RESUMO
In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. Here, we show that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. We observed similar roles for Fu during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Feminino , Células Germinativas/metabolismo , Humanos , Ovário/citologia , Ovário/metabolismo , Fosforilação , Células-Tronco/metabolismo , Ubiquitinação , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Relatively little is known about the in vivo functions of newly emerging genes, especially in metazoans. Although prior RNAi studies reported prevalent lethality among young gene knockdowns, our phylogenomic analyses reveal that young Drosophila genes are frequently restricted to the nonessential male reproductive system. We performed large-scale CRISPR/Cas9 mutagenesis of "conserved, essential" and "young, RNAi-lethal" genes and broadly confirmed the lethality of the former but the viability of the latter. Nevertheless, certain young gene mutants exhibit defective spermatogenesis and/or male sterility. Moreover, we detected widespread signatures of positive selection on young male-biased genes. Thus, young genes have a preferential impact on male reproductive system function.
Assuntos
Drosophila melanogaster/genética , Fertilidade/genética , Genes Essenciais/fisiologia , Genes de Insetos/fisiologia , Reprodução/genética , Animais , Sistemas CRISPR-Cas/genética , Evolução Molecular , Mutação da Fase de Leitura , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Letais/fisiologia , Infertilidade Masculina/genética , Masculino , Filogenia , Interferência de RNA , Espermatogênese/genética , Testículo/anatomia & histologia , Testículo/metabolismoRESUMO
BACKGROUND: Copy number variation (CNV) is a type of structural variation, which is a gain or loss event with abnormal changes in copy number. Methods to predict the pathogenicity of CNVs are required to realize the relationship between these variants and clinical phenotypes. ClassifyCNV, X-CNV, StrVCTVRE, etc. have been trained to predict the pathogenicity of CNVs, but few studies have been reported based on the deleterious significance of features. RESULTS: From single nucleotide polymorphism (SNP), gene and region dimensions, we collected 79 informative features that quantitatively describe the characteristics of CNV, such as CNV length, the number of protein genes, the number of three prime untranslated region. Then, according to the deleterious significance, we formulated quantitative methods for features, which fall into two categories: the first is variable type, including maximum, minimum and mean; the second is attribute type, which is measured by numerical sum. We used Gradient Boosted Trees (GBT) algorithm to construct dbCNV, which can be used to predict pathogenicity for five-tier classification and binary classification of CNVs. We demonstrated that the distribution of most feature values was consistent with the deleterious significance. The five-tier classification model accuracy for 0.85 and 0.79 in loss and gain CNVs, which proved that it has high discrimination power in predicting the pathogenicity of five-tier classification CNVs. The binary model achieved area under curve (AUC) values of 0.96 and 0.81 in the validation set, respectively, in gain and loss CNVs. CONCLUSION: The performance of the dbCNV suggest that functional deleteriousness-based model of CNV is a promising approach to support the classification prediction and to further understand the pathogenic mechanism.
Assuntos
Variações do Número de Cópias de DNA , Polimorfismo de Nucleotídeo Único , Virulência , Fenótipo , AlgoritmosRESUMO
BACKGROUND: Seldom performance evaluation and diagnosis comparison studies were reported for different chemiluminescent immunoassay (CLIA) kits approved under an emergency approval program for SARS-CoV-2 infection. METHODS: A total of 100 and 105 serum separately from non-infected populations and COVID-19 patients were detected with SARS-CoV-2 IgM and IgG kits. The characteristics including precision, functional sensitivity, linearity, and accuracy were evaluated for Axceed, iFlash, and Maglumi CLIA kits. RESULTS: Maglumi and iFlash had the best analytical sensitivity for IgM and IgG, respectively. Axceed kits had a linearity response in the detected concentration. The clinical sensitivity of Axceed, iFlash, and Maglumi was 68.0%, 64.9%, and 63.9% with a specificity of 99.0%, 96.0%, and 100% for IgM, 85.6%, 97.9%, and 94.8% with a specificity of 97.0% for IgG. ROC analysis indicated all kits had a diagnostic power greater than 0.9. Notably, either IgM or IgG kits obtained a poor agreement (Kappa value from 0.397 to 0.713) with others. Among 38 recovered patients, 94.7% had an effective immune response, and both seropositive IgM and IgG accounted for the biggest proportion (medium, 42 days onset), then followed by the single seropositive IgG (medium, 50 days onset) in Ab profile. CONCLUSION: The performance of CLIA kits satisfied the diagnosis of SARS-CoV-2 infection. Both positive of IgG and IgM contributes to improve the specificity, and a positive one will enhance the sensitivity.
Assuntos
Teste para COVID-19/métodos , COVID-19/etiologia , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Adulto , Idoso , Anticorpos Antivirais/sangue , Automação Laboratorial , COVID-19/diagnóstico , Feminino , Humanos , Luminescência , Gravidez , Complicações Infecciosas na Gravidez/etiologia , Complicações Infecciosas na Gravidez/terapia , Reprodutibilidade dos Testes , SARS-CoV-2/imunologia , Fatores de TempoRESUMO
BACKGROUND: We aimed to evaluate the analytical performance of five commercial RT-PCR kits (Genekey, Daan, BioGerm, Liferiver, and Yaneng) commonly used in China, since such comparison data are lacking. METHODS: A total of 20 COVID-19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT-PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross-reactivity. RESULTS: The positive detection rate was 100% for Genekey, Daan, and BioGerm,and 90% for Liferiver and Yaneng in 20 clinical SARS-CoV-2 infection. The coincidence rate of five kits in 10 negative samples was 100%. The detection rate of target genes for Daan, BioGerm, Liferiver, and Yaneng was 100% from Level 1 to Level 3. In Level 4, only Daan detection rate was 100%. In Level 5, five kits presented poor positive rate. The limit of detection declared by each manufacturer was verified. The repeatability for target genes was less than 5% and so did the total imprecision. There is no cross-reactivity of five kits with six human coronaviruses and four respiratory pathogens for ORF1ab and N gene. CONCLUSIONS: Five RT-PCR kits assessed in this study showed acceptable analytical performance characteristics and are useful tools for the routine diagnosis of SARS-CoV-2.
Assuntos
Teste para COVID-19/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Humanos , Limite de Detecção , Nasofaringe/virologia , Poliproteínas/genética , Reprodutibilidade dos Testes , Proteínas Virais/genéticaRESUMO
BACKGROUND: The XPEN60 CRP&SAA (hereafter XPEN60) is a new automated hematology analyzer that can rapidly detect C-reactive protein (CRP), serum amyloid A (SAA), and blood cell counts (CBC), including the 5-part differential of white blood cells (5-DIFF). The aim of this study was to evaluate the analytical performance of XPEN60. METHODS: The analytical performance of XPEN60 was evaluated on the basis of several parameters, including the limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), precision, accuracy, carryover, linearity, clinical reportable range (CRR), and interference test. In addition, method comparisons between CBC and 5-DIFF, CRP, and SAA were performed on several systems. RESULTS: Total imprecision and accuracy for all parameters fell within acceptable criteria, and excellent measurements were observed in the dilution linearity (coefficient of determination, R2 > .99). LoBs and LoDs (0 and 0.21 mg/L for CRP, 1.1 and 2.27 mg/L for SAA) satisfy the manufacturer's statement. LoQs were 0.61 and 3.62 mg/L for CRP and SAA, respectively. No significant carryover or interference tests (<10%) were observed in this study. The comparison analysis demonstrated strong agreement between XPEN60 results and those of Sysmex-XN1000 (XN1000), except for basophils (Bas) and eosinophils (Eos). The data correlated well with E601 and Mindray CRP-M100 for CRP. CONCLUSION: XPEN60 was demonstrated satisfactory analytical performance, which made it well-suited for use in clinical laboratories, emergency departments, and community hospitals.
Assuntos
Contagem de Células Sanguíneas , Análise Química do Sangue , Proteína C-Reativa/análise , Proteína Amiloide A Sérica/análise , Contagem de Células Sanguíneas/instrumentação , Contagem de Células Sanguíneas/normas , Análise Química do Sangue/instrumentação , Análise Química do Sangue/normas , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: To evaluate the effect of a new type of automatic glycated hemoglobin analyzer on the separation of abnormal hemoglobin. METHODS: Samples diagnosed as hemoglobin variants by capillary electrophoresis and gene testing were selected, and HbA1c analyzer was used for separation and detection. RESULTS: A total of 13 hemoglobin variants in 40 samples could be separated from the normal peaks. CONCLUSIONS: The variant mode of hemoglobin HbA1c can identify a variety of hemoglobin variants, and the type of variants can be preliminarily determined according to the retention time and characteristic peak shape of the variants.
Assuntos
Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Hemoglobinas Glicadas/genética , Hemoglobinas Glicadas/isolamento & purificação , Automação , Cromatografia Líquida de Alta Pressão , Humanos , Talassemia/sangue , Fatores de TempoRESUMO
BACKGROUND: In recent years, several high-risk human papillomavirus (HR-HPV) tests have been developed. The assay capabilities need to be systematically reviewed. Here, we compared the clinical sample performance of three novel HR-HPV assays (Liferiver, Yaneng, and Darui) based on different platforms with the widely adopted cobas4800 test. METHODS: A total of 346 cervical swabs from women who were screened for cervical cancer were analyzed for the presence of 14 HR-HPV genotypes. The distinction between the four assays was investigated by the genotyping and direct sequencing. RESULTS: The positive rates of the four assays ranged from 61.56% to 64.16%. The overall concordance was 88.15%. The Yaneng assays displayed the best sensitivity (100%) and specificity (98.43%). The sensitivity (98.17%) and specificity (98.43%) of the Darui assay were superior to those of the cobas4800 test (97.72% and 93.70%, respectively). The Liferiver assay displayed comparable sensitivity with the cobas4800 test (95.89% and 97.72%, respectively). The specificity of the cobas4800 was lower than that of the Liferiver assay (93.70% vs. 97.64%). CONCLUSIONS: The three novel HR-HPV assays displayed good agreement with the cobas4800 test. The analytical performance of all four fulfilled the requirements of sensitivity and specificity for HR-HPV detection.
Assuntos
Testes de DNA para Papilomavírus Humano , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Feminino , Testes de DNA para Papilomavírus Humano/métodos , Testes de DNA para Papilomavírus Humano/normas , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e EspecificidadeRESUMO
Post-transcriptional regulatory strategies that involve coupling between terminal uridyltransferase (TUTase) and exoribonuclease enzymes have recently been characterized in diverse species. Of note, the 3' exoribonuclease Dis3L2 has received substantial attention as a factor that metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of noncoding RNAs. To date, most studies of Dis3L2 have focused on fungi and mammalian cells. Here, we introduce Drosophila as a system that permits analysis of molecular mechanisms as well as the ability to interrogate organismal phenotypes. We started with a structure-function analysis of the Drosophila TUTase Tailor, which we recently identified to inhibit biogenesis of splicing-derived miRNA hairpins. Next, we show that Tailor/Dis3L2 form a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity, but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. We analyzed mutants of Tailor and Dis3L2, which are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. Dis3L2 defects are more severe than Tailor, and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis, causing male sterility. RNA-seq analysis from single- and double-mutant testes reveals aberrant gene expression programs and suggests that noncoding RNAs may be preferentially affected by Dis3L2. Overall, our studies of a new tailing/trimming complex reveal unexpectedly specific requirements during gametogenesis.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Exorribonucleases/genética , Infertilidade Masculina/genética , RNA Nucleotidiltransferases/genética , Espermatogênese/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Exorribonucleases/metabolismo , Feminino , Infertilidade Feminina/genética , Masculino , Mutação , RNA Nucleotidiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
Electrospun microfiber meshes have been shown to support the proliferation and differentiation of many types of stem cells, but the phenotypic fate of c-kit(+) human cardiac stem cells (hCSCs) have not been explored. To this end, we utilized thin (~5µm) elastomeric meshes consisting of aligned 1.7µm diameter poly (ester-urethane urea) microfibers as substrates to examine their effect on hCSC viability, morphology, proliferation, and differentiation relative to cells cultured on tissue culture polystyrene (TCPS). The results showed that cells on microfiber meshes displayed an elongated morphology aligned in the direction of fiber orientation, lower proliferation rates, but increased expressions of genes and proteins majorly associated with cardiomyocyte phenotype. The early (NK2 homeobox 5, Nkx2.5) and late (cardiac troponin I, cTnI) cardiomyocyte genes were significantly increased on meshes (Nkx=2.5 56.2±13.0, cTnl=2.9±0.56,) over TCPS (Nkx2.5=4.2±0.9, cTnl=1.6±0.5, n=9, p<0.05 for both groups) after differentiation. In contrast, expressions of smooth muscle markers, Gata6 and myosin heavy chain (SM-MHC), were decreased on meshes. Immunocytochemical analysis with cardiac antibody exhibited the similar pattern of above cardiac differentiation. We conclude that aligned microfiber meshes are suitable for guiding cardiac differentiation of hCSCs and may facilitate stem cell-based therapies for treatment of cardiac diseases.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Miocárdio/citologia , Poliésteres/farmacologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células-Tronco/citologia , Alicerces Teciduais/química , Biomarcadores/metabolismo , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Previous studies indicated that the fast-healer strain of MRL/MpJ-Fas(lpr)/J (MRL) mice demonstrated superior regenerative capabilities for digit wound healing and/or regeneration compared with the non-healer strain of C57BL/6 (C57) mice. These reports, however, mainly focused on morphological observations and analysis of gene expression with little attention on the role of angiogenesis in the amputated digits. By taking advantage of Laser Doppler Imaging and histological analysis, we examined the potential role(s) of angiogenesis in facilitating tissue regrowth/regeneration by comparing two strains of mice (MRL versus C57). The three middle digits on the mouse's right foot (RF) were amputated at the middle level of phalanx 2 (P2) on postnatal day 2 (Day 0), while the left foot (LF) remained intact and served as a control. Laser Doppler images and digital photographs were taken of both feet before, immediately after surgery, and on Day 7, 14, 21, and 28 to evaluate blood flow and overall length of digit regrowth. All measurements from the amputated digits of the RF were divided by those of the control LF to obtain normalized ratios for statistical comparisons between groups. It was found that MRL mice demonstrated an approximately 220% increase in regrowth ratios over that of C57 mice from Day 21-28 (p < 0.01, n = 13), while blood-flow increased by about 25% on Day 21 (p < 0.01, n = 13) compared to that in C57 mice. Histological analysis of both control and amputated limbs indicated an approximately 70% increase in the number of vessels (both arterial and venous) in MRL mice over that of the C57 mice (p < 0.05, n = 3). We conclude that higher blood flow and angiogenesis may play an important role in facilitating the fast regrowth ratios of amputated digits in MRL mice compared to C57 mice.
Assuntos
Neovascularização Fisiológica , Dedos do Pé/fisiologia , Cicatrização , Animais , Pé/diagnóstico por imagem , Fluxometria por Laser-Doppler , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Fluxo Sanguíneo Regional , Dedos do Pé/irrigação sanguínea , Dedos do Pé/diagnóstico por imagemRESUMO
BACKGROUND: The relatively high radiation dose from micro-CT is a cause for concern in preclinical research involving animal subjects. Interior region-of-interest (ROI) imaging was proposed for dose reduction, but has not been experimentally applied in micro-CT. OBJECTIVE: Our aim is to implement interior ROI imaging in a carbon nanotube (CNT) x-ray source based micro-CT, and present the ROI image quality and radiation dose reduction for interior cardiac micro-CT imaging of a mouse heart in situ. METHODS: An aperture collimator was mounted at the source-side to induce a small-sized cone beam (10âmm width) at the isocenter. Interior in situ micro-CT scans were conducted on a mouse carcass and several micro-CT phantoms. A GPU-accelerated hybrid iterative reconstruction algorithm was employed for volumetric image reconstruction. Radiation dose was measured for the same system operated at the interior and global micro-CT modes. RESULTS: Visual inspection demonstrated comparable image quality between two scan modes. Quantitative evaluation demonstrated high structural similarity index (up to 0.9614) with improved contrast-noise-ratio (CNR) on interior micro-CT mode. Interior micro-CT mode yielded significant reduction (up to 83.9%) for dose length product (DLP). CONCLUSIONS: This work demonstrates the applicability of using CNT x-ray source based interior micro-CT for preclinical imaging with significantly reduced radiation dose.
Assuntos
Coração/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Nanotubos de Carbono/química , Microtomografia por Raio-X/métodos , Algoritmos , Animais , Camundongos , Imagens de FantasmasRESUMO
BACKGROUND: The analytical performances of the NT-proBNP and BNP assays on the Cobas E601 and ADVIA Centaur were thoroughly evaluated. In addition, the values of BNP and NT-proBNP, which are heart failure markers, were compared in the diagnosis of HF patients with or without acute cerebral infarction since they could also be elevated in ischemic stroke. METHODS: Clinical and Laboratory Standards Institute (CLSI) documents were employed in the analytical evaluation of NT-proBNP and BNP assays on the Cobas E601 and ADVIA Centaur. Then 100 heart failure patients and 103 cerebral infarction complicated with heart failure patients, who had been diagnosed by clinical doctors blinded to NT-proBNP and BNP concentrations, were chosen to compare their values in the diagnosis of heart failure with or without acute cerebral infarction. RESULTS: The NT-proBNP and BNP methods are precise and accurate (total CV < 2.9%, deviation < 3.6%), have wide dynamic measuring ranges (8 pg/mL to 35 126 pg/mL and 2.0 pg/mL to 5094 pg/mL, respectively) with maximum dilutability of 1:2, and are free of common interferences. The most suitable sample types for NT-proBNP and BNP are serum and EDTA plasma, respectively, and both methods correlate well in simple-HF patients. Unlike BNP, the level of NT-proBNP is much higher in HF patients with acute cerebral infarction (p < 0.001). The Cobas E601 and ADVIA Centaur systems have good analytical performances. CONCLUSIONS: In HF patients with acute cerebral infarction, the NT-proBNP and BNP levels did not correlate and thus had implications for clinical diagnosis.
Assuntos
Biomarcadores/sangue , Insuficiência Cardíaca/sangue , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Infarto Cerebral/sangue , Infarto Cerebral/complicações , Feminino , Insuficiência Cardíaca/complicações , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Increasing evidence supports the idea that the regulation of stem cells requires both extrinsic and intrinsic mechanisms. However, much less is known about how intrinsic signals regulate the fate of stem cells. Studies on germline stem cells (GSCs) in the Drosophila ovary have provided novel insights into the regulatory mechanisms of stem cell maintenance. In this study, we demonstrate that a ubiquitin-dependent pathway mediated by the Drosophila eff gene, which encodes the E2 ubiquitin-conjugating enzyme Effete (Eff), plays an essential role in GSC maintenance. We show that Eff both physically and genetically interacts with dAPC2, a key component of the anaphase-promoting complex (APC), which acts as a multisubunit E3 ligase and plays an essential role in targeting mitotic regulators for degradation during exit from mitosis. This interaction indicates that Eff regulates the APC/C-mediated proteolysis pathway in GSCs. Moreover, we show that expression of a stable form of Cyclin A, but not full-length Cyclin A, results in GSC loss. Finally we show that, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Collectively, our data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila. Given that the regulation of mitotic Cyclins is evolutionarily conserved between flies and mammals, our study also implies that a similar mechanism may be conserved in mammals.
Assuntos
Ciclina A/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Células-Tronco/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Células Cultivadas , Ciclina A/química , Ciclina A/genética , Drosophila/citologia , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Hidrólise , Mitose/fisiologia , Mutação , Ovário/citologia , Ovário/fisiologia , Ligação Proteica , Células-Tronco/citologia , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , UbiquitinaçãoRESUMO
Epitranscriptomic modifications can impact behavior. Here, we used Drosophila melanogaster to study N6-methyladenosine (m6A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m6A binders. Assays of short term memory in m6A mutants reveal neural-autonomous requirements of m6A writers working via Ythdf, but not Ythdc1. Furthermore, m6A/Ythdf operate specifically via the mushroom body, the center for associative learning. We map m6A from wild-type and Mettl3 mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites that are highly enriched in 5' UTRs. Genomic analyses indicate that Drosophila m6A is preferentially deposited on genes with low translational efficiency and that m6A does not affect RNA stability. Nevertheless, functional tests indicate a role for m6A/Ythdf in translational activation. Altogether, our molecular genetic analyses and tissue-specific m6A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway.
Assuntos
Adenosina/análogos & derivados , Adenosina/metabolismo , Drosophila melanogaster/fisiologia , Aprendizagem/fisiologia , Memória/fisiologia , Regiões 5' não Traduzidas , Adenosina/genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Feminino , Proteínas Nucleares/metabolismo , Proteômica , Estabilidade de RNA , RNA Mensageiro/metabolismoRESUMO
CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction. Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection. Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.
