Construction of Human Neuromuscular Disease-Related Gene Site-Specific Mutant Cell Line by Cas9 Mutation System

Objective: to construct human neuromuscular disease-related gene site-specific mutant cell line by Cas9 mutation system. Methods: according to the principle of CRISPR/Cas9 target design, the exon region of CXCR4 gene sequence was found in the National Center for Biotechnology Information (NCBI) of the United States. Two sgRNAs were designed. Lenticrisprv2 was used as the vector to construct the lenticrisprv2-sgrna recombinant plasmid, which was transformed into the sensitive stbl3 strain. The monoclonal sequencing was selected to verify and expand the culture of the plasmid, then it was transferred to 293T cells for packaging to a slow virus. The virus was collected and infected with 4T1 cells. The monoclonal cells were isolated and cultured by puromycin screening and limited dilution method. The genomic DNA of the selected monoclonal cells was extracted and the DNA fragment near the knockout site was amplified by PCR and sequenced. Results: one cell line had 6 deletion mutations, including DYSF mutation site of neuromuscular disease gene and HEK293T cell model knocked out by DYSF mutation site of neuromuscular disease gene. Conclusion: the recombinant plasmid targeting CXCR4 gene was obtained by CRISPR/Cas9 system, and the human neuromuscular disease-related gene site-specific mutant cell line was successfully constructed

An RNN Model for Generating Sentences with a Desired Word at a Desired Position

Generating sentences with a desired word is useful in many natural language processing tasks. State-of-the-art recurrent neural network (RNN)-based models mainly generate sentences in a left-to-right manner, which does not allow explicit and direct constraints on the words at arbitrary positions in a sentence. To address this issue, we propose a generative model of sentences named Coupled-RNN. We employ two RNN\u27s to generate sentences backwards and forwards respectively starting from a desired word, and inject position embeddings into the model to solve the problem of position information loss. We explore two coupling mechanisms to optimize the reconstruction loss globally. Experimental results demonstrate that Coupled-RNN can generate high quality sentences that contain a desired word at a desired position

Prodromal dementia with lewy bodies and recurrent panic attacks as the first symptom : a case report

La demencia de inicio psiquiátrico con cuerpos de Lewy (DLB) puede incluir síntomas de depresión, alucinaciones, ansiedad y apatía. Aquí, reportamos un paciente con DLB con ataques de pánico recurrentes como su primer síntoma 5 años antes de un diagnóstico de base biológica de probable DCL. Proporcionamos una descripción ampliada de la presentación clínica y curso de DCL de inicio psiquiátrico a demencia en una mujer de 83 años. Este caso ilustra el diagnóstico erróneo común de DLB y la demora en tener un diagnóstico clínico y evaluación de biomarcadores para el diagnóstico estructurado. Con una descripción detallada de la clínica. presentación de este caso, las estrategias de tratamiento empírico y las perspectivas del paciente, Nuestro objetivo es concienciar a los médicos sobre los ataques de pánico dentro de la DCL de inicio psiquiátrico. Palabras clave: demencia con cuerpos de Lewy, ataques de pánico, reporte de caso, demencia prodrómica con cuerpos de Lewy, síntomas neuropsiquiátricosQ2Q2Psychiatric-onset dementia with Lewy bodies (DLB) might include symptoms of depression, hallucinations, anxiety, and apathy. Here, we report a patient with DLB with recurrent panic attacks as her first symptom 5 years before a biological-based diagnosis of probable DLB. We provide an extended description of the clinical presentation and course from psychiatric-onset DLB to dementia in an 83-year-old woman. This case illustrates the commonmisdiagnosis of DLB and the delay of having a detailed clinical and biomarker assessment for structured diagnosis. With a detailed description of the clinical presentation of this case, the empirical treatment strategies, and the patient perspectives, we aim to make clinicians aware of panic attacks within the psychiatric-onset DLB. Keywords: dementia with Lewy bodies, panic attacks, case report, prodromal dementia with Lewy bodies, neuropsychiatric symptomshttps://orcid.org/ 0000-0001-5832-0603https://scholar.google.es/citations?hl=es&user=MrICwaMAAAAJhttps://scienti.minciencias.gov.co/cvlac/visualizador/generarCurriculoCv.do?cod_rh=0001429659Revista Nacional - Indexad

Identification and validation of a novel angiogenesis-related gene signature for predicting prognosis in gastric adenocarcinoma

