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1.
PLoS One ; 16(6): e0252507, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34061896

RESUMO

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.


Assuntos
Teste para COVID-19/normas , COVID-19/diagnóstico , COVID-19/epidemiologia , Testes Diagnósticos de Rotina/normas , Indicadores e Reagentes/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/genética , COVID-19/virologia , Teste para COVID-19/métodos , Camarões/epidemiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Gana/epidemiologia , Humanos , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Indicadores e Reagentes/provisão & distribuição , Técnicas de Diagnóstico Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Biologia Sintética/métodos , Transformação Bacteriana , Reino Unido/epidemiologia
2.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064393

RESUMO

Although it is not yet universally accepted that all neurodegenerative diseases (NDs) are prion disorders, there is little disagreement that Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia (FTD), and other NDs are a consequence of protein misfolding, aggregation, and spread. This widely accepted perspective arose from the prion hypothesis, which resulted from investigations on scrapie, a common transmissible disease of sheep and goats. The prion hypothesis argued that the causative infectious agent of scrapie was a novel proteinaceous pathogen devoid of functional nucleic acids and distinct from viruses, viroids, and bacteria. At the time, it seemed impossible that an infectious agent like the one causing scrapie could replicate and exist as diverse microbiological strains without nucleic acids. However, aggregates of a misfolded host-encoded protein, designated the prion protein (PrP), were shown to be the cause of scrapie as well as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome (GSS), which are similar NDs in humans. This review discusses historical research on diseases caused by PrP misfolding, emphasizing principles of pathogenesis that were later found to be core features of other NDs. For example, the discovery that familial prion diseases can be caused by mutations in PrP was important for understanding prion replication and disease susceptibility not only for rare PrP diseases but also for far more common NDs involving other proteins. We compare diseases caused by misfolding and aggregation of APP-derived Aß peptides, tau, and α-synuclein with PrP prion disorders and argue for the classification of NDs caused by misfolding of these proteins as prion diseases. Deciphering the molecular pathogenesis of NDs as prion-mediated has provided new approaches for finding therapies for these intractable, invariably fatal disorders and has revolutionized the field.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Proteínas Priônicas/genética , Scrapie/genética , alfa-Sinucleína/genética , Proteínas tau/genética , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Síndrome de Creutzfeldt-Jakob/etiologia , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Demência Frontotemporal/etiologia , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Expressão Gênica , Doença de Gerstmann-Straussler-Scheinker/etiologia , Doença de Gerstmann-Straussler-Scheinker/genética , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Doença de Gerstmann-Straussler-Scheinker/patologia , Humanos , Camundongos , Mutação , Doença de Parkinson/etiologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Príons , Dobramento de Proteína , Scrapie/etiologia , Scrapie/metabolismo , Scrapie/patologia , Ovinos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo
3.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064452

RESUMO

Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.


Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Canais de Cátion TRPP/genética , Células A549 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Células-Tronco Mesenquimais/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Canais de Cátion TRPP/metabolismo , Transfecção , Transgenes
4.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068701

RESUMO

In addition to its canonical functions, vitamin D has been proposed to be an important mediator of the immune system. Despite ample sunshine, vitamin D deficiency is prevalent (>80%) in the Middle East, resulting in a high rate of supplementation. However, the underlying molecular mechanisms of the specific regimen prescribed and the potential factors affecting an individual's response to vitamin D supplementation are not well characterized. Our objective is to describe the changes in the blood transcriptome and explore the potential mechanisms associated with vitamin D3 supplementation in one hundred vitamin D-deficient women who were given a weekly oral dose (50,000 IU) of vitamin D3 for three months. A high-throughput targeted PCR, composed of 264 genes representing the important blood transcriptomic fingerprints of health and disease states, was performed on pre and post-supplementation blood samples to profile the molecular response to vitamin D3. We identified 54 differentially expressed genes that were strongly modulated by vitamin D3 supplementation. Network analyses showed significant changes in the immune-related pathways such as TLR4/CD14 and IFN receptors, and catabolic processes related to NF-kB, which were subsequently confirmed by gene ontology enrichment analyses. We proposed a model for vitamin D3 response based on the expression changes of molecules involved in the receptor-mediated intra-cellular signaling pathways and the ensuing predicted effects on cytokine production. Overall, vitamin D3 has a strong effect on the immune system, G-coupled protein receptor signaling, and the ubiquitin system. We highlighted the major molecular changes and biological processes induced by vitamin D3, which will help to further investigate the effectiveness of vitamin D3 supplementation among individuals in the Middle East as well as other regions.


Assuntos
Colecalciferol/genética , Imunomodulação/imunologia , Receptores de Lipopolissacarídeos/genética , Receptor 4 Toll-Like/genética , Vitamina D/genética , Adolescente , Adulto , Colecalciferol/administração & dosagem , Colecalciferol/imunologia , Suplementos Nutricionais , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Terapia Nutricional , Vitamina D/imunologia , Deficiência de Vitamina D/dietoterapia , Deficiência de Vitamina D/genética , Deficiência de Vitamina D/imunologia , Deficiência de Vitamina D/patologia , Adulto Jovem
5.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071702

RESUMO

Cisplatin is a chemotherapy drug that kills cancer cells by damaging their DNA. In human cells, this damage is repaired primarily by nucleotide excision repair. While cisplatin is generally effective, many cancers exhibit initial or acquired resistance to it. Here, we studied cisplatin resistance in a defined cell line system. We conducted a comprehensive genomic characterization of the cisplatin-sensitive A2780 ovarian cancer cell line compared to A2780cis, its resistant derivative. The resistant cells acquired less damage, but had similar repair kinetics. Genome-wide mapping of nucleotide excision repair showed a shift in the resistant cells from global genome towards transcription-coupled repair. By mapping gene expression changes following cisplatin treatment, we identified 56 upregulated genes that have higher basal expression in the resistant cell line, suggesting they are primed for a cisplatin response. More than half of these genes are novel to cisplatin- or damage-response. Six out of seven primed genes tested were upregulated in response to cisplatin in additional cell lines, making them attractive candidates for future investigation. These novel candidates for cisplatin resistance could prove to be important prognostic markers or targets for tailored combined therapy in the future.


Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Genoma/efeitos dos fármacos , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo
6.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071707

RESUMO

Cardiac connexins (Cxs) are proteins responsible for proper heart function. They form gap junctions that mediate electrical and chemical signalling throughout the cardiac system, and thus enable a synchronized contraction. Connexins can also individually participate in many signal transduction pathways, interacting with intracellular proteins at various cellular compartments. Altered connexin expression and localization have been described in diseased myocardium and the aim of this study is to assess the involvement of Cx43, Cx26, and some related molecules in ponatinib-induced cardiac toxicity. Ponatinib is a new multi-tyrosine kinase inhibitor that has been successfully used against human malignancies, but its cardiotoxicity remains worrisome. Therefore, understanding its signaling mechanism is important to adopt potential anti cardiac damage strategies. Our experiments were performed on hearts from male and female mice treated with ponatinib and with ponatinib plus siRNA-Notch1 by using immunofluorescence, Western blotting, and proteomic analyses. The altered cardiac function and the change in Cxs expression observed in mice after ponatinib treatment, were results dependent on the Notch1 pathway and sex. Females showed a lower susceptibility to ponatinib than males. The downmodulation of cardiac Cx43, Cx26 and miR-122, high pS368-Cx43 phosphorylation, cell viability and survival activation could represent some of the female adaptative/compensatory reactions to ponatinib cardiotoxicity.


Assuntos
Cardiomiopatias , Conexina 26 , Conexina 43 , Imidazóis , Piridazinas , Fatores Sexuais , Anormalidades Induzidas por Medicamentos , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Cardiomiopatias/etiologia , Cardiomiopatias/fisiopatologia , Cardiotoxicidade , Conexina 26/efeitos dos fármacos , Conexina 26/metabolismo , Conexina 43/efeitos dos fármacos , Conexina 43/metabolismo , Modelos Animais de Doenças , Feminino , Junções Comunicantes/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Imidazóis/efeitos adversos , Imidazóis/farmacologia , Masculino , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteômica , Piridazinas/efeitos adversos , Piridazinas/farmacologia , Receptor Notch1/metabolismo , Transdução de Sinais
7.
J Int Med Res ; 49(6): 3000605211019663, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34101510

RESUMO

OBJECTIVE: This study aimed to investigate whether differential expression of the retinoic acid receptor-related orphan receptor A (RORA) gene is related to occurrence of acute myocardial infarction (AMI). METHODS: This was a retrospective study. White blood cells of 93 patients with acute myocardial infarction and 74 patients with stable coronary artery disease were collected. Reverse transcription quantitative polymerase chain reaction and western blotting were used to measure RORA mRNA and protein expression, respectively. RESULTS: RORA mRNA expression levels in peripheral blood leukocytes in patients with AMI were 1.57 times higher than those in patients with stable coronary artery disease. Protein RORA levels in peripheral blood of patients with AMI were increased. Binary logistic regression analysis showed that high expression of RORA was an independent risk factor for AMI, and it increased the risk of AMI by 2.990 times. CONCLUSION: RORA expression levels in patients with AMI is significantly higher than that in patients with stable coronary artery disease. High expression of RORA is related to AMI and it may be an independent risk factor for AMI.


Assuntos
Infarto do Miocárdio , Receptores do Ácido Retinoico , Expressão Gênica , Humanos , Leucócitos , Infarto do Miocárdio/genética , Estudos Retrospectivos
8.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070066

RESUMO

Megalin has been proposed as an endocytic receptor for aminoglycosides as well as estrogen and androgen. We aimed to investigate the otoprotective effects of antiandrogens (flutamide, FM) on kanamycin (KM)-induced hearing loss in rats. Rats were divided into four groups. The KM group was administered KM (20 mg/kg/day) for 5 days, while the FM group received FM (15 mg/kg/day) for 10 days. In the KM + FM group, KM and FM (15 mg/kg/day) were simultaneously injected for 5 days and then FM was injected for 5 days. Auditory brainstem responses were measured. Western blotting and/or quantitative reverse transcriptase-polymerase chain reaction were performed for megalin, cytochrome P450 1A1 (Cyp1a1), Cyp1b1, metallothionein 1A (MT1A), MT2A, tumor necrosis factor (TNF)-α, caspase 3, and cleaved caspase 3. The FM + KM group showed attenuated auditory thresholds when compared with the KM group at 4, 8, 16, and 32 kHz (all p < 0.05). The KM + FM group showed lower megalin and Cyp1b1 levels than the KM group (all p < 0.05). The KM + FM group revealed lower MT1A, TNFα, and caspase 3 protein levels, compared with those in the KM group (all p < 0.05). Androgen receptor inhibition protects against cochlear injuries in KM-induced hearing loss rats by attenuating megalin expression, revealing anti-inflammatory and anti-apoptotic effects.


Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Perda Auditiva Neurossensorial/prevenção & controle , Animais , Antibacterianos/toxicidade , Limiar Auditivo/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/patologia , Cóclea/fisiopatologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Flutamida/farmacologia , Expressão Gênica/efeitos dos fármacos , Perda Auditiva Neurossensorial/induzido quimicamente , Perda Auditiva Neurossensorial/fisiopatologia , Canamicina/toxicidade , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Metalotioneína/genética , Metalotioneína/metabolismo , Substâncias Protetoras/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
9.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070219

RESUMO

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase (TERT) and ß-Klotho (KLB) in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of ß-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). TERT plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts' development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced KLB and FGFR1 expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances KLB expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase KLB expression and activate the FGFR1/ß-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.


Assuntos
Blastocisto/metabolismo , Bovinos/embriologia , Bovinos/genética , Proteínas de Membrana/genética , Prenhez/genética , Telomerase/genética , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Bovinos/fisiologia , Células Cultivadas , Fase de Clivagem do Zigoto/metabolismo , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Expressão Gênica , Células da Granulosa/metabolismo , Proteínas de Membrana/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Gravidez , Prenhez/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
10.
BMC Genomics ; 22(1): 423, 2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103008

RESUMO

BACKGROUND: The study of protein complexes and protein functional modules has become an important method to further understand the mechanism and organization of life activities. The clustering algorithms used to analyze the information contained in protein-protein interaction network are effective ways to explore the characteristics of protein functional modules. RESULTS: This paper conducts an intensive study on the problems of low recognition efficiency and noise in the overlapping structure of protein functional modules, based on topological characteristics of PPI network. Developing a protein function module recognition method ECTG based on Topological Features and Gene expression data for Protein Complex Identification. CONCLUSIONS: The algorithm can effectively remove the noise data reflected by calculating the topological structure characteristic values in the PPI network through the similarity of gene expression patterns, and also properly use the information hidden in the gene expression data. The experimental results show that the ECTG algorithm can detect protein functional modules better.


Assuntos
Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Algoritmos , Análise por Conglomerados , Expressão Gênica , Proteínas/genética , Proteínas/metabolismo
11.
BMC Res Notes ; 14(1): 207, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34103088

RESUMO

OBJECTIVE: Many C. elegans aging studies use the compound 5-fluro-2'-deoxyuridine (FUdR) to produce a synchronous population of worms. However, the effects of FUdR on the bacterial gene expression of OP50 E. coli, the primary laboratory C. elegans food source, is not fully understood. This is particularly relevant as studies suggest that intestinal microbes can affect C. elegans physiology. Therefore, it is imperative that we understand how exposure to FUdR can affect gene expression changes in OP50 E. coli. RESULTS: An RNAseq dataset comprised of expression patterns of 2900 E. coli genes in the strain OP50, which were seeded on either nematode growth media (NGM) plates or on FUdR (50 µM) supplemented NGM plates, was analyzed. Analysis showed differential gene expression in genes involved in general transport, amino acid biosynthesis, transcription, iron transport, and antibiotic resistance. We specifically highlight metabolic enzymes in the L-histidine biosynthesis pathway as differentially expressed between NGM and FUdR exposed OP50. We conclude that OP50 exposed to FUdR results in differential expression of many genes, including those in amino acid biosynthetic pathways.


Assuntos
Caenorhabditis elegans , Floxuridina , Animais , Caenorhabditis elegans/genética , Dieta , Escherichia coli/genética , Expressão Gênica
12.
BMC Genomics ; 22(1): 410, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34082698

RESUMO

BACKGROUND: There are two genetically distinct subspecies of cattle, Bos taurus taurus and Bos taurus indicus, which arose from independent domestication events. The two types of cattle show substantial phenotypic differences, some of which emerge during fetal development and are reflected in birth outcomes, including birth weight. We explored gene expression profiles in the placenta and four fetal tissues at mid-gestation from one taurine (Bos taurus taurus; Angus) and one indicine (Bos taurus indicus; Brahman) breed and their reciprocal crosses. RESULTS: In total 120 samples were analysed from a pure taurine breed, an indicine breed and their reciprocal cross fetuses, which identified 6456 differentially expressed genes (DEGs) between the two pure breeds in at least one fetal tissue of which 110 genes were differentially expressed in all five tissues examined. DEGs shared across tissues were enriched for pathways related to immune and stress response functions. Only the liver had a substantial number of DEGs when reciprocal crossed were compared among which 310 DEGs were found to be in common with DEGs identified between purebred livers; these DEGs were significantly enriched for metabolic process GO terms. Analysis of DEGs across purebred and crossbred tissues suggested an additive expression pattern for most genes, where both paternal and maternal alleles contributed to variation in gene expression levels. However, expression of 5% of DEGs in each tissue was consistent with parent of origin effects, with both paternal and maternal dominance effects identified. CONCLUSIONS: These data identify candidate genes potentially driving the tissue-specific differences between these taurine and indicine breeds and provide a biological insight into parental genome effects underlying phenotypic differences in bovine fetal development.


Assuntos
Domesticação , Alelos , Animais , Bovinos/genética , Feminino , Expressão Gênica , Gravidez
13.
BMC Res Notes ; 14(1): 222, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34082815

RESUMO

OBJECTIVE: The impact of psychosocial stress on a variety of negative health outcomes is well documented, with current research efforts directed at possible mechanisms. Here, we focused on a potential mechanism involving differential expression of mRNA and microRNA in response to acute psychosocial stress. We utilized a validated behavioral paradigm, the Trier Social Stress Test (TSST), to induce acute psychosocial stress in a cohort of volunteers. Stress reactivity was assessed repeatedly during the TSST using saliva samples that were analyzed for levels of cortisol. Peripheral blood mononuclear cells were extracted from blood drawn at baseline and at two time points following the stress paradigm. Total RNA was extracted, and mRNA and microRNA microarrays were utilized to assess within-subject changes in gene expression between baseline and the two post-stressor time points. RESULTS: For microarray gene expression analysis, we focused on 12 participants who showed a robust cortisol response to the task, as an indicator of robust HPA-axis activation. We discovered a set of mRNAs and miRNAs that exhibited dynamic expression change in response to the TSST in peripheral blood mononuclear cells, further characterizing the link between psychosocial stress and cellular response mechanisms.


Assuntos
MicroRNAs , Sistema Hipófise-Suprarrenal , Expressão Gênica , Humanos , Hidrocortisona , Sistema Hipotálamo-Hipofisário , Leucócitos Mononucleares , MicroRNAs/genética , Projetos Piloto , RNA Mensageiro/genética , Saliva , Estresse Psicológico/genética
14.
BMC Genomics ; 22(1): 396, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34044772

RESUMO

BACKGROUND: Transmission of pathogens by vector mosquitoes is intrinsically linked with mosquito's reproductive strategy because anautogenous mosquitoes require vertebrate blood to develop a batch of eggs. Each cycle of egg maturation is tightly linked with the intake of a fresh blood meal for most species. Mosquitoes that acquire pathogens during the first blood feeding can transmit the pathogens to susceptible hosts during subsequent blood feeding and also vertically to the next generation via infected eggs. Large-scale gene-expression changes occur following each blood meal in various tissues, including ovaries. Here we analyzed mosquito ovary transcriptome following a blood meal at three different time points to investigate blood-meal induced changes in gene expression in mosquito ovaries. RESULTS: We collected ovaries from Aedes aegypti that received a sugar meal or a blood meal on days 3, 10 and 20 post blood meal for transcriptome analysis. Over 4000 genes responded differentially following ingestion of a blood meal on day 3, and 660 and 780 genes on days 10 and 20, respectively. Proteins encoded by differentially expressed genes (DEGs) on day 3 include odorant binding proteins (OBPs), defense-specific proteins, and cytochrome P450 detoxification enzymes. In addition, we identified 580 long non-coding RNAs that are differentially expressed at three time points. Gene ontology analysis indicated that genes involved in peptidase activity, oxidoreductase activity, extracellular space, and hydrolase activity, among others were enriched on day 3. Although most of the DEGs returned to the nonsignificant level compared to the sugar-fed mosquito ovaries following oviposition on days 10 and 20, there remained differences in the gene expression pattern in sugar-fed and blood-fed mosquitoes. CONCLUSIONS: Enrichment of OBPs following blood meal ingestion suggests that these genes may have other functions besides being part of the olfactory system. The enrichment of immune-specific genes and cytochrome P450 genes indicates that ovaries become well prepared to protect their germ line from any pathogens that may accompany the blood meal or from environmental contamination during oviposition, and to deal with the detrimental effects of toxic metabolites.


Assuntos
Aedes , Aedes/genética , Animais , Feminino , Expressão Gênica , Mosquitos Vetores/genética , Ovário , Oviposição
15.
Nat Commun ; 12(1): 2702, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976201

RESUMO

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Iniciação da Transcrição Genética , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elongação da Transcrição Genética
16.
Nat Commun ; 12(1): 3074, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031373

RESUMO

Single-cell RNA sequencing combined with spatial information on landmark genes enables reconstruction of spatially-resolved tissue cell atlases. However, such approaches are challenging for rare cell types, since their mRNA contents are diluted in the spatial transcriptomics bulk measurements used for landmark gene detection. In the small intestine, enterocytes, the most common cell type, exhibit zonated expression programs along the crypt-villus axis, but zonation patterns of rare cell types such as goblet and tuft cells remain uncharacterized. Here, we present ClumpSeq, an approach for sequencing small clumps of attached cells. By inferring the crypt-villus location of each clump from enterocyte landmark genes, we establish spatial atlases for all epithelial cell types in the small intestine. We identify elevated expression of immune-modulatory genes in villus tip goblet and tuft cells and heterogeneous migration patterns of enteroendocrine cells. ClumpSeq can be applied for reconstructing spatial atlases of rare cell types in other tissues and tumors.


Assuntos
Transporte Biológico/genética , Transporte Biológico/fisiologia , Biologia Computacional/métodos , Intestinos/fisiologia , Animais , Diferenciação Celular , Enterócitos/metabolismo , Células Enteroendócrinas/metabolismo , Células Epiteliais/metabolismo , Epitélio , Expressão Gênica , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
17.
Plant Physiol Biochem ; 164: 21-26, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33940390

RESUMO

Sweetpotato (Ipomoea batatas [L.] Lam.) stem tips, which contain high concentrations of chlorogenic acid (CGA), are useful as a physiologically functional food to protect against some serious diseases. According to previous studies, exogenous application of phytohormones may be an effective agrotechnical measure to control CGA biosynthesis through the transcriptional regulation of pathway gene expressions. To understand the mechanism of CGA biosynthesis in sweetpotato, we investigated the effects of exogenous phytohormones on CGA metabolism in stem tips of sweetpotato. A significantly elevated CGA content was observed in salicylic acid (SA)-treated sweetpotato stem tips at 72 h, as well as in those subjected to abscisic acid (ABA) or gibberellic acid (GA) treatments. Dynamic expression change of seven enzyme genes involved in sweetpotato CGA biosynthesis were analyzed to determine correlations between transcript levels and CGA accumulation. As revealed by the differential expression of these genes under distinct phytohormone treatments, the regulation of specific pathway genes is a critical determinant of the accumulation of CGA in sweetpotato stem tips. We also found that several hormone-responsive sites, such as those for ABA, GA, SA, and jasmonic acid (JA), were present in the promoter regions of sweetpotato CGA biosynthestic pathway genes. Collectively, phytohormones can regulate the transcription of CGA synthesis-related genes and ultimately affect CGA accumulation in sweetpotato stem tips, whereas the regulatory differences are mirrored by cis-acting elements in the corresponding pathway gene promoters.


Assuntos
Ipomoea batatas , Ácido Clorogênico/farmacologia , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ipomoea batatas/genética , Reguladores de Crescimento de Plantas/farmacologia
18.
Nat Commun ; 12(1): 2923, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011956

RESUMO

Idiopathic pulmonary fibrosis (IPF) causes progressive fibrosis and worsening pulmonary function. Prognosis is poor and no effective therapies exist. We show that programmed cell death 5 (PDCD5) expression is increased in the lungs of patients with IPF and in mouse models of lung fibrosis. Lung fibrosis is significantly diminished by club cell-specific deletion of Pdcd5 gene. PDCD5 mediates ß-catenin/Smad3 complex formation, promoting TGF-ß-induced transcriptional activation of matricellular genes. Club cell Pdcd5 knockdown reduces matricellular protein secretion, inhibiting fibroblast proliferation and collagen synthesis. Here, we demonstrate the club cell-specific role of PDCD5 as a mediator of lung fibrosis and potential therapeutic target for IPF.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Proteínas de Neoplasias/genética , Idoso , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/metabolismo , Bronquíolos/metabolismo , Bronquíolos/patologia , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
J Med Microbiol ; 70(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34038343

RESUMO

Introduction. In vitro experimentation is intentionally contrived to isolate specific phenomena in the context of profound biological complexity. Mycoplasmas in the upper airway likely contribute to this complexity and play a largely unknown role in both health and disease. Similarly, the presence and role of mycoplasma in in vitro investigation are largely unknown.Hypothesis. We hypothesize mycoplasma in human vocal fold fibroblasts (VFF) will affect both basal gene-expression patterns as well as the cell response to exogenous stimuli.Aim. We sought to determine mycoplasma presence across vocal fold fibroblast cultures, basal transcriptional changes as a function of mycoplasma, and responsiveness to exogenous glucocorticoids in mycoplasma-positive and -negative VFF.Methodology. PCR-based mycoplasma detection was performed in an immortalized human VFF line as well as rat and rabbit primary VFF cultures and extracted rat laryngeal tissue. RNA sequencing was performed in mycoplasma-positive and -negative human cells at baseline and in response to dexamethasone.Results. Mycoplasma was identified in the human cell line as well as primary culture from rabbits. Mycoplasma was not detected in tissue or primary culture from rat vocal folds. Basal mRNA expression in human VFF differed significantly following mycoplasma treatment. In addition, differential responses to dexamethasone were observed across multiple pathways as a function of mycoplasma presence in these cells. Pathways including apoptosis, DNA damage repair, and G1 to S cell cycle signalling were significantly enriched in mycoplasma-positive cells.Conclusion. Variability of mycoplasma presence across culture conditions and differential responses to exogenous stimuli as a function of mycoplasma presence are potentially problematic for the translation of in vitro experimentation in the upper aerodigestive tract. It remains unclear if these findings represent contamination or the baseline state of this specialized tissue.


Assuntos
Fibroblastos , Glucocorticoides/farmacologia , Infecções por Mycoplasma , Prega Vocal , Animais , Apoptose , Ciclo Celular , Células Cultivadas , Reparo do DNA , Fibroblastos/efeitos dos fármacos , Fibroblastos/microbiologia , Expressão Gênica , Humanos , Mycoplasma , Infecções por Mycoplasma/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , Prega Vocal/metabolismo , Prega Vocal/patologia
20.
Adv Exp Med Biol ; 1269: 387-391, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33966247

RESUMO

The gene expression of Prox-1 and Hif-1a for the isolated primo vessels (PVs) and composite lymphatic vessels (LVs) containing PVs (LVs + PVs) was investigated by RNA-sequencing (Seq) and quantitative polymerase chain reaction (qRT-PCR) analysis. RNA-Seq on the passed 10 samples on RNA-QC for two experimental groups with PVs and PVs + LVs proceeded to the library construction stage automatically and analyzed differentially expressed genes (DEGs). From the real-time qRT-PCR analysis data, we found the marker genes of Prox-1 and Hif-1a were enriched and decreased in an isolated PVs compared to LVs, respectively. Based on mRNA transcriptional data, Prox-1 and Hif-1a were increased and decreased in PVs compared to LVs + PVs under lipopolysaccharide (LPS) treatment and relieved by acupuncture electric stimulation (AES), respectively. This finding indicates that high and low levels of Prox-1 and Hif-1a may be involved in the function of PVs and that pathophysiological and physiological condition could progress into inflamed lymphatic endothelial cells expanding the PV within the LV.


Assuntos
Células Endoteliais , Vasos Linfáticos , Animais , Expressão Gênica , Proteínas de Homeodomínio , Subunidade alfa do Fator 1 Induzível por Hipóxia , Lipopolissacarídeos , Coelhos , Análise de Sequência de RNA
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