RESUMO
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a malignant tumor with a strong tendency for metastasis and recurrence. Finding effective biomarkers for the early diagnosis of HNSCC is critical for the early treatment and prognosis of patients. METHODS: RNA sequencing data including long non-coding RNAs (lncRNAs), messenger RNA (mRNAs) and microRNAs (miRNAs) of 141 HNSCC and 44 adjacent normal tissues were obtained from the TCGA. Differentially expressed genes were analyzed using the R package DESeq. GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. A competing endogenous RNAs (ceRNA) network was constructed. The most differentially expressed genes in the main ceRNA network were chosen for nasopharyngeal carcinoma (NPC) cell lines and NPEC2 Bmi-1 cell line verification. A receiver operating characteristic (ROC) curve was constructed for 141 specimens of HNSCC tissues from 44 control samples. RESULTS: In our study, 79 HNSCC-associated abnormally expressed lncRNAs , 86 abnormally expressed miRNAs and 324 abnormally expressed mRNAs were identified. The public microarray results showed that LINC00958 and HOXC13-AS expression levels were upregulated in HNSCC tissues compared with the adjacent normal tissues in this study (p < 0.0001). LINC00958 and HOXC13-AS expression levels in NPC cell lines were higher than those in the NPEC2 Bmi-1 cell line (p < 0.05). The results showed that the area under the ROC curve (AUC) of LINC00958 reached up to 0.906 at a cutoff value of 7.96, with a sensitivity and specificity of 80.85% and 90.91%, respectively. The AUC of HOXC13-AS reached up to 0.898 at a cutoff value of 0.695, with sensitivity and specificity values of 86.23% and 83.78%, respectively. CONCLUSION: The current study indicates that LINC00958 and HOXC13-AS are new candidate diagnostic biomarkers for HNSCC patients.
RESUMO
BACKGROUND: Bladder cancer is one of the most common malignancies. So far, no effective biomarker for bladder cancer prognosis has been identified. Aberrant DNA methylation is frequently observed in the bladder cancer and holds considerable promise as a biomarker for predicting the overall survival (OS) of patients. MATERIALS AND METHODS: We downloaded the DNA methylation and transcriptome data for bladder cancer from The Cancer Genome Atlas (TCGA), a public database, screened hypo-methylated and up-regulated genes, similarly, hyper-methylation with low expression genes, then retrieved the relevant methylation sites. Cox regression analysis was used to identify a nine-methylation site signature of a training group. Predictive ability was validated in a test group by receiver operating characteristic (ROC) analysis. RESULTS: We identified nine bladder cancer-specific methylation sites as potential prognostic biomarkers and established a risk score system based on the methylation site signature to evaluate the OS. The performance of the signature was accurate, with area under curve was 0.73 in the training group and 0.71 in the test group. Taking clinical features into consideration, we constructed a nomogram consisting of the nine-methylation site signature and patients' clinical variables, and found that the signature was an independent risk factor. CONCLUSIONS: Overall, the significant nine methylation sites could be novel prediction biomarkers, which could aid in treatment and also predict the overall survival likelihoods of bladder cancer patients.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Técnicas de Apoio para a Decisão , Perfilação da Expressão Gênica , Nomogramas , Proteoma , Neoplasias da Bexiga Urinária/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapiaRESUMO
The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m6A pathway. We first apply miCLIP to map m6A across embryogenesis, characterize its m6A 'writer' complex, validate its YTH 'readers' CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.
Assuntos
Adenosina/análogos & derivados , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/metabolismo , Processos de Determinação Sexual , Adenosina/química , Processamento Alternativo , Motivos de Aminoácidos , Animais , Comportamento Animal , Metilação de DNA , Proteínas de Drosophila/metabolismo , Feminino , Íntrons , Masculino , Espectrometria de Massas , Modelos Genéticos , Família Multigênica , Mutagênese , Mutação , Ovário/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
Uniquely identifying research animals is a widespread and essential procedure. Potential disadvantages of commonly used identification methods such as toe clipping, ear punching, and ear tagging include tissue loss and adverse effects in physiologic homeostasis and animal behavior. In addition, the labels produced by using these methods can become unreadable, potentially leading to misidentification. In this study, we proposed a combined approach involving ear, tail, and toe tattooing that can be used to permanently identify mice regardless of their age. Four groups (neonatal and adult C57BL/6J [black] and CD1 [white] mice) were used. Single- or 2-color tattooing (ear, tail, or toe or combinations thereof) was performed to identify a defined or unlimited number of mice, respectively. Tail tattooing using both green and red pastes was suitable for identifying white-haired neonatal mice as early as postnatal day 1, whereas toe tattooing with green paste was an effective alternative approach for labeling black-haired mouse pups. In comparison, single-color (green) or 2-color (green and red) ear tattooing identified both white and black adult mice older than 3 wk. Ear tattooing can be adapted to labeling an unlimited number of adult mice by adding the cage number. We conclude that tattooing various combinations of the ears, tail, and toes provides an easy and permanent approach for identifying mice of all ages with minimal disturbance to the animals.