BackgroundAngiogenesis is a major promotor of tumor progression and metastasis in gastric adenocarcinoma (STAD). We aimed to develop a novel lncRNA gene signature by identifying angiogenesis-related genes to better predict prognosis in STAD patients.MethodsThe expression profiles of angiogenesis-related mRNA and lncRNA genes were collected from The Cancer Genome Atlas (TCGA). Then, the “limma” package was used to identify differentially expressed genes (DEGs). The expression profiles of angiogenesis-related genes were clustered by consumusclusterplus. The Pearson correlation coefficient was further used to identify lncRNAs coexpressed with angiogenesis-related clustere genes. We used Lasso Cox regression analysis to construct the angiogenesis-related lncRNAs signature. Furthermore, the diagnostic accuracy of the prognostic risk signature were validated by the TCGA training set, internal test sets and external test set. We used multifactor Cox analysis to determine that the risk score is an independent prognostic factor different from clinical characteristics. Nomogram has been used to quantitatively determine personal risk in a clinical environment. The ssGSEA method or GSE176307 data were used to evaluate the infiltration state of immune cells or predictive ability for the benefit of immunotherapy by angiogenesis-related lncRNAs signature. Finally, the expression and function of these signature genes were explored by RT–PCR and colony formation assays.ResultsAmong angiogenesis-related genes clusters, the stable number of clusters was 2. A total of 289 DEGs were identified and 116 lncRNAs were screened to have a significant coexpression relationship with angiogenic DEGs (P value<0.001 and |R| >0.5). A six-gene signature comprising LINC01579, LINC01094, RP11.497E19.1, AC093850.2, RP11.613D13.8, and RP11.384P7.7 was constructed by Lasso Cox regression analysis. The multifactor Cox analysis and Nomogram results showed that our angiogenesis-related lncRNAs signature has good predictive ability for some different clinical factors. For immune, angiogenesis-related lncRNAs signature had the ability to efficiently predict infiltration state of 23 immune cells and immunotherapy. The qPCR analysis showed that the expression levels of the six lncRNA signature genes were all higher in gastric adenocarcinoma tissues than in adjacent tissues. The functional experiment results indicated that downregulation of the expression of these six lncRNA signature genes suppressed the proliferation of ASG and MKN45 cells.ConclusionSix angiogenesis-related genes were identified and integrated into a novel risk signature that can effectively assess prognosis and provide potential therapeutic targets for STAD patients

Metformin improves healthspan and lifespan in mice

Metformin is a drug commonly prescribed to treat patients with type 2 diabetes. Here we show that long-term treatment with metformin (0.1% w/w in diet) starting at middle age extends healthspan and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimics some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced LDL and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increases AMP-activated protein kinase activity and increases antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation. Our results indicate that these actions may contribute to the beneficial effects of metformin on healthspan and lifespan. These findings are in agreement with current epidemiological data and raise the possibility of metformin-based interventions to promote healthy aging

Opposite Polarity Monospore Genome De Novo Sequencing and Comparative Analysis Reveal the Possible Heterothallic Life Cycle of Morchella importuna

Morchella is a popular edible fungus worldwide due to its rich nutrition and unique flavor. Many research efforts were made on the domestication and cultivation of Morchella all over the world. In recent years, the cultivation of Morchella was successfully commercialized in China. However, the biology is not well understood, which restricts the further development of the morel fungus cultivation industry. In this paper, we performed de novo sequencing and assembly of the genomes of two monospores with a different mating type (M04M24 and M04M26) isolated from the commercially cultivated strain M04. Gene annotation and comparative genome analysis were performed to study differences in CAZyme (Carbohydrate-active enzyme) enzyme content, transcription factors, duplicated sequences, structure of mating type sites, and differences at the gene and functional levels between the two monospore strains of M. importuna. Results showed that the de novo assembled haploid M04M24 and M04M26 genomes were 48.98 and 51.07 Mb, respectively. A complete fine physical map of M. importuna was obtained from genome coverage and gene completeness evaluation. A total of 10,852 and 10,902 common genes and 667 and 868 endemic genes were identified from the two monospore strains, respectively. The Gene Ontology (GO) and KAAS (KEGG Automatic Annotation Serve) enrichment analyses showed that the endemic genes performed different functions. The two monospore strains had 99.22% collinearity with each other, accompanied with certain position and rearrangement events. Analysis of complete mating-type loci revealed that the two monospore M. importuna strains contained an independent mating-type structure and remained conserved in sequence and location. The phylogenetic and divergence time of M. importuna was analyzed at the whole-genome level for the first time. The bifurcation time of morel and tuber was estimated to be 201.14 million years ago (Mya); the two monospore strains with a different mating type represented the evolution of different nuclei, and the single copy homologous genes between them were also different due to a genetic differentiation distance about 0.65 Mya. Compared with truffles, M. importuna had an extension of 28 clusters of orthologous genes (COGs) and a contraction of two COGs. The two different polar nuclei with different degrees of contraction and expansion suggested that they might have undergone different evolutionary processes. The different mating-type structures, together with the functional clustering and enrichment analysis results of the endemic genes of the two different polar nuclei, imply that M. importuna might be a heterothallic fungus and the interaction between the endemic genes may be necessary for its complete life history. Studies on the genome of M. importuna facilitate a better understanding of morel biology and evolution

Chromosome-Wide Characterization of Intragenic Crossover in Shiitake Mushroom, <i>Lentinula edodes</i>

Meiotic crossover plays a critical role in generating genetic variations and is a central component of breeding. However, our understanding of crossover in mushroom-forming fungi is limited. Here, in Lentinula edodes, we characterized the chromosome-wide intragenic crossovers, by utilizing the single-nucleotide polymorphisms (SNPs) datasets of an F1 haploid progeny. A total of 884 intragenic crossovers were identified in 110 single-spore isolates, the majority of which were closer to transcript start sites. About 71.5% of the intragenic crossovers were clustered into 65 crossover hotspots. A 10 bp motif (GCTCTCGAAA) was significantly enriched in the hotspot regions. Crossover frequencies around mating-type A (MAT-A) loci were enhanced and formed a hotspot in L. edodes. Genome-wide quantitative trait loci (QTLs) mapping identified sixteen crossover-QTLs, contributing 8.5–29.1% of variations. Most of the detected crossover-QTLs were co-located with crossover hotspots. Both cis- and trans-QTLs contributed to the nonuniformity of crossover along chromosomes. On chr2, we identified a QTL hotspot that regulated local, global crossover variation and crossover hotspot in L. edodes. These findings and observations provide a comprehensive view of the crossover landscape in L. edodes, and advance our understandings of conservation and diversity of meiotic recombination in mushroom-forming fungi

Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions