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This article draws a perspective on the increasingly unavoidable question of whether steps can be taken in genomics and biology at large to move them more rapidly towards more analytical and deductive biology, akin to similar developments that occurred in other natural sciences, such as physics and chemistry, centuries ago. It provides a summary of recent advances in other relevant sciences in the last 3 decades that are likely to pull it in that direction in the next decade or so, as well as what methods and tools will make it possible.
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PURPOSE: Kyphosis involves spines curving excessively backward beyond their physiological curvature. Although the normal structure of the spinal vertebrae is extremely important for maintaining posture and the normal function of the thoracic and abdominal organs, our knowledge concerning the pathogenesis of the disease is insufficient. We herein report that the downregulation of the calcium signaling pathway is involved in the pathogenesis of congenital kyphosis. METHODS: The third to fifth lumbar spine segments, the kyphotic region of Ishibashi (IS) rats, which are used as a model of congenital kyphoscoliosis, were collected. A DNA microarray, quantitative PCR, Western blotting, and immunohistochemistry were used to measure the expression of genes and proteins related to intracellular calcium signaling. RESULTS: We found that the expression of calcium-sensing receptor (CaSR) and transient receptor potential vanilloid 1 (Trpv1)-two receptors involved in the calcium signaling-was decreased in the lumbar spine of IS rats. We also observed that the number of CaSR-immunoreactive and Trpv1-immunoreactive cells in the lumbar spine of IS rats was lower than in wild-type rats. Furthermore, the expression of intracellular molecules downstream of these receptors, such as phosphorylated protein kinase C, c-Jun N-terminal kinase, and neural EGFL-like 1, was also reduced. In fact, the calcium content in the lumbar spine of IS rats was significantly lower than that in wild-type rats. CONCLUSION: These results indicate that adequate calcium signaling is extremely important for the regulation of normal bone formation and may also be a key factor for understanding the pathogenesis of congenital kyphosis.
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Cifose , Escoliose , Ratos , Animais , Cálcio , Cifose/patologia , Vértebras Lombares/patologia , Escoliose/genética , Postura/fisiologia , Vértebras Torácicas/patologiaRESUMO
Spinal kyphosis involves the vertebrae curving excessively backward, beyond their physiological curvature. Although the normal structure of the spinal vertebrae is extremely important for maintaining posture, the normal function of the thoracic and abdominal organs, and cosmetics, our knowledge concerning the pathogenesis of this disease is lacking. Furthermore, the responsible gene has not yet been identified. In this short review, we summarize the current state of kyphosis research and introduce the molecular and cellular mechanisms associated with the pathogenesis of this disease, based on findings obtained using rats that develop kyphosis.
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Cifose , Animais , Cifose/genética , Cifose/patologia , Postura/fisiologia , Ratos , Coluna Vertebral/patologiaRESUMO
Whole-genome transcriptional analyses performed on microorganisms are traditionally based on a small number of samples. To map transient expression variations, and thoroughly characterize gene expression throughout the growth curve of the widely used model organism Lactococcus lactis MG1363, gene expression data were collected with unprecedented time resolution. The resulting gene expression patterns were globally analyzed in several different ways to demonstrate the richness of the data and the ease with which novel phenomena can be discovered. When the culture moves from one growth phase to another, gene expression patterns change to such an extent that we suggest that those patterns can be used to unequivocally distinguish growth phases from each other. Also, within the classically defined growth phases, subgrowth phases were distinguishable with a distinct expression signature. Apart from the global expression pattern shifts seen throughout the growth curve, several cases of short-lived transient gene expression patterns were clearly observed. These could help explain the gene expression variations frequently observed in biological replicates. A method was devised to estimate a measure of unnormalized/absolute gene expression levels and used to determine how global transcription patterns are influenced by nutrient starvation or acidification of the medium. Notably, we inferred that L. lactis MG1363 produces proteins with on average lower pIs and lower molecular weights as the medium acidifies and nutrients get scarcer. IMPORTANCE This data set is a rich resource for microbiologists interested in common mechanisms of gene expression, regulation and in particular the physiology of L. lactis. Thus, similar to the common use of genome sequence data by the scientific community, the data set constitutes an extensive data repository for mining and an opportunity for bioinformaticians to develop novel tools for in-depth analysis.
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Lactococcus lactis , Adaptação Fisiológica , Concentração de Íons de Hidrogênio , Lactococcus lactis/metabolismo , Nutrientes , TranscriptomaRESUMO
BACKGROUND: The benefits of postoperative chemotherapy in patients with estrogen receptor (ER)-positive breast cancer remain unclear. The use of tumor grade, Ki-67, or ER expression failed to provide an accurate prognosis of the risk of relapse after surgery in patients. This study aimed to evaluate whether a multigene assay Curebest™ 95GC Breast (95GC) can identify the risk of recurrence and provide more insights into the requirements for chemotherapy in patients. METHODS: This single-arm retrospective multicenter joint study included patients with ER-positive, node-negative breast cancer who were treated at five facilities in Japan and had received endocrine therapy alone as adjuvant therapy. The primary lesion specimens obtained during surgery were analyzed using the 95GC breast cancer multigene assay. Based on the 95GC results, patients were classified into low-risk (95GC-L) and high-risk (95GC-H) groups. RESULTS: The 10-year relapse-free survival rates were 88.4 and 59.6% for the 95GC-L and 95GC-H groups, respectively. Histologic grade, Ki-67, and PAM50 exhibited a significant relationship with the 95GC results. The segregation into 95GC-L and 95GC-H groups within established clinical factors can identify subgroups of patients using histologic grade or PAM50 classification with good prognosis without receiving chemotherapy. CONCLUSIONS: Based on the results of our retrospective study, 95GC could be used to evaluate the long-term prognosis of ER-positive, node-negative breast cancer. Even though further prospective validation is necessary, the inclusion of 95GC in clinical practice could help to select optimal treatments for breast cancer patients and identify those who do not benefit from the addition of chemotherapy, thus avoiding unnecessary treatment.
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Neoplasias da Mama/genética , Expressão Gênica , Recidiva Local de Neoplasia/genética , Receptores de Estrogênio , Análise Serial de Tecidos/métodos , Adulto , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/química , Neoplasias da Mama/classificação , Neoplasias da Mama/terapia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Japão , Antígeno Ki-67/análise , Linfonodos , Pessoa de Meia-Idade , Gradação de Tumores , Estudos Retrospectivos , Risco , Taxa de Sobrevida , Fatores de TempoRESUMO
To investigate the biological processes affected by long-term iron supplementation, newly hatched silkworms were exposed to high iron mulberry diet (10 and 100â¯ppm) and its effect on silkworm transcriptom was determined. The results showed that the silkworm was responsive to iron by increasing iron concentration and ferritin levels in the hemolymph and by regulating the expression of many other genes. A total of 523 and 326 differentially expressed genes were identified in 10 and 100â¯ppm Fe group compared to the control, respectively. Of these genes, 249 were shared between in both the 10â¯ppm and 100â¯ppm Fe group, including 152 up-regulated and 97 down-regulated genes. These shared genes included 19 known Fe regulated, 24 immune-related, 12 serine proteases and serine proteases homologs, 41 cuticular and cuticle genes. Ten genes (carboxypeptidases A, serine protease homologs 85, fibrohexamerin/P25, transferrin, sex-specific storage-protein 2, fungal protease inhibitor F, insect intestinal mucin, peptidoglycan recognition protein B, cuticle protein CPH45, unknown gene) were involved in the regulation of iron overload responses.
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Bombyx/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Insetos/genética , Sobrecarga de Ferro/genética , Ferro da Dieta/administração & dosagem , Ferro/administração & dosagem , Transcriptoma/efeitos dos fármacos , Animais , Bombyx/efeitos dos fármacos , Feminino , Proteínas de Insetos/metabolismo , Sobrecarga de Ferro/fisiopatologia , MasculinoRESUMO
After a vertebrate dies, many of its organ systems, tissues, and cells remain functional while its body no longer works as a whole. We define this state as the "twilight of death" - the transition from a living body to a decomposed corpse. We claim that the study of the twilight of death is important to ethical, legal and medical science. We examined gene expression at the twilight of death in the zebrafish and mouse reaching the conclusion that apparently thousands of transcripts significantly increase in abundance from life to several hours/days postmortem relative to live controls. Transcript dynamics of different genes provided "proof-of-principle" that models accurately predict an individual's elapsed-time-of-death (i.e. postmortem interval). While many transcripts were associated with survival and stress compensation, others were associated with epigenetic factors, developmental control, and cancer. Future studies are needed to determine whether the high incidence of cancer in transplant recipients is due to the postmortem processes in donor organs.
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Expressão Gênica/genética , Neoplasias/genética , Animais , Morte , Ciências Forenses/métodos , Humanos , Pesquisa , Transplante/métodosRESUMO
Microglia activation is central to the pathophysiology of retinal degenerative disorders. Resveratrol, a naturally occurring non-flavonoid phenolic compound present in red wine has potent anti-inflammatory and immunomodulatory properties. However, molecular mechanisms by which resveratrol influences microglial inflammatory pathways and housekeeping functions remain unclear. Here, we first studied the immuno-modulatory effects of resveratrol on BV-2 microglial cells at the transcriptome level using DNA-microarrays and selected qRT-PCR analyses. We then analyzed resveratrol effects on microglia morphology, phagocytosis and migration and estimated their neurotoxicity on 661â¯W photoreceptors by quantification of caspase 3/7 levels. We found that resveratrol effectively blocked gene expression of a broad spectrum of lipopolysaccharide (LPS)-induced pro-inflammatory molecules, including cytokines and complement proteins. These transcriptomic changes were accompanied by potent inhibition of LPS-induced nitric oxide secretion and reduced microglia-mediated apoptosis of 661â¯W photoreceptor cultures. Our findings highlight novel targets involved in the anti-inflammatory and neuroprotective action of resveratrol against neuroinflammatory responses.
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Microglia/efeitos dos fármacos , Microglia/metabolismo , Estilbenos/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Camundongos , Microglia/citologia , Modelos Neurológicos , Degeneração Neural/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fagocitose/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real , ResveratrolRESUMO
Oligonucleotides containing photo-caged dienes were prepared and shown to react quantitatively in a light-induced Diels-Alder cycloaddition with functional maleimides in aqueous solution within minutes. Due to its high yield and fast rate, the reaction was exploited for DNA surface patterning with sub-micrometer resolution employing direct laser writing (DLW). Functional DNA arrays were written by direct laser writing (DLW) in variable patterns, which were further encoded with fluorophores and proteins through DNA directed immobilization. This mild and efficient light-driven platform technology holds promise for the fabrication of complex bioarrays with sub-micron resolution.
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DNA/química , Corantes Fluorescentes/química , Maleimidas/química , Química Click , Reação de Cicloadição , Lasers , LuzRESUMO
OBJECTIVE: To develop a multiplex oligonucleotide ligation-polymerase chain reaction( MOL-PCR) based universal microarray assay for multiplexed detection of foodborne pathogens. METHODS: Eight common foodborne pathogens causing bacterial food poisoning were selected as detection models. An upstream and downstream adjacent detection probes were designed within specific primer pair for each of eight pathogens. Target fragments of the eight pathogens were enriched by multiplex PCR and used as ligation templates. Abundant fluorescently labeled single-stranded amplicons containing anti-tag sequences were gained by multiplex ligase detection reaction and asymmetric PCR labeling with universal primers. The products could be detected by hybridization with corresponding tag sequences immobilized on DNA microarrays. RESULTS: The results indicated that the assay could specifically identify all eight pathogens in single and multiple infections. The detection limits were( 1. 1- 8. 5) × 10~2 CFU /mL of pure bacterial cultures. The microarray results for 96 food poisoning and clinical diarrheal samples were consistent with that of traditional culture, biochemical identification and real-time PCR. CONCLUSION: The assay provides a novel platform for rapid, accurate, sensitive and high-throughput detection of pathogenic bacteria of foodborne diseases.
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Microbiologia de Alimentos/métodos , Doenças Transmitidas por Alimentos/genética , Doenças Transmitidas por Alimentos/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Primers do DNA , DNA Bacteriano , Microbiologia de Alimentos/instrumentação , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oligonucleotídeos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: RNA is often targeted to be localized to the specific subcellular compartments. Specific localization of mRNA is believed to be an important mechanism for targeting their protein products to the locations, where their function is required. RESULTS: In this study we performed the genome wide transcriptome analysis of peroxisome preparations from the mouse liver using microarrays. We demonstrate that RNA is absent inside peroxisomes, however it is associated at their exterior via the noncovalent contacts with the membrane proteins. We detect enrichment of specific sets of transcripts in two preparations of peroxisomes, purified with different degrees of stringency. Importantly, among these were mRNAs encoding bona fide peroxisomal proteins, such as peroxins and peroxisomal matrix enzymes involved in beta-oxidation of fatty acids and bile acid biosynthesis. The top-most enriched mRNA, whose association with peroxisomes we confirm microscopically was Hmgcs1, encoding 3-hydroxy-3-methylglutaryl-CoA synthase, a crucial enzyme of cholesterol biosynthesis pathway. We observed significant representation of mRNAs encoding mitochondrial and secreted proteins in the peroxisomal fractions. CONCLUSIONS: This is a pioneer genome-wide study of localization of mRNAs to peroxisomes that provides foundation for more detailed dissection of mechanisms of RNA targeting to subcellular compartments.
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Estudo de Associação Genômica Ampla , Peroxissomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Perfilação da Expressão Gênica , Espaço Intracelular , Espectrometria de Massas , Camundongos , Transporte de RNA , TranscriptomaRESUMO
BACKGROUND: Based in epidemiological evidence, repetitive ovulation has been proposed to play a role in the origin of ovarian cancer by inducing an aberrant wound rupture-repair process of the ovarian surface epithelium (OSE). Accordingly, long term cultures of isolated OSE cells undergo in vitro spontaneous transformation thus developing tumorigenic capacity upon extensive subcultivation. In this work, C57BL/6 mouse OSE (MOSE) cells were cultured up to passage 28 and their RNA and DNA copy number profiles obtained at passages 2, 5, 7, 10, 14, 18, 23, 25 and 28 by means of DNA microarrays. Gene ontology, pathway and network analyses were focused in passages earlier than 20, which is a hallmark of malignancy in this model. RESULTS: At passage 14, 101 genes were up-regulated in absence of significant DNA copy number changes. Among these, the top-3 enriched functions (>30 fold, adj p < 0.05) comprised 7 genes coding for centralspindlin, chromosome passenger and minichromosome maintenance protein complexes. The genes Ccnb1 (Cyclin B1), Birc5 (Survivin), Nusap1 and Kif23 were the most recurrent in over a dozen GO terms related to the mitotic process. On the other hand, Pten plus the large non-coding RNAs Malat1 and Neat1 were among the 80 down-regulated genes with mRNA processing, nuclear bodies, ER-stress response and tumor suppression as relevant terms. Interestingly, the earliest discrete segmental aneuploidies arose by passage 18 in chromosomes 7, 10, 11, 13, 15, 17 and 19. By passage 23, when MOSE cells express the malignant phenotype, the dysregulated gene expression repertoire expanded, DNA imbalances enlarged in size and covered additional loci. CONCLUSION: Prior to early aneuploidies, overexpression of genes coding for the mitotic apparatus in passage-14 pre-malignant MOSE cells indicate an increased proliferation rate suggestive of replicative stress. Concomitant down-regulation of nuclear bodies and RNA processing related genes suggests altered control of nuclear RNA maturation, features recently linked to impaired DNA damage response leading to genome instability. These results, combined with cytogenetic analysis by other authors in this model, suggest that transcriptional profile at passage 14 might induce cytokinesis failure by which tetraploid cells approach a near-tetraploid stage containing primary chromosome aberrations that initiate the tumorigenic drive.
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Transformação Celular Neoplásica/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Mitose/genética , Neoplasias Ovarianas/genética , Lesões Pré-Cancerosas/genética , Animais , Transformação Celular Neoplásica/metabolismo , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Fenótipo , Mapas de Interação de Proteínas , TranscriptomaRESUMO
Esophageal cancer is often diagnosed at an advanced stage. Diagnostic markers are needed for achieving a cure in esophageal cancer detecting and treating tumor cells earlier. In patients with locally advanced squamous cell carcinoma of the esophagus (ESCC), we profiled the gene expression of ESCC compared to corresponding normal biopsies for diagnostic markers by genome microarrays. Profiling of gene expression identified 4844 genes differentially expressed, 2122 upregulated and 2722 downregulated in ESCC. Twenty-three overexpressed candidates with best scores from significance analysis have been selected for further analysis by TaqMan low-density array-technique using a validation cohort of 40 patients. The verification rate was 100 % for ESCC. Twenty-two markers were additionally overexpressed in adenocarcinoma of the esophagus (EAC). The markers significantly overexpressed already in earlier tumor stages (pT1-2) of both histological subtypes (n = 19) have been clustered in a "diagnostic signature": PLA2G7, PRAME, MMP1, MMP3, MMP12, LIlRB2, TREM2, CHST2, IGFBP2, IGFBP7, KCNJ8, EMILIN2, CTHRC1, EMR2, WDR72, LPCAT1, COL4A2, CCL4, and SNX10. The marker signature will be translated to clinical practice to prove its diagnostic impact. This diagnostic signature may contribute to the earlier detection of tumor cells, with the aim to complement clinical techniques resulting in the development of better detection of concepts of esophageal cancer for earlier therapy and more favorite prognosis.
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Biomarcadores Tumorais/biossíntese , Neoplasias Esofágicas/diagnóstico , Proteínas de Neoplasias/biossíntese , Transcriptoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos , PrognósticoRESUMO
DNA barcoding, microarray technology and next generation sequencing have emerged as promising tools for the elucidation of plant genetic diversity and its conservation. They are proving to be immensely helpful in authenticating the useful medicinal plants for herbal drug preparations. These newer versions of molecular markers utilize short genetic markers in the genome to characterize the organism to a particular species. This has the potential not only to classify the known and yet unknown species but also has a promising future to link the medicinally important plants according to their properties. The newer trends being followed in DNA chips and barcoding pave the way for a future with many different possibilities. Several of these possibilities might be: characterization of unknown species in a considerably less time than usual, identification of newer medicinal properties possessed by the species and also updating the data of the already existing but unnoticed properties. This can assist us to cure many different diseases and will also generate novel opportunities in medicinal drug delivery and targeting.
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Código de Barras de DNA Taxonômico , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Medicinais , Variação Genética , Plantas Medicinais/classificação , Plantas Medicinais/genéticaRESUMO
Congenital scoliosis is a condition characterized by spinal curvature beyond the physiological norm. The molecular mechanisms underlying the pathogenesis of congenital scoliosis are beginning to be clarified; however, the genes related to congenital scoliosis are still unknown. We herein report the results of a comprehensive analysis of gene expression in the spines from a rat model of congenital kyphoscoliosis obtained using DNA microarrays. The rats (Ishibashi rats, IS) showed decreased expression levels of genes associated with bone formation, such as those associated with retinol metabolism and type I collagen. Interestingly, the flexion sites of the IS rats showed low expression levels of tropomyosin receptor kinases (Trks: TrkA, TrkB, and TrkC), which belong to the neurotrophic receptor tyrosine kinase family. Moreover, this phenomenon was observed only in the flexion sites of the spine, and the expression levels of Trks in other parts of the spine in these rats were normal. The decreased expression levels of Trks were observed at both the mRNA and protein levels. We also observed that the number of Trk-immunopositive cells in the lumbar spine in the IS rats was lower than that in wild-type rats. These findings indicate that the Trks have an important function in regulating normal bone formation, and provide a molecular explanation for the pathogenesis of congenital kyphoscoliosis.
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Regulação para Baixo , Cifose/congênito , Vértebras Lombares/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Escoliose/congênito , Animais , Cifose/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Wistar , Escoliose/metabolismo , Transcrição GênicaRESUMO
Monoclonal gammopathy of undetermined significance (MGUS) is a benign condition with an approximate 1% annual risk of symptomatic plasma cell disorder development, mostly to multiple myeloma (MM). We performed genomewide screening of copy-number alterations (CNAs) in 90 MGUS and 33 MM patients using high-density DNA microarrays. We identified CNAs in a smaller proportion of MGUS (65.6%) than in MM (100.0%, P = 1.31 × 10-5 ) and showed median number of CNAs is lower in MGUS (3, range 0-22) than in MM (13, range 4-38, P = 1.82 × 10-10 ). In the MGUS cohort, the most frequent losses were located at 1p (5.6%), 6q (6.7%), 13q (30.0%), 14q (14.4%), 16q (8.9%), 21q (5.6%), and gains at 1q (23.3%), 2p (6.7%), 6p (13.3%), and Xq (7.8%). Hyperdiploidy was detected in 38.9% of MGUS cases, and the most frequent whole chromosome gains were 3 (25.6%), 5 (23.3%), 9 (37.8%), 15 (23.3%), and 19 (32.2%). We also identified CNAs such as 1p, 6q, 8p, 12p, 13q, 16q losses, 1q gain and hypodiploidy, which are potentially associated with an adverse prognosis in MGUS. In summary, we showed that MGUS is similar to MM in that it is a genetically heterogeneous disorder, but overall cytogenetic instability is lower than in MM, which confirms that genetic abnormalities play important role in monoclonal gammopathies.
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Variações do Número de Cópias de DNA , Estudo de Associação Genômica Ampla , Genômica , Gamopatia Monoclonal de Significância Indeterminada/genética , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Progressão da Doença , Feminino , Instabilidade Genômica , Genômica/métodos , Humanos , Masculino , Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genéticaRESUMO
γ-Glutamyl transferase (GGT) is useful as a marker in pathological conditions, including several types of cancer. We optimized the histochemical detection of GGT to assay the gene expression profiles of phenotype-specific cells selected by laser capture microdissection (LCM). For optimization, we used the livers of rats subjected to hepatocarcinogenesis. This model induced nodules of hepatocytes and tumors with GGT activity. To obtain sufficient high-quality RNA after histochemistry and LCM, we included an RNase inhibitor and air-dried the tissue sections. This optimization allowed the visualization of GGT activity in situ and a yield of 1.4 to 2.0 µg of total RNA from 15 to 18 mm² of microdissected tissue (20 µm thickness). The average RNA integrity number in GGT-positive tissue, determined by chip-capillary electrophoresis, was 6.9, and the 28S/18S ribosomal RNA (rRNA) ratio was 1.4. The RNAs were processed for the Rat Gene 1.0 ST Array (Affymetrix). Comparable quality control metrics, such as signal intensity and RNA degradation plots, were found between the LCM samples and non-LCM tissue. The increased expression of Ggt1 expected in GGT-positive tissue was confirmed by microarrays and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). This optimization provided a suitable method for whole-transcript analysis of GGT-positive tissue isolated using LCM.
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Perfilação da Expressão Gênica/métodos , Imuno-Histoquímica/métodos , Microdissecção e Captura a Laser/métodos , gama-Glutamiltransferase/metabolismo , Animais , Fígado/citologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , gama-Glutamiltransferase/genéticaRESUMO
Cri-du-chat is a rare congenital syndrome characterized by intellectual disability, severe speech/developmental delay, dysmorphic features, and additional syndromic findings. The etiology of this disorder is well known, and is attributed to a large deletion on chromosome 5 that typically ranges from band 5p15.2 to the short arm terminus. This region contains CTNND2, a gene encoding a neuronal-specific protein, delta-catenin, which plays a critical role in cellular motility and brain function. The exact involvement of CTNND2 in the cognitive functionality of individuals with Cri-du-chat has not been fully deciphered, but it is thought to be significant. This report describes an 8-year-old African-American female with a complex chromosome 5 abnormality and a relatively mild case of cri-du-chat syndrome. Because of the surprisingly mild cognitive phenotype, although a karyotype had confirmed the 5p deletion at birth, an oligo-SNP microarray was obtained to further characterize her deletion. The array revealed a complex rearrangement, including a breakpoint in the middle of CTNND2, which resulted in a partial deletion and partial duplication of that gene. The array also verified the expected 5p terminal deletion. Although the patient has a significant deletion in CTNND2, half of the gene (including the promoter region) is not only preserved, but is duplicated. The patient's milder cognitive and behavioral presentation, in conjunction with her atypical 5p alteration, provides additional evidence for the role of CTNND2 in the cognitive phenotype of individuals with Cri-du-chat.
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Cateninas/genética , Deleção Cromossômica , Cromossomos Humanos Par 5 , Síndrome de Cri-du-Chat/diagnóstico , Síndrome de Cri-du-Chat/genética , Duplicação Gênica , Fenótipo , Criança , Hibridização Genômica Comparativa , Fácies , Feminino , Humanos , delta CateninaRESUMO
Escherichia coli is used intensively for recombinant protein production, but one key challenge with recombinant E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high concentrations of inactive recombinant protein that require recovery steps to salvage a functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. In this study, DNA microarrays were used to compare the E. coli gene expression response dynamics to soluble and insoluble recombinant protein production. As expected and previously reported, the classical heat-shock genes had increased expression due to IB formation, including protein folding chaperones and proteases. Gene expression levels for protein synthesis-related and energy-synthesis pathways were also increased. Many transmembrane transporter and corresponding catabolic pathways genes had decreased expression for substrates not present in the culture medium. Additionally, putative genes represented over one-third of the genes identified to have significant expression changes due to IB formation, indicating many important cellular responses to IB formation still need to be characterized. Interestingly, cells grown in 3% ethanol had significantly reduced gene expression responses due to IB formation. Taken together, these results indicate that IB formation is complex, stimulates the heat-shock response, increases protein and energy synthesis needs, and streamlines transport and catabolic processes, while ethanol diminished all of these responses.
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Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Corpos de Inclusão/metabolismo , Proteínas Recombinantes/metabolismo , Biotecnologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/metabolismo , Etanol/farmacologia , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Redes e Vias Metabólicas/genética , Análise de Sequência com Séries de Oligonucleotídeos , SolubilidadeRESUMO
An Escherichia coli ΔpfkA mutant lacking the major phosphofructokinase possesses a partially cyclized pentose phosphate pathway leading to an increased NADPH per glucose ratio. This effect decreases the amount of glucose required for NADPH regeneration in reductive biotransformations, such as the conversion of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation. DNA microarray analysis revealed amongst other things increased expression of soxS, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of soxS. Furthermore, several target genes of the ArcAB two-component system showed a lower mRNA level in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2 /Q ratio in the reference strain. This prompted us to analyze yields and productivities of MAA reduction to MHB under different oxygen regimes in a bioreactor. Under anaerobic conditions, the specific MHB production rates of both strains were comparable (7.4 ± 0.2 mmolMHB h(-1) gcdw (-1) ) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h(-1) gcdw (-1) ) and of the ΔpfkA mutant (11.0 mmol h(-1) gcdw (-1) ) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference strain increased after the addition of MAA, presumably due to the oxidation of the acetate accumulated before MAA addition, the OTR of the ΔpfkA strain strongly decreased, indicating a very low respiration rate despite sufficient oxygen supply. The latter effect can likely be attributed to a restricted conversion of NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is outcompeted in the presence of MAA by the recombinant NADPH-dependent alcohol dehydrogenase. The differences in respiration rates can explain the suggested higher ArcAB activity in the reference strain.