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1.
Nat Commun ; 12(1): 4132, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226556

RESUMO

Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits - Equalizers - that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.


Assuntos
Variações do Número de Cópias de DNA , Dosagem de Genes , Regulação da Expressão Gênica , Animais , Linhagem Celular , Expressão Gênica , MicroRNAs , Plasmídeos , Transfecção
2.
Nat Commun ; 12(1): 3946, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168152

RESUMO

Alpha-fetoprotein producing gastric carcinoma (AFPGC) is a rare and aggressive subtype of gastric cancer. However, little is known about the genomic features of this disease. We perform whole-exome sequencing analysis of AFPGC, and identify 34 significantly mutated genes. Somatic copy number alterations analysis reveals several significant focal amplifications (e.g. 19q12, 17q12) and focal deletions (e.g. 1p36.11, 9p21.3), and some of these negatively affect the patient prognosis. Comparative analyses reveal that AFPGC has distinct genomic features from gastric cancer of The Cancer Genome Atlas as well as four molecular subtypes. Several frequently altered genes with potential as therapeutic targets are identified in AFPGC. Further analysis reveals that AFPGC with amplification of CCNE1 at 19q12 and/or ERBB2 at 17q12 show poorer survival and more aggressive. Subsequently, based on our established patient-derived xenograft models for AFPGC, translational research is performed and the therapeutic value of targeting CCNE1 and ERBB2 is validated. In this work, we provide an understanding of genomic characteristics of AFPGC and propose a platform to explore and validate the genome-guided personalized treatment for this disease.


Assuntos
Mutação , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidade , alfa-Fetoproteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ciclina E/genética , Feminino , Dosagem de Genes , Humanos , Imidazóis/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Proteínas Oncogênicas/genética , Prognóstico , Pirimidinas/farmacologia , Receptor ErbB-2/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Sequenciamento Completo do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Nat Protoc ; 16(7): 3522-3546, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34089021

RESUMO

Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6-15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4-7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.


Assuntos
Dosagem de Genes , Imunoensaio/métodos , Proteínas/metabolismo , Análise de Célula Única , Anticorpos/metabolismo , Calibragem , Linhagem Celular Tumoral , Sobrevivência Celular , Análise de Dados , Ouro/química , Humanos , Proteínas Imobilizadas/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Coloração e Rotulagem
4.
Aging (Albany NY) ; 13(12): 16834-16858, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34166224

RESUMO

The beneficial effects of calorie restriction (CR) are numerous. However, there is no scientific evidence about how a high-calorie diet (HCD) background influences the mechanisms underlying CR on skeletal muscles in an experimental mouse model. Herein we present empirical evidence showing significant interactions between HCD (4 months) and CR (3 months). Pectoralis major and quadriceps femoris vastus medialis, in the experimental and control groups, displayed metabolic and physiologic heterogeneity and remarkable plasticity, according to the dietary interventions. HCD-CR not only altered genetic activation patterns of satellite SC markers but also boosted the expression of myogenic regulatory factors and key activators of mitochondrial biogenesis, which in turn were also associated with metabolic fiber transition. Our data prompt us to theorize that the effects of CR may vary according to the physiologic, metabolic, and genetic peculiarities of the skeletal muscle described here and that INTM/IM lipid infiltration and tissue-specific fuel-energy status (demand/supply) both hold dependent-interacting roles with other key anti-aging mechanisms triggered by CR. Systematic integration of an HCD with CR appears to bring potential benefits for skeletal muscle function and energy metabolism. However, at this stage of our research, an optimal balance between the two dietary conditions, where anti-aging effects can be accomplished, is under intensive investigation in combination with other tissues and organs at different levels of organization within the organ system.


Assuntos
Restrição Calórica , Dieta , Músculo Esquelético/patologia , Adenilato Quinase/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Animais , Biomarcadores/metabolismo , DNA Mitocondrial/genética , Modelos Animais de Doenças , Feminino , Dosagem de Genes , Regulação da Expressão Gênica , Lipídeos/química , Camundongos Endogâmicos ICR , Biogênese de Organelas , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Sirtuína 1/metabolismo , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Telomerase/metabolismo
5.
Toxicon ; 199: 68-71, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34087288

RESUMO

Paralytic shellfish toxin (PST) content in the dinoflagellate Gymnodinium catenatum changes with culture age, with a higher toxin concentration in the logarithmic phase that decreases when the culture ages. The gene copy number (GCN) of domains sxtA1 and sxtA4 was higher in the lag and stationary phase, and lag phase, respectively. No relationship was found between the GCN of the domains sxtA4 and sxtA1 with the PST content in G. catenatum.


Assuntos
Dinoflagelados , Intoxicação por Frutos do Mar , Toxinas Biológicas , Dinoflagelados/genética , Dosagem de Genes , Humanos , Frutos do Mar
6.
Nat Commun ; 12(1): 3418, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103502

RESUMO

The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.


Assuntos
Cryptococcus/efeitos dos fármacos , Farmacorresistência Fúngica , Flucitosina/farmacologia , Cromossomos Fúngicos/genética , Células Clonais , Cryptococcus/genética , Cryptococcus/crescimento & desenvolvimento , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Duplicação Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Supressores , Variação Genética , Genoma Fúngico , Espaço Intracelular/metabolismo , Testes de Sensibilidade Microbiana , Mutação/genética , Reprodutibilidade dos Testes , Uridina Difosfato Ácido Glucurônico/metabolismo
7.
Nat Commun ; 12(1): 2751, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980847

RESUMO

Sequence variants in gene regulatory regions alter gene expression and contribute to phenotypes of individual cells and the whole organism, including disease susceptibility and progression. Single-nucleotide variants in enhancers or promoters may affect gene transcription by altering transcription factor binding sites. Differential transcription factor binding in heterozygous genomic loci provides a natural source of information on such regulatory variants. We present a novel approach to call the allele-specific transcription factor binding events at single-nucleotide variants in ChIP-Seq data, taking into account the joint contribution of aneuploidy and local copy number variation, that is estimated directly from variant calls. We have conducted a meta-analysis of more than 7 thousand ChIP-Seq experiments and assembled the database of allele-specific binding events listing more than half a million entries at nearly 270 thousand single-nucleotide polymorphisms for several hundred human transcription factors and cell types. These polymorphisms are enriched for associations with phenotypes of medical relevance and often overlap eQTLs, making candidates for causality by linking variants with molecular mechanisms. Specifically, there is a special class of switching sites, where different transcription factors preferably bind alternative alleles, thus revealing allele-specific rewiring of molecular circuitry.


Assuntos
Alelos , Genoma Humano , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Bases de Dados Genéticas , Dosagem de Genes , Regulação da Expressão Gênica/genética , Estudo de Associação Genômica Ampla , Humanos , Motivos de Nucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Locos de Características Quantitativas
8.
Nat Commun ; 12(1): 2892, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001903

RESUMO

Flying insects have invaded all the aerial space on Earth and this astonishing radiation could not have been possible without a remarkable morphological diversification of their flight appendages. Here, we show that characteristic spatial expression profiles and levels of the Hox genes Antennapedia (Antp) and Ultrabithorax (Ubx) underlie the formation of two different flight organs in the fruit fly Drosophila melanogaster. We further demonstrate that flight appendage morphology is dependent on specific Hox doses. Interestingly, we find that wing morphology from evolutionary distant four-winged insect species is also associated with a differential expression of Antp and Ubx. We propose that variation in the spatial expression profile and dosage of Hox proteins is a major determinant of flight appendage diversification in Drosophila and possibly in other insect species during evolution.


Assuntos
Proteína do Homeodomínio de Antennapedia/genética , Proteínas de Drosophila/genética , Voo Animal , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Proteína do Homeodomínio de Antennapedia/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dosagem de Genes , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Asas de Animais/anatomia & histologia , Asas de Animais/metabolismo
9.
Harmful Algae ; 103: 102008, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33980448

RESUMO

Recent increase of Harmful Algal Blooms (HAB) causes world-wide ecological, economical, and health issues, and more attention is paid to frequent coastal monitoring for the early detection of HAB species to prevent or reduce such impacts. Use of molecular tools in addition to traditional microscopy-based observation has become one of the promising methodologies for coastal monitoring. However, as ribosomal RNA (rRNA) genes are commonly targeted in molecular studies, variability in the rRNA gene copy number within and between species must be considered to provide quantitative information in quantitative PCR (qPCR), digital PCR (dPCR), and metabarcoding analyses. Currently, this information is only available for a limited number of species. The present study utilized a dPCR technology to quantify copy numbers of rRNA genes per single cell in 16 phytoplankton species, the majority of which are toxin-producers, using a newly developed universal primer set accompanied by a labeled probe with a fluorophore and a double-quencher. In silico PCR using the newly developed primers allowed the detection of taxa from 8 supergroups, demonstrating universality and broad coverage of the primer set. Chelex buffer was found to be suitable for DNA extraction to obtain DNA fragments with suitable size to avoid underestimation of the copy numbers. The study successfully demonstrated the first comparison of absolute quantification of 18S rRNA copy numbers per cell from 16 phytoplankton species by the dPCR technology.


Assuntos
Variações do Número de Cópias de DNA , Proliferação Nociva de Algas , Dosagem de Genes , Genes de RNAr , Fitoplâncton/genética
10.
Biomed Res Int ; 2021: 5568845, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33981770

RESUMO

The flora compositions of nitrogen-fixing bacteria in roots of Pennisetum giganteum z.x.lin at different growth stages and the expression and copy number of nitrogen-fixing gene nifH were studied by Illumina Miseq second-generation sequencing technology and qRT-PCR. The results showed that there were more than 40,000~50,000 effective sequences in 5 samples from the roots of P. giganteum, with Proteobacteria and Cyanobacteria as the dominant nitrogen-fixing bacteria based on the OTU species annotations for each sample and Bradyrhizobium as the core bacterial genera. The relative expression and quantitative change of nifH gene in roots of P. giganteum at different growth stages were consistent with the changes in the flora compositions of nitrogen-fixing microbia. Both revealed a changing trend with an initial increase and a sequential decrease, as well as changing order as jointing stage>maturation stage>tillering stage>seedling stage>dying stage. The relative expression and copy number of nifH gene were different in different growth stages, and the difference among groups basically reached a significant level (p < 0.05). The relative expression and copy number of nifH gene at the jointing stage were the highest, and the 2-△△CT value was 4.43 folds higher than that at the seedling stage, with a copy number of 1.32 × 107/g. While at the dying stage, it was the lowest, and the 2-△△CT value was 0.67 folds, with a copy number of 0.31 × 107/g.


Assuntos
Proteínas de Bactérias , Bactérias Fixadoras de Nitrogênio , Oxirredutases , Pennisetum/microbiologia , Raízes de Plantas/microbiologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dosagem de Genes/genética , Genes Bacterianos/genética , Bactérias Fixadoras de Nitrogênio/classificação , Bactérias Fixadoras de Nitrogênio/genética , Bactérias Fixadoras de Nitrogênio/metabolismo , Oxirredutases/análise , Oxirredutases/genética , Oxirredutases/metabolismo , Microbiologia do Solo
11.
J Med Microbiol ; 70(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33999798

RESUMO

Introduction. New Delhi metallo-ß-lactamase (NDM)-producing Klebsiella pneumoniae has become a serious global health concern.Hypothesis/Gap Statement. Due to the high genetic diversity among NDM-positive K. pneumoniae, we need further surveillance and studies to better understand the relationships between them. In addition, the coexistence of several plasmid replicon types in NDM-positive K. pneumoniae may affect the copy number of bla NDM, the MIC level to antibiotics, as well as increasing the chance of horizontal gene transfer.Aim. The aim of this study was to determine incompatible plasmid groups and copy numbers of bla NDM, and to investigate the genetic relationship of 37 NDM-positive K. pneumoniae in Kerman, Iran.Methodology. The bla NDM-1 gene was detected and confirmed by PCR-sequencing. The plasmid replicon types were determined by PCR-based replicon typing (PBRT) and the copy number of bla NDM-1 was determined by quantitaive real time-PCR (qPCR). Random amplified polymorphic DNA (RAPD)-PCR typing was used to detect genetic relationships between the strains.Results. In this study, 10 different replicon types, including Frep [n=25 (67.5 %)], FIIAs [n=11 (29.7 %)], FIA [n=5 (13.5 %)], FIB [n=3 (8.1 %)], I1-Iγ [n=2 (5.4 %)], L/M [n=7 (18.9 %)], A/C [n=7 (18.9 %)], Y [n=3 (8.1 %)], P [n=1 (2.7 %)] and FIC [n=1 (2.7 %)] were reported. The copy numbers of the bla NDM-1 gene varied from 30.00 to 5.0×106 and no statistically significant correlation was observed between a rise of the MIC to imipenem and the copy numbers of bla NDM-1 (P>0.05). According to RAPD typing results, 35 strains were divided into five clusters, while two strains were non-typeable.Conclusion. The spread of NDM-1-producing K. pneumoniae strains that carry several plasmid replicon types increases the chance of horizontal transfer of antibiotic resistance genes in hospital settings. In this study, 10 different replicon types were identified. We could not find any relationship between the increase of MIC levels to imipenem and the copy numbers of bla NDM-1. Therefore, due to the identification of different replicon types in this study, the type and genetic characteristics of bla NDM-1-carrying plasmids, and other factors such as antibiotic selective pressure, probably affect the copy number of bla NDM-1 and change the MIC level to imipenem.


Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Dosagem de Genes , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Plasmídeos , beta-Lactamases/genética , Humanos , Irã (Geográfico) , Klebsiella pneumoniae/classificação , Tipagem Molecular , Técnica de Amplificação ao Acaso de DNA Polimórfico , Replicon , Resistência beta-Lactâmica
12.
J Virol Methods ; 294: 114174, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33984396

RESUMO

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste de Ácido Nucleico para COVID-19/normas , RNA Viral/genética , SARS-CoV-2/genética , Manejo de Espécimes/normas , COVID-19/diagnóstico , COVID-19/virologia , Dosagem de Genes , Genes Essenciais , Humanos , Limite de Detecção , RNA Viral/isolamento & purificação , Padrões de Referência , Ribonuclease P/genética , Manejo de Espécimes/métodos , Carga Viral
13.
eNeuro ; 8(3)2021.
Artigo em Inglês | MEDLINE | ID: mdl-33958373

RESUMO

Mutations of the gene encoding the RET tyrosine kinase causes Hirschsprung's disease (HSCR) and medullary thyroid carcinoma (MTC). Current consensus holds that HSCR and MTC are induced by inactivating and activating RET mutations, respectively. However, it remains unknown whether activating mutations in the RET gene have adverse effects on ENS development in vivo We addressed this issue by examining mice engineered to express RET51(C618F), an activating mutation identified in MTC patients. Although Ret51(C618F)/51(C618F) mice displayed hyperganglionosis of the ENS, Ret51(C618F)/- mice exhibited severe intestinal aganglionosis because of premature neuronal differentiation. Reduced levels of glial cell-derived neurotrophic factor (GDNF), a RET-activating neurotrophic factor, ameliorated the ENS phenotype of Ret51(C618F)/- mice, demonstrating that GDNF-mediated activation of RET51(C618F) is responsible for severe aganglionic phenotype. The RET51(C618F) allele showed genetic interaction with Ednrb gene, one of modifier genes for HSCR. These data reveal that proliferation and differentiation of ENS precursors are exquisitely controlled by both the activation levels and total dose of RET. Increased RET activity coupled with a decreased gene dosage can cause intestinal aganglionosis, a finding that provides novel insight into HSCR pathogenesis.


Assuntos
Doença de Hirschsprung , Proteínas Proto-Oncogênicas c-ret , Animais , Dosagem de Genes , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Doença de Hirschsprung/genética , Humanos , Camundongos , Mutação/genética , Proteínas Proto-Oncogênicas c-ret/genética
14.
Nat Genet ; 53(5): 650-662, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33972799

RESUMO

In cancer cells, enhancer hijacking mediated by chromosomal alterations and/or increased deposition of acetylated histone H3 lysine 27 (H3K27ac) can support oncogene expression. However, how the chromatin conformation of enhancer-promoter interactions is affected by these events is unclear. In the present study, by comparing chromatin structure and H3K27ac levels in normal and lymphoma B cells, we show that enhancer-promoter-interacting regions assume different conformations according to the local abundance of H3K27ac. Genetic or pharmacological depletion of H3K27ac decreases the frequency and the spreading of these interactions, altering oncogene expression. Moreover, enhancer hijacking mediated by chromosomal translocations influences the epigenetic status of the regions flanking the breakpoint, prompting the formation of distinct intrachromosomal interactions in the two homologous chromosomes. These interactions are accompanied by allele-specific gene expression changes. Overall, our work indicates that H3K27ac dynamics modulates interaction frequency between regulatory regions and can lead to allele-specific chromatin configurations to sustain oncogene expression.


Assuntos
Alelos , Cromatina/química , Loci Gênicos , Histonas/metabolismo , Conformação de Ácido Nucleico , Oncogenes , Acetilação , Pareamento de Bases/genética , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Epigênese Genética , Dosagem de Genes , Humanos , Lisina/metabolismo , Regiões Promotoras Genéticas
15.
Int J Mol Sci ; 22(6)2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808720

RESUMO

Using a murine model of chronic ischemic cardiomyopathy caused by an old myocardial infarction (MI), we have previously found that three doses of 1 × 106 c-kit positive cardiac cells (CPCs) are more effective than a single dose of 1 × 106 cells. The goal of this study was to determine whether the beneficial effects of three doses of CPCs (1 × 106 cells each) can be fully replicated by a single combined dose of 3 × 106 CPCs. Mice underwent a 60-min coronary occlusion; after 90 days of reperfusion, they received three echo-guided intraventricular infusions at 5-week intervals: (1) vehicle × 3; (2) one combined dose of CPCs (3 × 106) and vehicle × 2; or (3) three doses of CPCs (1 × 106 each). In the combined-dose group, left ventricular ejection fraction (LVEF) improved after the 1st CPC infusion, but not after the 2nd and 3rd (vehicle) infusions. In contrast, in the multiple-dose group, LVEF increased after each CPC infusion; at the final echo, LVEF averaged 35.2 ± 0.6% (p < 0.001 vs. the vehicle group, 27.3 ± 0.2%). At the end of the study, the total cumulative change in EF from pretreatment values was numerically greater in the multiple-dose group (6.6 ± 0.6%) than in the combined-dose group (4.8 ± 0.8%), although the difference was not statistically significant (p = 0.08). Hemodynamic studies showed that several parameters of LV function in the multiple-dose group were numerically greater than in the combined-dose group (p = 0.08 for the difference in LVEF). Compared with vehicle, cardiomyocyte cross-sectional area was reduced only in the multiple-dose group (-32.7%, 182.6 ± 15.1 µm2 vs. 271.5 ± 27.2 µm2, p < 0.05, in the risk region and -28.5%, 148.5 ± 12.1 µm2 vs. 207.6 ± 20.5 µm2, p < 0.05, in the noninfarcted region). LV weight/body weight ratio and LV weight/tibia length ratios were significantly reduced in both cell treated groups vs. the vehicle group, indicating the attenuation of LV hypertrophy; however, the lung weight/body weight ratio was significantly reduced only in the multiple-dose group, suggesting decreased pulmonary congestion. Taken together, these results indicate that in mice with chronic ischemic cardiomyopathy, the beneficial effects of three doses of CPCs on LV function and hypertrophy cannot be fully replicated with a single dose, notwithstanding the fact that the total number of cells delivered with one or three doses is the same. Thus, it is the multiplicity of doses, and not the total number of cells, that accounts for the superiority of the repeated-dose paradigm. This study supports the idea that the efficacy of cell therapy in heart failure can be augmented by repeated administrations.


Assuntos
Cardiomiopatias/etiologia , Dosagem de Genes , Isquemia Miocárdica/complicações , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Biomarcadores , Biópsia , Pesos e Medidas Corporais , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Cardiomiopatias/terapia , Células Cultivadas , Modelos Animais de Doenças , Ecocardiografia , Fibrose , Testes de Função Cardíaca , Hemodinâmica , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Camundongos , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Isquemia Miocárdica/etiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo
16.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809541

RESUMO

Liquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles, representing functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integrating the data on LLPS-associated proteins from dedicated databases revealed only modest agreement between them and yielded a high-confidence dataset of 89 human LLPS drivers. Analysis of the supporting evidence for our dataset uncovered a systematic and potentially concerning difference between protein concentrations used in a good fraction of the in vitro LLPS experiments, a key parameter that governs the phase behavior, and the proteomics-derived cellular abundance levels of the corresponding proteins. Closer scrutiny of the underlying experimental data enabled us to offer a sound rationale for this systematic difference, which draws on our current understanding of the cellular organization of the proteome and the LLPS process. In support of this rationale, we find that genes coding for our human LLPS drivers tend to be dosage-sensitive, suggesting that their cellular availability is tightly regulated to preserve their functional role in direct or indirect relation to condensate formation. Our analysis offers guideposts for increasing agreement between in vitro and in vivo studies, probing the roles of proteins in LLPS.


Assuntos
Dosagem de Genes , Genes , Transição de Fase , Proteínas/química , Bases de Dados Factuais , Humanos , Anotação de Sequência Molecular , Organelas , Proteoma/metabolismo
17.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810107

RESUMO

Transmembrane proteins are involved in an array of stress responses, particularly in thermo-sensation and thermo-regulation. In this study, we performed a genome-wide identification and characterization of the Transient Receptor Potential (TRP) genes in the Pacific oyster (Crassostrea gigas) and investigated their expression profiles after heat stress to identify critical TRPs potentially associated with thermal regulation. A total of 66 TRP genes were identified in the C. gigas, which showed significant gene expansion and tandem duplication. Meta-analysis of the available RNA-Seq data generated from samples after acute heat stress revealed a set of heat-inducible TRPs. Further examination of their expression profiles under chronic heat stress, and comparison between C. gigas and C. angulata, two oyster species with different tolerance levels to heat stress, led to the identification of TRPC3.6, TRPC3.7, and TRPV4.7 as important TRPs involved in thermal regulation in oysters. This work provided valuable information for future studies on the molecular mechanism of TRP mediated thermal tolerance, and identification of diagnostic biomarker for thermal stress in the oysters.


Assuntos
Crassostrea/fisiologia , Resposta ao Choque Térmico/genética , Transcriptoma , Canais de Potencial de Receptor Transitório/genética , Animais , Biologia Computacional/métodos , Crassostrea/classificação , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Invertebrados , Fenótipo , Filogenia , Estresse Fisiológico/genética , Vertebrados
18.
DNA Cell Biol ; 40(5): 643-651, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902329

RESUMO

Mitochondria play a critical role in cell function and embryo development. Recently, increasing studies have investigated whether mitochondrial DNA (mtDNA) can be used as a predictive biomarker of embryo implantation. However, the results of its effect on implantation are still controversial. To further understand the clinical application value of mtDNA content for reproductive potential, we analyzed the influence of relative mtDNA quantity on embryo quality and transfer outcomes based on the results of second-generation sequencing of preimplantation genetic testing patients in our center. Biopsied trophectoderm (TE) from aneuploid blastocysts contained much larger amounts of mtDNA than those from euploid blastocysts (p < 0.000). In an analysis of only euploid blastocysts (n = 769), female age had no effect on mtDNA content (p = 0.216). TE cells biopsied on day 5 (n = 355) contained significantly higher amounts of mtDNA compared to those biopsied on day 6 (n = 388) or day 7 (n = 26) (p < 0.000). Higher quality trophoblast was associated with lower mtDNA content (p = 0.026), but quality of inner cell mass was not correlated with quantity of mtDNA (p = 0.112). For transferred embryos, the biopsied date and mtDNA content were significantly associated with embryo implantation and live birth outcomes. Day-5 euploid blastocysts with lower quantities of mtDNA exhibited higher implantation rate and live birth rate. However, our data indicated that mtDNA content may not be considered an independent predictive marker, it may be a useful reference for the selection of day-5 transferred euploid blastocysts.


Assuntos
Blastocisto/metabolismo , DNA Mitocondrial/metabolismo , Ectoderma/metabolismo , Transferência Embrionária , Trofoblastos/metabolismo , Biópsia , Cromossomos Humanos/genética , Implantação do Embrião , Dosagem de Genes , Humanos , Nascido Vivo , Modelos Logísticos , Idade Materna , Curva ROC
19.
BMC Genomics ; 22(1): 234, 2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33823803

RESUMO

BACKGROUND: For multicellular organisms, much remains unknown about the dynamics of synonymous codon and amino acid use in highly expressed genes, including whether their use varies with expression in different tissue types and sexes. Moreover, specific codons and amino acids may have translational functions in highly transcribed genes, that largely depend on their relationships to tRNA gene copies in the genome. However, these relationships and putative functions are poorly understood, particularly in multicellular systems. RESULTS: Here, we studied codon and amino acid use in highly expressed genes from reproductive and nervous system tissues (male and female gonad, somatic reproductive system, brain and ventral nerve cord, and male accessory glands) in the cricket Gryllus bimaculatus. We report an optimal codon, defined as the codon preferentially used in highly expressed genes, for each of the 18 amino acids with synonymous codons in this organism. The optimal codons were mostly shared among tissue types and both sexes. However, the frequency of optimal codons was highest in gonadal genes. Concordant with translational selection, a majority of the optimal codons had abundant matching tRNA gene copies in the genome, but sometimes obligately required wobble tRNAs. We suggest the latter may comprise a mechanism for slowing translation of abundant transcripts, particularly for cell-cycle genes. Non-optimal codons, defined as those least commonly used in highly transcribed genes, intriguingly often had abundant tRNAs, and had elevated use in a subset of genes with specialized functions (gametic and apoptosis genes), suggesting their use promotes the translational upregulation of particular mRNAs. In terms of amino acids, we found evidence suggesting that amino acid frequency, tRNA gene copy number, and amino acid biosynthetic costs (size/complexity) had all interdependently evolved in this insect model, potentially for translational optimization. CONCLUSIONS: Collectively, the results suggest a model whereby codon use in highly expressed genes, including optimal, wobble, and non-optimal codons, and their tRNA abundances, as well as amino acid use, have been influenced by adaptation for various functional roles in translation within this cricket. The effects of expression in different tissue types and the two sexes are discussed.


Assuntos
Aminoácidos , Gryllidae , Aminoácidos/metabolismo , Animais , Códon/genética , Feminino , Dosagem de Genes , Gryllidae/genética , Gryllidae/metabolismo , Masculino , Biossíntese de Proteínas , RNA de Transferência/genética , RNA de Transferência/metabolismo
20.
J Clin Exp Hematop ; 61(2): 71-77, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33883344

RESUMO

For this study, we investigated comprehensive expression of conjoined genes (CGs) in non-Hodgkin B-cell lymphoma (B-NHL) cell line KPUM-UH1 by using paired-end RNA sequencing. Furthermore, we analyzed the expression of these transcripts in an additional 21 cell lines, 37 primary samples of various malignancies and peripheral blood mononuclear cells of four normal individuals. Seventeen CGs were detected in KPUM-UH1: CTBS-GNG5, SRP9-EPHX1, RMND5A-ANAPC, OTX1-EHBP1, ATF2-CHN1, PRKAA1-TTC33, LARP1-MRPL22, LOC105379697-BAK1, TIAM2-SCAF8, SPAG1-VPS13B, WBP1L-CNNM2, NARS2-GAB2, CTSC-RAB38, VAMP1-CD27-AS1, LRRC37A2-NSF, UBA2-WTIP and ZNF600-ZNF611. To our knowledge, 10 of these genes have not been previously reported. The various characteristics of the CGs included in- and out-of-frame fusions, chimeras involving non-coding RNA and transcript variants. A finding of note was that LARP1-MRPL2 was characterized as in-frame fusion and was recurrently expressed in B-NHL samples. In this study, variety of CGs was expressed both in malignant and normal cells, some of which might be specific to lymphoma.


Assuntos
Linfoma de Células B/genética , Fusão Oncogênica , Proteínas de Fusão Oncogênica/genética , Sequência de Bases , Linhagem Celular Tumoral , Dosagem de Genes , Humanos , Análise de Sequência de RNA , Células Tumorais Cultivadas
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