthracnose management.

Abstract: Infectious bronchitis virus (IBV) induces respiratory and urogenital disease in chickens. Although IBV replicates in the gastrointestinal tract, enteric lesions are uncommon. We have reported a case of runting-stunting syndrome in commercial broilers from which an IBV variant was isolated from the intestines. The isolate, CalEnt, demonstrated an enteric tissue tropism in chicken embryos and SPF chickens experimentally. Here, we determined the full genome of CalEnt and compared it to other IBV strains, in addition to comparing the pathobiology of CalEnt and M41 in commercial broilers. Despite the high whole-genome identity to other IBV strains, CalEnt is rather unique in nucleotide composition. The S gene phylogenetic analyses showed great similarity between CalEnt and Cal 99. Clinically, vent staining was slightly more frequent in CalEnt-infected birds than those challenged with M41. Furthermore, IBV IHC detection was more evident and the viral shedding in feces was overall higher with the CalEnt challenge compared with M41. Despite underlying intestinal lesions caused by coccidiosis and salmonellosis vaccination, microscopic lesions in CalEnt-infected chickens were more severe than in M41-infected chickens or controls, supporting the enteric tropism of CalEnt. Further studies in SPF chickens are needed to determine the pathogenesis of the virus, its molecular mechanisms for the enteric tropism, and its influence in intestinal health.

Astrocytes perform a wide variety of essential functions defining normal operation of the nervous system, and are active contributors to the pathogenesis of neurodegenerative disorders such as Alzheimer among others. Recent data provide compelling evidence that distinct reactive astrocyte states are associated with specific stages of Alzheimer´s disease. The advent of transcriptomics technologies enables rapid progress in the characterisation of such pathological astrocyte states. In this review, we provide an overview of the origin, main functions, molecular and morphological features of astrocytes in physiological as well as pathological conditions related to Alzheimer´s disease. We will also explore the main roles of astrocytes in the pathogenesis of Alzheimer´s disease and summarize main transcriptional changes and altered molecular pathways observed in astrocytes during the course of the disease.

Germline mutations in predisposition genes account for only 20% of all familial colorectal cancer (CRC) and the remaining genetic burden may be due to rare high-to-moderate-penetrance germline variants that are not explored. With the aim of identifying such potential cancer predisposing variants, we performed whole exome sequencing on three CRC cases and three unaffected members of a Polish family and identified two novel heterozygous variants; a coding variant in APC down-regulated 1 gene (APCDD1, p.R299H) and a non-coding variant in the 5’ untranslated region (UTR) of histone deacetylase 5 gene (HDAC5). Sanger sequencing confirmed the variants segregating with the disease and Taqman assays revealed 8 additional APCDD1 variants in a cohort of 1705 familial CRC patients and no further HDAC5 variants. Proliferation assays indicated an insignificant proliferative impact for the APCDD1 variant. Luciferase reporter assays using the HDAC5 variant resulted in an enhanced promoter activity. Targeting of transcription factor binding sites of SNAI-2 and TCF4 interrupted by HDAC5 variant showed a significant impact of TCF4 on promoter activity of mutated HDAC5. Our findings contribute not only to the identification of unrecognized genetic causes of familial CRC but also underline the importance of 5´UTR variants affecting transcriptional regulation and the pathogenesis of complex disorders.

Kidneys of mice, rats and humans possess progenitors that maintain daily homeostasis and take part in endogenous regenerative processes following injury, owing to their capacity to proliferate and differentiate. In the glomerular and tubular compartments of the nephron, consistent studies demonstrated that well-characterized, distinct populations of progenitor cells, localized in the parietal epithelium of Bowman capsule and scattered in the proximal and distal tubules, could generate segment-specific cells in physiological conditions and following tissue injury. However, defective or abnormal regenerative responses of these progenitors can contribute to pathologic conditions. The molecular characteristics of renal progenitors have been extensively studied, revealing that numerous classical and evolutionarily conserved pathways, such as Notch or Wnt/β-catenin, play a major role in cell regulation. Others, such as retinoic acid, renin-angiotensin-aldosterone system, TLR2 (Toll-Like Receptor 2) and leptin, are also important in this process. In this review, we summarize the plethora of molecular mechanisms directing renal progenitor responses during homeostasis and following kidney injury. Finally, we will explore how single cell RNA sequencing could bring the characterization of renal progenitors to the next level, while knowing their molecular signature is gaining relevance in the clinic.

Transposable elements (TEs) are mobile genetic elements capable of rapidly altering the genome through their movements. The importance of TE activity has been documented in many biological processes, such as introducing genetic instability, altering patterns of gene expression, and accelerating genome evolution. Increasing appreciation of TEs results in the growing number of bioinformatics software to identify insertion events. However, the application of existing TE finding tools is limited by either narrow-focused design of the package, too many dependencies on other tools, or prior knowledge required as input files that may not be readily available to all users. Here, we report a simple pipeline, TEfinder, developed for the detection of new TE insertions with minimal software dependencies using four inputs that can be easily generated with popular variant calling pipelines. The external software requirements are BEDTools, SAMtools, and Picard. Necessary inputs include TEs present in the reference genome, binary paired-end alignment, reference genome index, and a list of TE names. We tested TEfinder pipeline among several evolving populations of Fusarium oxysporum generated through a short-term adaptation study. Our results demonstrate that this easy-to-use tool can effectively detect new TE insertion events, making it accessible and practical for TE analysis.

The rapid evolution of Next Generation Sequencing in clinical settings and the resulting challenge of variants interpretation in the light of constantly updated information, requires robust data management systems and organized approaches to variant reinterpretation. In this paper, we present iVar: a freely available and highly customizable tool provided with a user-friendly web interface. It represents a platform for the unified management of variants identified by different sequencing technologies. iVar accepts, as input, VCF files and text annotation files and elaborates them, optimizing data organization and avoiding redundancies. Updated annotations can be periodically re-uploaded and associated to variants as historicize attributes. Data can be visualized through variant-centered and sample-centered interfaces. A customizable search functionality can be exploited to periodically check if pathogenicity related data of a variant are changed over time. Patient recontacting ensuing from variant reinterpretation is made easier by iVar through the effective identification of all patients present in the database and carrying a specific variant. We tested iVar by uploading 4171 VCF files and 1463 annotation files, obtaining a database of 4166 samples and 22569 unique variants. iVar has proven to be a useful tool with good performances for collecting and managing data from medium-throughput

Molecular identification via Internal Transcribed Spacer region (nrDNA-ITS) sequencing of Fusarium spp. isolates from wheat originated from Stara Zagora region, were performed for the first time in Bulgaria. А total of 60 wheat samples (Triticum aestivum) were morphologically identified at the genus level as Fusarium spp. in advance. The rDNA-ITS region of all isolates was successfully amplified and the PCR products obtained were directly sequenced. After a comparison of detected sequences with NCBI database, members of three different fungal genera (Fusarium, Chaetomium, and Alternaria) were identified. Among Fusarium isolates, the F. tricinctum was prevailing, followed by F. poae. A total of three isolate F. proliferatum, F. graminearum and F. equiseti were presented with a single probe. The lowest genetic distance (0.004) was detected between F. tricinctum isolates. On the base of genetic distances, fungal isolates were grouped in two main clusters – one comprising F. tricinctum isolates and F. proliferatum, and second including F. equiseti, F. graminearum and F. poae. It could be concluded that the rDNA-ITS genome region of the genus Fusarium may be used as a suitable marker of early detection, accurate and reliable identification of Fusarium spp. contamination of wheat. The timely and accurate information would assist in the selection of appropriate approaches for control of fusarium infections and possible mycotoxins contamination of agricultural production.

The KARS gene encodes the aminoacyl-tRNA synthetase (aaRS) which activates and joins the lysin with its corresponding transfer RNA (tRNA), through the ATP-dependent aminoacylation of the amino acid. The KARS gene mutations have been linked to diverse neurologic phenotypes such as: neurosensorial hearing loss, leukodistrophy, microcephaly, developmental delay or regression, peripheral neuropathy, cardiomyopathy, impairment of the mitochondrial respiratory chain, hyperlactatemia, among others. This article presents the case of a Colombian pediatric patient with two pathological missense variants in a compound heterozygous state in the KARS gene.

