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1.
BMC Genomics ; 25(1): 141, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38311722

RESUMO

BACKGROUND: Schizochytrium limacinum holds significant value utilized in the industrial-scale synthesis of natural DHA. Nitrogen-limited treatment can effectively increase the content of fatty acids and DHA, but there is currently no research on chromatin accessibility during the process of transcript regulation. The objective of this research was to delve into the workings of fatty acid production in S. limacinum by examining the accessibility of promoters and profiling gene expressions. RESULTS: Results showed that differentially accessible chromatin regions (DARs)-associated genes were enriched in fatty acid metabolism, signal transduction mechanisms, and energy production. By identifying and annotating DARs-associated motifs, the study obtained 54 target transcription factor classes, including BPC, RAMOSA1, SPI1, MYC, and MYB families. Transcriptomics results revealed that several differentially expressed genes (DEGs), including SlFAD2, SlALDH, SlCAS1, SlNSDHL, and SlDGKI, are directly related to the biosynthesis of fatty acids, meanwhile, SlRPS6KA, SlCAMK1, SlMYB3R1, and SlMYB3R5 serve as transcription factors that could potentially influence the regulation of fatty acid production. In the integration analysis of DARs and ATAC-seq, 13 genes were identified, which were shared by both DEGs and DARs-associated genes, including SlCAKM, SlRP2, SlSHOC2, SlTN, SlSGK2, SlHMP, SlOGT, SlclpB, and SlDNAAF3. CONCLUSIONS: SlCAKM may act as a negative regulator of fatty acid and DHA synthesis, while SlSGK2 may act as a positive regulator, which requires further study in the future. These insights enhance our comprehension of the processes underlying fatty acid and DHA production in S. limacinum. They also supply a foundational theoretical framework and practical assistance for the development of strains rich in fatty acids and DHA.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Estramenópilas , Humanos , RNA-Seq , Nitrogênio/metabolismo , Ácidos Graxos/metabolismo , Cromatina/metabolismo , Ácidos Docosa-Hexaenoicos , Estramenópilas/genética , Estramenópilas/metabolismo
2.
BMC Bioinformatics ; 25(1): 54, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302873

RESUMO

BACKGROUND: Transcriptome assembly from RNA-sequencing data in species without a reliable reference genome has to be performed de novo, but studies have shown that de novo methods often have inadequate ability to reconstruct transcript isoforms. We address this issue by constructing an assembly pipeline whose main purpose is to produce a comprehensive set of transcript isoforms. RESULTS: We present the de novo transcript isoform assembler ClusTrast, which takes short read RNA-seq data as input, assembles a primary assembly, clusters a set of guiding contigs, aligns the short reads to the guiding contigs, assembles each clustered set of short reads individually, and merges the primary and clusterwise assemblies into the final assembly. We tested ClusTrast on real datasets from six eukaryotic species, and showed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novo assemblers, at a moderate reduction in precision. For recall, ClusTrast was on top in the lower end of expression levels (<15% percentile) for all tested datasets, and over the entire range for almost all datasets. Reference transcripts were often (35-69% for the six datasets) reconstructed to at least 95% of their length by ClusTrast, and more than half of reference transcripts (58-81%) were reconstructed with contigs that exhibited polymorphism, measuring on a subset of reliably predicted contigs. ClusTrast recall increased when using a union of assembled transcripts from more than one assembly tool as primary assembly. CONCLUSION: We suggest that ClusTrast can be a useful tool for studying isoforms in species without a reliable reference genome, in particular when the goal is to produce a comprehensive transcriptome set with polymorphic variants.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Transcriptoma , Análise de Sequência , RNA-Seq , Análise de Sequência de RNA , Isoformas de Proteínas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos
3.
Cell Transplant ; 33: 9636897231218382, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38314688

RESUMO

Iron dysregulation is a crucial factor in the development of neurological diseases, leading to the accumulation of reactive oxygen species (ROS) and oxidative stress, triggering inflammatory responses, and ultimately causing neurological impairment. Pachymic acid (PA) is an active ingredient extracted from the medicinal fungus Poria cocos, which has been reported with multiple pharmacological effects, including anti-inflammatory, anti-ischemia/reperfusion, and anticancer actions. In this study, we test whether PA have neuroprotection effect aganist ferrous ions induced toxicity in SH-SY5Y cells. It was found that pre-treatment with PA reduced intracellular ROS levels, increased mitochondrial membrane potential, and protected cells from apoptotic death. RNA-seq and qRT-PCR results indicated that PA can regulate the key genes IL1B, CXCL8, CCL7, and LRP1 on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, such as NF-κB signaling pathway, IL-17 signaling pathway, to prevent Fe2+-induced apoptotic cell death. Our research indicated that PA has potential therapeutic effects on the neuroprotection by regulating neuroinflammation and oxidative stress damage.


Assuntos
Neuroblastoma , Fármacos Neuroprotetores , Triterpenos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Ferro/farmacologia , Neuroproteção , RNA-Seq , Linhagem Celular Tumoral , Morte Celular , Estresse Oxidativo , Apoptose , Fármacos Neuroprotetores/farmacologia
4.
Cell Mol Life Sci ; 81(1): 99, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38386071

RESUMO

Proneural genes play a crucial role in neuronal differentiation. However, our understanding of the regulatory mechanisms governing proneural genes during neuronal differentiation remains limited. RFX4, identified as a candidate regulator of proneural genes, has been reported to be associated with the development of neuropsychiatric disorders. To uncover the regulatory relationship, we utilized a combination of multi-omics data, including ATAC-seq, ChIP-seq, Hi-C, and RNA-seq, to identify RFX4 as an upstream regulator of proneural genes. We further validated the role of RFX4 using an in vitro model of neuronal differentiation with RFX4 knock-in and a CRISPR-Cas9 knock-out system. As a result, we found that RFX4 directly interacts with the promoters of POU3F2 and NEUROD1. Transcriptomic analysis revealed a set of genes associated with neuronal development, which are highly implicated in the development of neuropsychiatric disorders, including schizophrenia. Notably, ectopic expression of RFX4 can drive human embryonic stem cells toward a neuronal fate. Our results strongly indicate that RFX4 serves as a direct upstream regulator of proneural genes, a role that is essential for normal neuronal development. Impairments in RFX4 function could potentially be related to the development of various neuropsychiatric disorders. However, understanding the precise mechanisms by which the RFX4 gene influences the onset of neuropsychiatric disorders requires further investigation through human genetic studies.


Assuntos
Perfilação da Expressão Gênica , Fator Intrínseco , Humanos , Regiões Promotoras Genéticas/genética , RNA-Seq , Fatores de Transcrição Hélice-Alça-Hélice Básicos
5.
Am J Hum Genet ; 111(2): 323-337, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38306997

RESUMO

Genome-wide association studies (GWASs) have uncovered susceptibility loci associated with psychiatric disorders such as bipolar disorder (BP) and schizophrenia (SCZ). However, most of these loci are in non-coding regions of the genome, and the causal mechanisms of the link between genetic variation and disease risk is unknown. Expression quantitative trait locus (eQTL) analysis of bulk tissue is a common approach used for deciphering underlying mechanisms, although this can obscure cell-type-specific signals and thus mask trait-relevant mechanisms. Although single-cell sequencing can be prohibitively expensive in large cohorts, computationally inferred cell-type proportions and cell-type gene expression estimates have the potential to overcome these problems and advance mechanistic studies. Using bulk RNA-seq from 1,730 samples derived from whole blood in a cohort ascertained from individuals with BP and SCZ, this study estimated cell-type proportions and their relation with disease status and medication. For each cell type, we found between 2,875 and 4,629 eGenes (genes with an associated eQTL), including 1,211 that are not found on the basis of bulk expression alone. We performed a colocalization test between cell-type eQTLs and various traits and identified hundreds of associations that occur between cell-type eQTLs and GWASs but that are not detected in bulk eQTLs. Finally, we investigated the effects of lithium use on the regulation of cell-type expression loci and found examples of genes that are differentially regulated according to lithium use. Our study suggests that applying computational methods to large bulk RNA-seq datasets of non-brain tissue can identify disease-relevant, cell-type-specific biology of psychiatric disorders and psychiatric medication.


Assuntos
Estudo de Associação Genômica Ampla , Lítio , Humanos , Estudo de Associação Genômica Ampla/métodos , RNA-Seq , Locos de Características Quantitativas/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença
6.
Integr Cancer Ther ; 23: 15347354241233258, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38369762

RESUMO

BACKGROUND: Soothing the liver (called Shu Gan Jie Yu in Chinese, SGJY) is a significant therapeutic method for breast cancer in TCM. In this study, 3 liver-soothing herbs, including Cyperus rotundus L., Citrus medica L. var. sarcodactylis Swingle and Rosa rugosa Thunb. were selected and combined to form a SGJY herbal combinatory. THE AIM OF THE STUDY: To investigate the inhibiting effect of SGJY on breast cancer in vivo and vitro, and to explore the potential mechanisms. MATERIALS AND METHODS: SGJY herbal combination was extracted using water. A breast cancer rat model was developed by chemical DMBA by gavage, then treated with SGJY for 11 weeks. The tumor tissue was preserved for RNA sequencing and analyzed by IPA software. The inhibition effects of SGJY on MCF-7 and T47D breast cancer cells were investigated by SRB assay and cell apoptosis analysis, and the protein expression levels of SNCG, ER-α, p-AKT and p-ERK were measured by western blotting. RESULTS: SGJY significantly reduced the tumor weight and volume, and the level of estradiol in serum. The results of IPA analysis reveal SGJY upregulated 7 canonical pathways and downregulated 16 canonical pathways. Estrogen receptor signaling was the key canonical pathway with 9 genes downregulated. The results of upstream regulator analysis reveal beta-estradiol was the central target; the upstream regulator network scheme showed that 86 genes could affect the expression of the beta-estradiol, including SNCG, CCL21 and MB. Additionally, SGJY was verified to significantly alter the expression of SNCG mRNA, CCL21 mRNA and MB mRNA which was consistent with the data of RNA-Seq. The inhibition effects of SGJY exhibited a dose-dependent response. The apoptosis rates of MCF7 and T47D cells were upregulated. The protein expression of SNCG, ER-α, p-AKT and p-ERK were all significantly decreased by SGJY on MCF-7 and T47D cells. CONCLUSION: The results demonstrate that SGJY may inhibit the growth of breast cancer. The mechanism might involve downregulating the level of serum estradiol, and suppressing the protein expression in the SNCG/ER-α/AKT-ERK pathway.


Assuntos
Neoplasias da Mama , Sistema de Sinalização das MAP Quinases , Humanos , Ratos , Animais , Feminino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células MCF-7 , RNA-Seq , Receptores de Estrogênio/metabolismo , Estradiol , RNA Mensageiro/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia , gama-Sinucleína/genética , gama-Sinucleína/metabolismo , gama-Sinucleína/farmacologia
7.
Commun Biol ; 7(1): 200, 2024 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-38368460

RESUMO

Common mitochondrial DNA (mtDNA) deletions are large structural variants in the mitochondrial genome that accumulate in metabolically active tissues with age and have been investigated in various diseases. We applied the Splice-Break2 pipeline (designed for high-throughput quantification of mtDNA deletions) to human RNA-Seq datasets and describe the methodological considerations for evaluating common deletions in bulk, single-cell, and spatial transcriptomics datasets. A robust evaluation of 1570 samples from 14 RNA-Seq studies showed: (i) the abundance of some common deletions detected in PCR-amplified mtDNA correlates with levels observed in RNA-Seq data; (ii) RNA-Seq library preparation method has a strong effect on deletion detection; (iii) deletions had a significant, positive correlation with age in brain and muscle; (iv) deletions were enriched in cortical grey matter, specifically in layers 3 and 5; and (v) brain regions with dopaminergic neurons (i.e., substantia nigra, ventral tegmental area, and caudate nucleus) had remarkable enrichment of common mtDNA deletions.


Assuntos
Encéfalo , Substância Negra , Humanos , RNA-Seq , Encéfalo/metabolismo , Substância Negra/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/genética
8.
Front Immunol ; 15: 1301877, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38370405

RESUMO

Visceral white spot disease (VWND) caused by Pseudomonas plecoglossicida poses a major threat to the sustainable development of large yellow croaker (Larimichthys crocea) aquaculture. Genome-wide association analysis (GWAS) and RNA-seq research indicated that LcCD82a play an important role in resistance to visceral white spot disease in L. crocea, but the molecular mechanism of LcCD82a response to P. plecoglossicida infection is still unclear. In this study, we cloned and validated the Open Reading Frame (ORF) sequence of LcCD82a and explored the expression profile of LcCD82a in various tissues of L.crocea. In addition, two different transcript variants (LcCD82a-L and LcCD82a-S) of LcCD82a were identified that exhibit alternative splicing patterns after P. plecoglossicida infection, which may be closely related to the immune regulation during pathogenetic process of VWND. In order to explore the function of LcCD82a, we purified the recombinant protein of LcCD82a-L and LcCD82a-S. The bacterial agglutination and apoptosis function analysis showed that LcCD82a may involve in extracellular bacterial recognition, agglutination, and at the same time participate in the process of antigen presentation and induction of cell apoptosis. Collectively, our studies demonstrate that LcCD82a plays a crucial role in regulating apoptosis and antimicrobial immunity.


Assuntos
Perciformes , Infecções por Pseudomonas , Animais , Estudo de Associação Genômica Ampla , RNA-Seq , Proteínas Recombinantes , Perciformes/genética
9.
Artigo em Inglês | MEDLINE | ID: mdl-38306949

RESUMO

Growth is a crucial economic trait of all aquaculture species. It is important to explore the molecular regulation on growth, which could help improve the growth rate of species. Mining the growth-related genes is the foundation for revealing its molecular regulation on growth. Presently, the molecular regulation of growth in Procambarus clarkii is not clear, and the study on exploring growth-related genes is limited. In this study, RNA-Seq was used to compare gene expression profiles of the individuals with different growth rates involved in four groups including Big Male (BM), Big Female (BF), Small male (SM), and Small Female (SF) from one P. clarkii family, and the analyses were performed in combination with sex. Meanwhile, whole-genome resequencing data was used to get growth-specific SNP (Single Nucleotide Polymorphism)/InDel (Insertion/Deletion) sites information. Totally, we identified 16,127 genes, of which 9065 were successfully annotated in the GO database. Among these, 1328 DEGs were identified in BM vs. SM, with 357 up-regulated and 971 down-regulated. Additionally, 3507 DEGs were identified in BF vs. SF, with 241 up-regulated and 3266 down-regulated. 96 DEGs were up-regulated and 820 DEGs were down-regulated in Growth-related Group. The expression levels of nine DEGs were validated by RT-qPCR to verify the analysis results of sequencing. 684,040 growth-related SNPs and 182,050 growth-related InDels were obtained after screened. These findings provide candidate growth-related genes and growth-specific SNP/InDel sites for regulation of growth traits in P. clarkii, and new insight into the molecular regulation of P. clarkii growth.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Humanos , Feminino , Masculino , Animais , Astacoidea/genética , Genoma , RNA-Seq
10.
Cell Biochem Funct ; 42(2): e3943, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38379015

RESUMO

Dapagliflozin (DAPA) are clinically effective in improving diabetic nephropathy (DN). However, whether and how chromatin accessibility changed by DN responds to DAPA treatment is unclear. Therefore, we performed ATAC-seq, RNA-seq, and weighted gene correlation network analysis to identify the chromatin accessibility, the messenger RNA (mRNA) expression, and the correlation between clinical phenotypes and mRNA expression using kidney from three mouse groups: db/m mice (Controls), db/db mice (case group), and those treated with DAPA (treatment group). RNA-Seq and ATAC-seq conjoint analysis revealed many overlapping pathways and networks suggesting that the transcriptional changes of DN and DAPA intervention largely occured dependently on chromatin remodeling. Specifically, the results showed that some key signal transduction pathways, such as immune dysfunction, glucolipid metabolism, oxidative stress and xenobiotic and endobiotic metabolism, were repeatedly enriched in the analysis of the RNA-seq data alone, as well as combined analysis with ATAC-seq data. Furthermore, we identified some candidate genes (UDP glucuronosyltransferase 1 family, Dock2, Tbc1d10c, etc.) and transcriptional regulators (KLF6 and GFI1) that might be associated with DN and DAPA restoration. These reversed genes and regulators confirmed that pathways related to immune response and metabolism pathways were critically involved in DN progression.


Assuntos
Compostos Benzidrílicos , Diabetes Mellitus , Nefropatias Diabéticas , Glucosídeos , Camundongos , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , RNA-Seq , Cromatina , RNA Mensageiro/metabolismo
11.
Gene ; 893: 147944, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38381510

RESUMO

Tannic acid (TA), a significant plant secondary metabolite, is contained in the daily food of Brandt's voles. Its adverse effect on gut function has been shown in earlier research, but the underlying molecular mechanisms remain uncertain. In this study, male Brandt's vole (13 weeks old) were divided into two groups and given 0 (control) or 1,200 (TA-treated) mg•kg-1 TA for 18 days. Then RNA sequencing was used to conduct a thorough transcriptome analysis on the duodenum, jejunum, and ileum of Brandt's voles. Results showed that TA significantly increased serum total cholesterol concentration (P < 0.05) and decreased the nutrient digestibility (P < 0.05) of Brandt's voles. Furthermore, there were 174 differentially expressed genes (DEGs) in the duodenum, 96 DEGs in the jejunum, and 88 DEGs in the ileum between the control and TA-treated groups. Enrichment analysis revealed that many genes associated with bile secretion, fat digestion and absorption, innate immune response, and tight junction such as ABCG2, ABCG8, PEAK1, and IFR2, etc. were altered after TA treatment, which were verified by quantitative real-time PCR. These findings suggested that TA can change the expression of intestinal genes, thereby, altering nutrition metabolism and immunological function, eventually hindering the growth of Brandt's voles. The results of this study provide a theoretical basis for explaining how TA affects the gut function of Brandt's voles at the molecular level.


Assuntos
Arvicolinae , Perfilação da Expressão Gênica , Polifenóis , Animais , RNA-Seq , Análise de Sequência de RNA , Arvicolinae/genética
12.
Bioinformatics ; 40(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38310330

RESUMO

MOTIVATION: The advancement of long-read RNA sequencing technologies leads to a bright future for transcriptome analysis, in which clustering long reads according to their gene family of origin is of great importance. However, existing de novo clustering algorithms require plenty of computing resources. RESULTS: We developed a new algorithm GeLuster for clustering long RNA-seq reads. Based on our tests on one simulated dataset and nine real datasets, GeLuster exhibited superior performance. On the tested Nanopore datasets it ran 2.9-17.5 times as fast as the second-fastest method with less than one-seventh of memory consumption, while achieving higher clustering accuracy. And on the PacBio data, GeLuster also had a similar performance. It sets the stage for large-scale transcriptome study in future. AVAILABILITY AND IMPLEMENTATION: GeLuster is freely available at https://github.com/yutingsdu/GeLuster.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Perfilação da Expressão Gênica/métodos , Algoritmos , RNA-Seq , Análise por Conglomerados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Análise de Sequência de DNA/métodos
13.
Sci Rep ; 14(1): 910, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-38195813

RESUMO

Protection of the Critically Endangered East Asian Pangolin species is hampered by the vulnerability of captive individuals to infection. Studies have previously shown the pangolin to have a unique pseudogenisation of many immunity genes (including IFNE, IFIH1, cGAS, STING, TLR5, and TLR11), and we suspected that these losses could account for this vulnerability. Here we used RNA-Seq data to show the effect of these gene losses on the transcriptional response to a viral skin infection in a deceased pangolin. This virus is very closely related to the one causing the current COVID-19 pandemic in the human population (SARS-CoV2), and we found the most upregulated pathway was the same one previously identified in the lungs of SARS-CoV2-infected humans. As predicted, we found that the pathways downstream of the lost genes were not upregulated. For example, the pseudogenised interferon epsilon (IFNE) is known to be particularly important in epithelial immunity, and we show that interferon-related responses were not upregulated in the infected pangolin skin. We suggest that the pangolin's innate gene pseudogenisation is indeed likely to be responsible for the animal's vulnerability to infection.


Assuntos
Pandemias , Pangolins , Animais , Humanos , RNA Viral , RNA-Seq , Espécies em Perigo de Extinção , Interferons
14.
Genet Res (Camb) ; 2024: 5539065, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38205232

RESUMO

Background: The association between acute myeloid leukemia (AML) and macrophage remains to be deeply explored. Methods: Gene expression profiles and clinical variable characteristics of AML patients were collected from TCGA, GEO, and TARGET databases. Consensus clustering was employed to construct the macrophage-related clusters. The macrophage-related index (MRI) was constructed using the LASSO and multivariate Cox analysis. The GSE71014 and TARGET datasets were utilized as external validation sets. Single-cell sequencing data for AML (GSE116256) was adopted to analyze modeled gene expression levels in cells. Results: Two macrophage-related clusters with different prognostic and immune infiltration characteristics were constructed in AML. Cluster B had a poorer prognosis, more cancer-promoting pathway enrichment, and an immunosuppressive microenvironment. Relied on the MRI, patients of different groups showed different levels of immune infiltration, different mutations, and prognoses. LGALS1 and BCL2A1 may play roles in promoting cancer in AML, while ELANE may have a significant effect on suppressing cancer. Conclusion: Macrophage-related genes (MRGs) had significant impacts on the occurrence and progression of AML. MRI may better evaluate the prognosis and immune features of AML patients.


Assuntos
Leucemia Mieloide Aguda , Macrófagos Associados a Tumor , Humanos , RNA-Seq , Análise da Expressão Gênica de Célula Única , Macrófagos , Leucemia Mieloide Aguda/genética , Microambiente Tumoral/genética
15.
Microb Cell Fact ; 23(1): 14, 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38183013

RESUMO

BACKGROUND: Escherichia coli is a cost-effective expression system for production of antibody fragments like Fabs. Various yield improvement strategies have been applied, however, Fabs remain challenging to produce. This study aimed to characterize the gene expression response of commonly used E. coli strains BL21(DE3) and HMS174(DE3) to periplasmic Fab expression using RNA sequencing (RNA-seq). Two Fabs, Fabx and FTN2, fused to a post-translational translocation signal sequence, were produced in carbon-limited fed-batch cultivations. RESULTS: Production of Fabx impeded cell growth substantially stronger than FTN2 and yields of both Fabs differed considerably. The most noticeable, common changes in Fab-producing cells suggested by our RNA-seq data concern the cell envelope. The Cpx and Psp stress responses, both connected to inner membrane integrity, were activated, presumably by recombinant protein aggregation and impairment of the Sec translocon. The data additionally suggest changes in lipopolysaccharide synthesis, adjustment of membrane permeability, and peptidoglycan maturation and remodeling. Moreover, all Fab-producing strains showed depletion of Mg2+, indicated by activation of the PhoQP two-component signal transduction system during the early stage and sulfur and phosphate starvation during the later stage of the process. Furthermore, our data revealed ribosome stalling, caused by the Fabx amino acid sequence, as a contributor to low Fabx yields. Increased Fabx yields were obtained by a site-specific amino acid exchange replacing the stalling sequence. Contrary to expectations, cell growth was not impacted by presence or removal of the stalling sequence. Considering ribosome rescue is a conserved mechanism, the substantial differences observed in gene expression between BL21(DE3) and HMS174(DE3) in response to ribosome stalling on the recombinant mRNA were surprising. CONCLUSIONS: Through characterization of the gene expression response to Fab production under industrially relevant cultivation conditions, we identified potential cell engineering targets. Thereby, we hope to enable rational approaches to improve cell fitness and Fab yields. Furthermore, we highlight ribosome stalling caused by the amino acid sequence of the recombinant protein as a possible challenge during recombinant protein production.


Assuntos
Escherichia coli , Escherichia coli/genética , RNA-Seq , Análise de Sequência de RNA , Proteínas Recombinantes , Expressão Gênica
16.
Sci Rep ; 14(1): 1187, 2024 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-38216639

RESUMO

Chagas disease affects approximately 7 million people worldwide in Latin America and is a neglected tropical disease. Twenty to thirty percent of chronically infected patients develop chronic Chagas cardiomyopathy decades after acute infection. Identifying biomarkers of Chagas disease progression is necessary to develop better therapeutic and preventive strategies. Circulating microRNAs are increasingly reliable biomarkers of disease and therapeutic targets. To identify new circulating microRNAs for Chagas disease, we performed exploratory small RNA sequencing from the plasma of patients and performed de novo miRNA prediction, identifying potential new microRNAs. The levels of the new microRNAs temporarily named miR-Contig-1519 and miR-Contig-3244 and microRNAs that are biomarkers for nonchagasic cardiomyopathies, such as miR-148a-3p and miR-224-5p, were validated by quantitative reverse transcription. We found a specific circulating microRNA signature defined by low miR-Contig-3244, miR-Contig-1519, and miR-148a-3 levels but high miR-224-5p levels for patients with chronic Chagas disease. Finally, we predicted in silico that these altered circulating microRNAs could affect the expression of target genes involved in different cellular pathways and biological processes, which we will explore in the future.


Assuntos
Doença de Chagas , MicroRNA Circulante , Cardiopatias , MicroRNAs , Humanos , RNA-Seq , MicroRNAs/metabolismo , Biomarcadores/metabolismo , Doença Crônica , Doença de Chagas/diagnóstico , Doença de Chagas/genética
17.
BMC Genomics ; 25(1): 62, 2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38225547

RESUMO

BACKGROUND: Vesicular stomatitis virus (VSV) is a typical non-segmented negative-sense RNA virus of the genus Vesiculovirus in the family Rhabdoviridae. VSV can infect a wide range of animals, including humans, with oral blister epithelial lesions. VSV is an excellent model virus with a wide range of applications as a molecular tool, a vaccine vector, and an oncolytic vector. To further understand the interaction between VSV and host cells and to provide a theoretical basis for the application prospects of VSV, we analyzed the expression of host differentially expressed genes (DEGs) during VSV infection using RNA-Seq. RESULTS: Our analyses found a total of 1015 differentially expressed mRNAs and 161 differentially expressed LncRNAs in BHK-21 cells infected with VSV for 24 h compared with controls. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment showed that the differentially expressed lncRNAs and their target genes were mainly concentrated in pathways related to apoptosis, cancer, disease, and immune system activation, including the TNF, P53, MAPK, and NF-kappaB signaling pathways. The differentially expressed lncRNA can modulate immune processes by regulating genes involved in these signaling transmissions. Ten randomly selected DEGs, namely, Il12rb2, F2, Masp2, Mcl1, FGF18, Ripk1, Fas, BMF, POLK, and JAG1, were validated using RT-qPCR. As predicted through RNA-Seq analysis, these DEGs underwent either up- or downregulation, suggesting that they may play key regulatory roles in the pathways mentioned previously. CONCLUSIONS: Our study showed that VSV infection alters the host metabolic network and activates immune-related pathways, such as MAPK and TNF. The above findings provide unique insights for further study of the mechanism of VSV-host interactions and, more importantly, provide a theoretical basis for VSV as an excellent vaccine carrier.


Assuntos
RNA Longo não Codificante , Vacinas , Animais , Humanos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Perfilação da Expressão Gênica , RNA-Seq , Transcriptoma
18.
PLoS Comput Biol ; 20(1): e1011785, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38181047

RESUMO

Single-cell RNA sequencing (scRNA-seq) is a powerful technology to investigate the transcriptional programs in stromal, immune, and disease cells, like tumor cells or neurons within the Alzheimer's Disease (AD) brain or tumor microenvironment (ME) or niche. Cell-cell communications within ME play important roles in disease progression and immunotherapy response and are novel and critical therapeutic targets. Though many tools of scRNA-seq analysis have been developed to investigate the heterogeneity and sub-populations of cells, few were designed for uncovering cell-cell communications of ME and predicting the potentially effective drugs to inhibit the communications. Moreover, the data analysis processes of discovering signaling communication networks and effective drugs using scRNA-seq data are complex and involve a set of critical analysis processes and external supportive data resources, which are difficult for researchers who have no strong computational background and training in scRNA-seq data analysis. To address these challenges, in this study, we developed a novel open-source computational tool, sc2MeNetDrug (https://fuhaililab.github.io/sc2MeNetDrug/). It was specifically designed using scRNA-seq data to identify cell types within disease MEs, uncover the dysfunctional signaling pathways within individual cell types and interactions among different cell types, and predict effective drugs that can potentially disrupt cell-cell signaling communications. sc2MeNetDrug provided a user-friendly graphical user interface to encapsulate the data analysis modules, which can facilitate the scRNA-seq data-based discovery of novel inter-cell signaling communications and novel therapeutic regimens.


Assuntos
Análise de Célula Única , Software , RNA-Seq , Análise de Sequência de RNA , Perfilação da Expressão Gênica , Transdução de Sinais/genética
19.
Genes (Basel) ; 15(1)2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38254978

RESUMO

In this study, we elucidate the contribution of repetitive DNA sequences to the establishment of social structures in honeybees (Apis mellifera). Despite recent advancements in understanding the molecular mechanisms underlying the formation of honeybee castes, primarily associated with Notch signaling, the comprehensive identification of specific genomic cis-regulatory sequences remains elusive. Our objective is to characterize the repetitive landscape within the genomes of two honeybee subspecies, namely A. m. mellifera and A. m. ligustica. An observed recent burst of repeats in A. m. mellifera highlights a notable distinction between the two subspecies. After that, we transitioned to identifying differentially expressed DNA elements that may function as cis-regulatory elements. Nevertheless, the expression of these sequences showed minimal disparity in the transcriptome during caste differentiation, a pivotal process in honeybee eusocial organization. Despite this, chromatin segmentation, facilitated by ATAC-seq, ChIP-seq, and RNA-seq data, revealed a distinct chromatin state associated with repeats. Lastly, an analysis of sequence divergence among elements indicates successive changes in repeat states, correlating with their respective time of origin. Collectively, these findings propose a potential role of repeats in acquiring novel regulatory functions.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Cromatina , Abelhas/genética , Animais , Cromatina/genética , Genômica , RNA-Seq , Transdução de Sinais
20.
BMC Bioinformatics ; 25(1): 19, 2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38216877

RESUMO

In experiments with significant perturbations to transcription, nascent RNA sequencing protocols are dependent on external spike-ins for reliable normalization. Unlike in RNA-seq, these spike-ins are not standardized and, in many cases, depend on a run-on reaction that is assumed to have constant efficiency across samples. To assess the validity of this assumption, we analyze a large number of published nascent RNA spike-ins to quantify their variability across existing normalization methods. Furthermore, we develop a new biologically-informed Bayesian model to estimate the error in spike-in based normalization estimates, which we term Virtual Spike-In (VSI). We apply this method both to published external spike-ins as well as using reads at the [Formula: see text] end of long genes, building on prior work from Mahat (Mol Cell 62(1):63-78, 2016. https://doi.org/10.1016/j.molcel.2016.02.025 ) and Vihervaara (Nat Commun 8(1):255, 2017. https://doi.org/10.1038/s41467-017-00151-0 ). We find that spike-ins in existing nascent RNA experiments are typically under sequenced, with high variability between samples. Furthermore, we show that these high variability estimates can have significant downstream effects on analysis, complicating biological interpretations of results.


Assuntos
RNA , RNA/genética , Teorema de Bayes , Análise de Sequência de RNA , RNA-Seq
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