Bacteria and archaea participate in and are influenced by processes of substance circulation and energy exchanges in natural environment. Generally, the community changes of bacteria and archaea in sediment are mainly driven by seasonality in mid-latitude regions. But in our study, water diversion to Fen river played a more important role on OTU number, diversity and community structure of bacteria and archaea in sediment than seasonal variation, which was found by 16S rRNA high-throughput sequencing technology. This phenomenon might be caused by external transferred water on the physicochemical water environment and accelerated release of positive nitrogen from sediment caused by rise of water level. Changes of carbon-nitrogen cycle and increase of electrical conductivity (EC) value induced more diversion-responders than season-responders both for bacteria and archaea. Seasonal changes have been influencing bacteria and archaea mildly throughout the whole study reach. After water diversion, the environment indicators relating to bacteria community obviously changed from nutrients to salinity while that for archaea almost disappeared. Our research showed the effects of human activities on the communities of bacteria and archaea outweigh the forcing from natural seasonal changes in mid-latitude regions and revealed the mechanism, highlighting different responses of bacteria and archaea to environmental changes.

In addition to the risk of vaccine failure caused by strain mismatch, the production of inactivated FMD vaccines is dangerous if adequate biosafety cannot be maintained. Using a high-throughput sequencing protocol optimized for short nucleic acid fragments, the composition of a local inactivated vaccine was analyzed in depth. The serotype O strain identified in the vaccine was genetically identical to viruses found in recent FMD outbreaks in Egypt.

The 'big data revolution' has enabled novel types of analyses in the life sciences, facilitated by public sharing and reuse of datasets. Here, we review the prodigious potential of reusing publicly available datasets and the challenges, limitations and risks associated with it. Possible solutions to issues and research integrity considerations are also discussed. Due to the prominence, abundance and wide distribution of sequencing data, we focus on the reuse of publicly available sequence datasets. We define ‘successful reuse’ as the use of previously published data to enable novel scientific findings and use selected examples of such reuse from different disciplines to illustrate the enormous potential of the practice, while acknowledging their respective limitations and risks. A checklist to determine the reuse value and potential of a particular dataset is also provided. The open discussion of data reuse and the establishment of the practice as a norm has the potential to benefit all stakeholders in the life sciences.

Molecular analysis is an easier means to identify and isolate a specific gene which has imperative function for growth, body composition , fat deposition, metabolic and skeletal traits as well as the molecular genetics selection on individual genes is a very efficient method to genetically improve economically important traits in chickens. Insulin- like growth factor 1 ( IGF-1)is a member of a heterogeneous group of peptides with important growth.Transforming growth factorβ ( TGF-β) belongs to a large family of growth and differentiation factors that play a pivotal role in a great variety of biological activities including morphogenesis, development and differentiation. DNA was extracted from 48 chickens sampled from three strains Lohman (17) , Sinai (24) and Gimmizah (7) IGF-1 gene and TGFβ2 gene were amplified using PCR protocol. Electrophoresis was carried out on the products of PCR , bands viewed on transilluminator. The size of IGF-1 gene was 675 bp while the size of TGFβ2 gene was 188bp. Sharp bands were purified and sequenced and used the dendrogram to show the relationships between other vertebrate species

Although it is known that the gut microbiota (GM) can be modulated by diet, the efficacy of specific dietary interventions in determining its composition and diversity in obese patients remains to be ascertained. The present work aims to evaluate the impact of a moderately hypocaloric Mediterranean diet on the GM of obese and overweight patients (OB). The GM of 23 OB patients (F/M= 20/3) was compared before (T0) and after 3 months (T3) of the nutritional intervention (NI). Fecal samples were analyzed by Illumina MiSeq sequencing of the 16S rRNA gene. At baseline, the GM characterization confirmed the typical obesity-associated dysbiosis. After 3 months of NI, patients presented a statistically significant reduction of the body weight and fat mass, along with changes in the relative abundance of many microbial patterns. In fact, we observed an increased abundance in several Bacteroidetes taxa (i.e. Sphingobacteriaceae, Sphingobacterium, Bacteroides spp., Prevotella stercorea) and depletion of many Firmicutes taxa (i.e. Lachnospiraceae members, Ruminococcaceae and Ruminococcus, Veillonellaceae, Catenibacterium, Megamonas). In addition, the phylum Proteobacteria showed an increased abundance, while the genus Sutterella, within the same phylum, decreased after the intervention. Metabolic pathways, predicted by bioinformatic analyses, showed a decrease in membrane transport and cell motility after NI. The present study extends our knowledge of the GM profiles in OB, highlighting the potential benefit of a moderate caloric restriction in counteracting the gut dysbiosis.

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

Whole-genome DNA sequencing of Lactobacillus D75 and D76 strains (Vitaflor, Russia) was performed using the PacBio RS II platform, followed by de novo assembly with SMRT Portal 2.3.0. The average nucleotide identity (ANI) test showed that both strains belong to the Lactobacillus helveticus, but not the L. acidophilus as previously assumed. 31 exopolysaccharide (EPS) production genes (nine of which form a single genetic cluster), 13 adhesion genes, 38 milk protein and 11 milk sugar utilization genes, 13 genes for and against specific antagonistic activity, aight antibiotic resistance genes, and also three CRISPR blocks and eight Cas I-B system genes were identified in the genomes of the both strains. The expression of some genes was confirmed. In fact, the presence of identified genes suggests that L. helveticus D75 and D76 are able to form biofilms on the outer mucin layer, inhibit the growth of pathogens and pathobionts, utilize milk substrates with the formation of digestible milk sugars and bioactive peptides, resist bacteriophages and show some genome-determined resistance to antibiotics, stimulate the host’s immune system. Pathogenicity genes have not been identified. The study results confirm the safety and high probiotic potential of the strains.

Appropriate vaccine selection is crucial in the control of foot-and-mouth disease (FMD). Vaccination can prevent clinical disease and reduces viral shedding, but there is a lack of cross-protection between the seven serotypes and their sublineages, making the selection of an adequately protective vaccine difficult. Since the exact composition of their vaccines is not consistently disclosed by all manufacturers, incompatibility of the strains used for vaccination with regionally circulating strains can cause vaccination campaigns to fail. Here, we present a deep sequencing approach for polyvalent inactivated FMD vaccines that can identify all component strains by their genome sequences. The genomes of all strains of a commercial pentavalent FMD vaccine were de-novo assembled and the vaccine composition determined semi-quantitatively. The genome assembly required high stringency parameters to prevent misassemblies caused by conserved regions of the genome shared by related strains. In contrast, reference-guided assembly is only recommended in cases where the number of strains is previously known and appropriate reference sequences are available. The presented approach can be applied not only to any inactivated whole-virus FMD vaccine, but also to vaccine quality testing in general and allows for better decision-making for vaccines with unknown composition.

An outbreak of a virus-like disease has caused severe damage to noni plants (Morinda citrifolia L.) in Xishuangbanna area of China's southwestern Yunnan province since 2015. The diseased plants displayed typical mosaic symptom with light and dark green patches on leaves. Flexuous filamentous virus particles of about 800 nm in length were observed from the leaf saps by transmission electron microscope. Illumina transcriptomic sequencing further revealed the presence of a potyvirus and its near complete genome was obtained from de novo assembly. The complete genome of 9,659 nts was obtained by Sanger sequencing of eight amplicons generate by RT-PCR and 5’ and 3’ RACE. BLASTp analysis of the polyprotein sequence showed that the virus was most closely related to Tobacco vein banding mosaic virus (TVBMV), but these two viruses only shared 50.7% amino acid sequence similarity. Both phylogenetic analyses of the polyprotein and CP amino acid sequences indicated that this virus is a member of genus Potyvirus. However, the low sequence homology with all known potyviruses established this virus as a new species in the genus, tentatively named as Noni mosaic virus (NoMV). Our field surveys showed that 100% of the symptomatic samples and 28.57% of the asymptomatic samples were infected with this novel potyvirus. Aphids collected from diseased leaves were also detected carrying the virus. In summary, our data indicated that a novel species of potyvirus, NoMV, is prevalent in Yunnan, China and is associated with an emerging mosaic disease on M. citrifolia.

Evidence of familial inheritance in non-medullary thyroid cancer (NMTC) has accumulated over the last few decades. However, known variants account for a very small percentage of the genetic burden. Here, we focused on the identification of common pathways and networks enriched in NMTC families to better understand its pathogenesis with the final aim of identifying one novel high/moderate-penetrance germline predisposition variant segregating with the disease in each studied family. We performed whole genome sequencing on 23 affected and 3 unaffected family members from five NMTC-prone families and prioritized the identified variants using our Familial Cancer Variant Prioritization Pipeline (FCVPPv2). In total, 31 coding variants and 39 variants located in upstream, downstream, 5′ or 3′ untranslated regions passed FCVPPv2 filtering. Altogether, 210 genes affected by variants that passed the first three steps of the FCVPPv2 were analyzed using Ingenuity Pathway Analysis software. These genes were enriched in tumorigenic signaling pathways mediated by receptor tyrosine kinases and G-protein coupled receptors, implicating a central role of PI3K/AKT and MAPK/ERK signaling in familial NMTC. Our approach can facilitate the identification and functional validation of causal variants in each family as well as the screening and genetic counseling of other individuals at risk of developing NMTC.

Background: Advancements in the next-generation sequencing (NGS) techniques have allowed for efficient genetic variant detection at reduced costs. Methods: We describe an ad hoc NGS-based custom designed resequencing gene panel to identify genetic variants in 8 patients with dementing disorders. Results: We found variants of TREM2 and APP genes in three patients; these have been previously identified as pathogenic or likely pathogenic and, therefore, considered as “Disease Causing”. In the remaining subjects, the pathogenicity was evaluated on the in silico analysis, according to the guidelines of the American College of Medical Genetics. In one patient, the p.R205W variant was causative of the disease, thus considered as “Possibly Disease Causing”. The variants found from the other four subjects in the CSF1R, SERPINI1, GRN, and APP genes revealed discordant in silico results and, therefore, it was not possible to assign a definitive pathogenicity. Conclusions: Notwithstanding the limitations of a customized panel, we detected some rare genetic variants with a probable disease association. The future application of NGS techniques and the further replication of these experimental data will replace the so-called “gene by gene” approach with a “panel of genes” strategy, that offers promising perspectives in the diagnosis and management of neurodegenerative disorders.

Purpose. Moraxella is an ocular bacterial pathogen isolated in cases of keratitis, conjunctivitis, and endophthalmitis. Gram-negative brick-shaped diplobacilli from ocular specimens, and slow growth in culture, are early indications of Moraxella ocular infection; however, identifying Moraxella to species can be complex and inconsistent. Methods. In this study, bacteria consistent with Moraxella were identified to species using: 1) DNA sequencing coupled with vancomycin susceptibility, 2) MALDI-TOF Mass Spectrometry, and 3) Biolog ID System. Study samples consisted of 9 ATCC Moraxella controls, 82 isolates from keratitis, 21 isolates from conjunctivitis, and 4 isolates from endophthalmitis. Results. The ATCC controls were correctly identified. For keratitis, 66 (80.5%) were identified as M. nonliquefaciens, 7 (9.0%) as M. lacunata, 5 (6%) as M. osloensis, 2 (2.5%) as Acinetobacter lwoffi, 1 (1.0%) as M. bovis/nonliquefaciens, and 1 (1.0%) as M. osloensis/nonliquefaciens. For conjunctivitis, 9 (43.0%) were identified as M. osloensis, 6 (29.0%) as M. nonliquefaciens, 3 (14.3%) as Roseomonas, 2 (9.5%) as Acinetobacter (parvus, junii), and 1 (4.5%) as M. catarrhalis/M. nonliquefaciens. From endophthalmitis, 3 of 4 of the isolates were M. nonliquefaciens. Overall, M. nonliquefaciens and M. osloensis were identified in 70% (75 of 107) and 13% (14 of 107) of cases, respectively, totaling 83% (89 of 107). Conclusions. M. nonliquefaciens and M. osloensis are important bacterial pathogens of the eye as determined by DNA sequencing, MALDI-TOF MS, and Biolog. Although Moraxella catarrhalis is a clinical pathogen, other species of Moraxella appear to have a prominent role in eye infections.

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Typically, mouse-adaptation studies are conducted in inbred laboratory strains BALB/c and C57BL/6, which have defects in the antiviral Mx1 gene that results in increased susceptibility to infection and disease severity. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (a.k.a. CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L, and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering these three adaptive PB1 and PA mutations into the parental CA/07 strain, we demonstrated that adaptive mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions, and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

A novel binary toxin-positive non-027, non-078 Clostridium difficile strain designated LC693 whose sequence type was ST201 was isolated from the fecal sample of a patient with severe diarrhea in China. To understand the pathogenesis basis of C. difficile ST201, this recently recovered isolate LC693 was then chosen for whole genome sequencing. The project finally generated an estimated genome size of approximately 4.07 Mbp. The genome sequence was then analyzed together with the other two ST201 strains VL-0104 and VL-0391 and compared to the epidemic 027/ST1 and 078/ST11 strains. Phylogenetic analysis demonstrated that the ST201 strains belonged to clade 3. Genome size of the three ST201 strains ranged from 4.07 Mb~4.16 Mb, with an average GC content between 28.5%~28.9%. The ST201 genomes contained more than 40 antibiotic resistance genes and 15 of them were predicted to be associated with vancomycin-resistance, suggesting that they may have a strong antibiotic resistance. The ST201 strains contained a typical clade 3 specific PaLoc with a Tn6218 element inserted, and those genes harbored on their PaLoc that participated in the toxin expression and regulation were highly homologues to the epidemic 027 and 078 strains, with the exception of tcdC. A truncated TcdC was found in the ST201 strains, which is suggestive to have a contribution to the toxin production of the ST201 strains. In addition, the ST201 strains contained intact binary toxin coding and regulation genes, which is also proposed to contribute to the virulence. Genome comparison of the ST201 strains with the epidemic 027 and 078 strain identified 641 genes specific for the C. difficile ST201, and a number of them were predicted as fitness and virulence associated genes. The identification of those genes also contributes to the pathogenesis of the ST201 strain. To our knowledge, this is the first study that the genome sequence of C. difficile ST201 was discussed in detail, and the present study would have a contribution to understanding the pathogenesis basis of C. difficile ST201.

The classical Lotka-Volterra predator-prey model is globally stable and uniformly persistent. However, in real-life biosystems extinction of species due to stochastic effects is possible. In this paper, we consider the classical Lotka-Volterra predator-prey model under stochastic perturbations. For this model, using an analytical technique based on the direct Lyapunov method and a development of the ideas of R.Z. Khasminskii, we find the precise sufficient conditions for the stochastic extinction of one and both species and the precise necessary conditions for the stochastic system persistence. The stochastic extinction occurs via a process known as the stabilization by noise of the Khasminskii type, and, in order to establish the sufficient conditions for extinction, we found the conditions for the stabilization. The analytical results are illustrated by numerical simulations. {\bf Keywords:} stochastic perturbations, white noise, Ito's stochastic differential equation, the Lyapunov functions method, stability in probability, stabilization by noise, stochastic extinction, persistence.

To secure computers and information systems from attackers taking advantage of vulnerabilities in the system to commit cybercrime, several methods have been proposed for real-time detection of vulnerabilities to improve security around information systems. Of all the proposed methods, machine learning had been the most effective method in securing a system with capabilities ranging from early detection of software vulnerabilities to real-time detection of ongoing compromise in a system. As there are different types of cyberattacks, each of the existing state-of-the-art machine learning models depends on different algorithms for training which also impact their suitability for detection of a particular type of cyberattack. In this research, we analyzed each of the current state-of-theart machine learning models for different types of cyberattack detection from the past 10 years with a major emphasis on the most recent works for comparative study to identify the knowledge gap where work is still needed to be done with regard to detection of each category of cyberattack

Encapsulation is a valuable strategy to protect and deliver anthocyanins (ACNs), phenolic com-pounds with outstanding antioxidant capacity but limited stability. In this study, coacervation was used to encapsulate an ACNs-rich red cabbage extract (RCE). Two agri-food by-products polymers, whey protein isolate (WPI) and apple high-methoxyl pectin (HMP), were blended at pH 4.0 in a specific ratio to induce the formation of nanoparticles (NPs). The process optimisation yielded a monodispersed population (PDI < 0.200) of negatively charged (-17 mV) NPs with an average diameter of 380 nm. RCE concentration influenced size, charge, and antioxidant capacity in a dose-dependent manner. NPs were also sensitive to pH increase from 4 to 7, showing a progressive breakdown. The encapsulation efficiency was 30%, with a retention of ACNs within the polymeric matrix being influenced by their chemical structure: diacylated and/or C3-triglucoside forms were more efficiently encapsulated than monoacylated C3-diglucosides. In conclusion, we report a promising, simple, and sustainable method to produce monodispersed NPs for ACNs encapsu-lation and delivery. Evidence of differential binding of ACNs to NPs, dependent on specific ac-ylation/glycosylation patterns, indicate that care must be taken in the choice of the appropriate NPs formulation for the encapsulation of phenolic compounds.

The plethora of bio-functional compounds present in fermented alcoholic beverages like wine, as well as the valorization of bioactives from their wineries/breweries by-products like grape pomace and grape seed, has gained significant interest in the functional foods sector. Wine has always accompanied humanity, for religion or for health. For example, Christians are using wine in the “Holy Communion”, following what Jesus Christ himself did and said during the Last Supper, while the benefits of the wine consumption in moderation was embedded in all Mediterranean civilizations, as it is mentioned by the wise King-Prophet David in his psalms, but also by Hip-pocrates and other wise men, in a Greek-based philosophy of “All things in Moderation” (Cleobulus of Lindos; 6th century BC). In modern times, and after the outbreak of research on wine benefits since the 90’s, when the term “French Paradox” was introduced in the US public during a CBS show, recent evidence has outlined that these benefits are derived by the synergisms of its bio-functional compounds and their digestion-derived metabolites. Within this article, the proposed health benefits of moderate wine consumption, as a functional component of a balanced diet (i.e. the Mediterra-nean Diet), against inflammation-related chronic disorders, is thoroughly reviewed. The various bio-functional compounds of both wine and wineries’ by-products, such as their bioactive phe-nolics, unsaturated fatty acids, polar lipids, and dietary fibers, and their functional antioxidant, anti-inflammatory and antithrombotic health promoting properties are also thoroughly evaluated. The mechanisms of actions and synergism, by which the health benefits are elicited, are also ex-plored. Functional properties of non-alcoholic wine products are also introduced. Emphasis is also given to applications of wineries’ by-products bioactives, as ingredients of bio-functional foods, supplements and nutraceuticals. Limitations and future perspectives for this popular functional alcoholic beverage (wine) ant its rich in bioactives by-products are also addressed. .

In order to harmonize supply and demand of green energy, new future-proof technologies are needed. Here, hydrogen plays a key role. Within the current framework conditions, the production of green hydrogen is not yet economically viable. The use of the oxygen produced and the possible increase in efficiency associated with it mostly remains unconsidered. The aim is to demonstrate that the economic efficiency of a power-to-gas (PtG) project can be increased by using the by-product oxygen. In this research project, a water electrolyser connected to grid is powered to supply hydrogen to a hydrogen refuelling station. By utilising the by-product oxygen from water electrolysis for a wastewater treatment plant (WWTP), it is shown that the net present value (NPV) of the project can be improved by up to 13 % compared to the initial scenario. If a photovoltaic (PV) system is used in addition to grid electricity for higher green hydrogen production, the NPV can be further improved by up to 58 %. The levelized cost of hydrogen (LCOH) is calculated for different scenarios with and without oxygen configuration. A sensitivity analysis is then performed to find important parameters.

The goal of newborn screening is to improve health outcomes by identifying and treating affected newborns. This manuscript provides an overview of a data tool to facilitate the longitudinal collection of health information on newborns diagnosed with a condition through NBS. The Newborn Screening Translational Research Network (NBSTRN) developed the Longitudinal Pediatric Data Resource (LPDR) to capture, store, analyze, visualize, and share genomic and phenotypic data over the lifespan of NBS identified newborns to facilitate understanding of genetic disease, and to assess the impact of early identification and treatment. NBSTRN developed a consensus-based process using clinical care experts to create, maintain, and evolve question and answer sets organized into common data elements (CDEs). The LPDR contains 24,172 core and disease-specific CDEs for 118 rare genetic diseases, and the CDEs are being made available through the NIH CDE Repository. The number of CDEs for each condition average of 2,200 with a range from 69 to 7,944. The LPDR is used by state NBS programs, clinical researchers, and community-based organizations. Case level, de-identified data sets are available for secondary research and data mining. The development of the LPDR for longitudinal data gathering, sharing, and analysis supports research and facilitates the translation of new discoveries into clinical practice.

New insights into the causes and mechanisms of congenital heart disease (CHD) have been gained through the use of next-generation sequencing. Examination of the whole exome sequence detects detrimental gene variations modifying single or contiguous nucleotides, that are characterised as pathogenicity based on statistical assessments of families. and correlates with congenital heart disease, elevated expression during heart development, and reduction of harmful protein-coding mutations in the general population. Patients with CHD and extracardiac abnormalities enriched for gene classes meeting these criteria. CHDs and extracardiac defects, supporting a common set of pathways in the organogenesis of CHDs. Single-cell transcriptomics data reveal the expression of genes associated with CHD in specific cell lineages, and emerging evidence suggests that genetic variants disrupt multicellular genes essential for cardiogenesis. Metrics and units are being tracked in whole genome sequence studies.

In this work, Density Functional Theory (DFT) calculations were employed to study the photocatalytic CO2 to CO reduction by a series of Pt(II) square planar complexes with general formula [Pt(5-R-dpb)Cl] (dpb = 1,3-di(2-pyridyl)benzene anion, R = H, N,N-dimethylaniline,T thiophene, diazaborinine). The CO2 to CO conversion process is thought to proceed via two main steps, namely the photocatalytic/reduction step and the main catalytic step. The simulated absorption spectra exhibit strong bands in the 200 – 400 nm UV-Vis region. The calculated excited state reduction potentials are in the range 0.32 — -0.36 V revealing that the reductive quenching of the T1 state of the complexes could be modulated with suitable changes of the N^C^N pincer ligands. The CO2 fixation and activation by the ‘real’ three coordinated Pt(II) catalyst/intermediate is predicted to be favorable with Pt-CO2 bond dissociation energies, D0 in the range -36.9—-10.3 kcal/mol. The nature of the Pt-CO2 bond is complex with covalent, hyperconjugative and H-bonding interactions prevailing the repulsive electrostatic interactions. The main catalytic cycle is estimated to be a favorable exergonic process.

This paper presents a novel real time control strategy on four-wheel independent steering system for electric vehicle with stepper motors using neural networks combined with fuzzy logic. The control of the four wheels is based on a variation of Hopfield Neural Network (VHNN) method, in which the input is the error of each steering motor and the output is processed by a hyperbolic tangent function (HTF) feeding the fuzzy logic controller (FLC), which ultimately drives the stepper motor. The whole system is made up of the four aforementioned blocks where they work in sync and inseparable from each other with the common goal of driving all the steering stepper motors at the same time. Simulation results demonstrate that the proposed control method has strong robustness and practicability under different angles and speeds.