Introdução/objetivos: As gastroenterites agudas (GEA) são doenças que se constituem como a segunda maior causa de morte em crianças de todo mundo, especialmente em crianças menores de cinco anos, ...ocorrendo principalmente em países de baixa e média renda. Os agentes virais como rotavírus e norovírus têm sido demonstrados como as causas mais frequentes, no entanto alguns membros da família Picornaviridae também foram associados à diarreia em humanos. Diante disto, objetivou-se a realização da vigilância expandida dos picornavírus em amostras fecais de crianças com quadro de gastroenterite aguda em estados da região Norte e Nordeste do Brasil entre os anos de 2020 e 2021. Métodos: Para isso, foram utilizadas suspensões fecais provenientes de crianças com quadro de gastroenterite aguda e encaminhadas ao Laboratório de Enterovírus do Instituto Evandro Chagas através das redes de vigilância de rotavírus e póliovirus nos anos de 2020 e 2021. Para detecção foi realizada a extração do RNA viral utilizando o QIAmp Viral RNA Mini Kit (QIAGEN), posteriormente foi realizada a reação de RT-qPCR utilizando o kit comercial GoTaq Probe 1-Step RT-qPCR System (Promega) e oligonucleotídeos e sondas específicos para detecção de Parechovirus, Aichivirus e Cosavirus. Foram analisadas 419 amostras de suspensão fecal, nas quais 31,7% (133) foram positivas para parechovirus (HpeV), aichivírus (AiV) e cosavírus (CosV). Resultados: Com isso, foi observado à prevalência dos CosV, os quais estavam presentes em 57,1% (76/133) das amostras, seguido pelo HpeV com 37% (49/133) e por fim os AiV com 6% (8/133). Foram identificadas 18 codetecções entre os vírus investigados, sendo possível observar de HpeV com CosV em 13 amostras, duas codetecções entre AiV e CosV e três codetecções entre os três vírus investigados. Conclusão: Diante do exposto, conclui-se que é importante a vigilância desses vírus para fomentar estudos que visem comprovar sua atuação e comportamento como agentes causadores de gastroenterite, visto que sua prevalência em amostras vem se tornando cada vez mais prevalente e a escassez de estudos no mundo e no Brasil com esse objetivo acabam postergando esses resultados, por isso torna-se de extrema importância à continuidade dessa pesquisa nas regiões norte e nordeste, por conta de sua alta prevalência, para saber sua atuação e comportamento como agentes gastroentéricos.
Small non-coding RNAs (sncRNAs) present in the conditioned medium (CM) of bovine preimplantation embryos are potential noninvasive biomarkers for assessing embryo quality. Accurate quantification of ...sncRNA levels in the spent CM is of utmost importance in this regard. RT-qPCR is considered as the gold standard for quantifying RNA. In order to standardize RT-qPCR data in the sample type under investigation, the use of suitable stable sncRNAs is essential. Here, we selected 10 sncRNAs from small RNA sequencing of CM samples derived from both bovine blastocysts and degenerate embryos, and evaluated their expression stability together with that of cel-miR-39 as a spike and the often-used U6 small nuclear RNA at different embryo developmental stages. In CM of 2-cell embryos, rsRNA-1044 showed the most stable expression, while tDR-1:32-Gly–CCC–1 was the most stable expressed sncRNA in CM of the stages beyond the 2-cell stage. Next, tDR-1:32-Gly–CCC–1 was used for normalizing the RT-qPCR data from the CM of blastocysts and degenerate embryos. Bta-miR-155 and tDR-39:75-Arg-CCG-2 were found to be significantly up-regulated in the CM of blastocysts compared to that of the degenerated embryos (P = 0.028 and P = 0.017, respectively), suggesting their expression levels are related to embryo development stage. In conclusion, tDR-1:32-Gly–CCC–1 can serve as a suitable reference sncRNA for normalization of RT-qPCR data of the CM from bovine blastocysts.
•tsRNAs but not microRNAs are abundant in bovine preimplantation embryo conditioned medium (CM).•rsRNA-1044 is the most stable small non-coding RNA in the CM of the 2-cell embryo.•tDR-1:32-Gly–CCC–1 exhibited the best expression stability in the stages beyond the 2-cell stage.•bta-miR-155 and tDR-39:75-Arg-CCG-2 are significantly up-regulated in the CM of blastocysts compared to degenerates.
The SARS‐CoV‐2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate ...the virus. Together, this calls for the development of safer methods for SARS‐CoV‐2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two‐step RT‐qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one‐step RT‐qPCR against SARS‐CoV‐2 using NP and OP samples. We furthermore tested a SARS‐CoV‐2 dilution series to determine the detection threshold. The method enables downstream detection of SARS‐CoV‐2 by RT‐qPCR with high sensitivity (~4 viral RNA copies per RT‐qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT‐qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR‐ready viral RNA and circumvents the need for commercial RNA purification kits.
Introdução: A identificação precisa e ágil das variantes circulantes do SARS-CoV-2 é fundamental para uma vigilância epidemiológica eficaz e para direcionar medidas de contenção da disseminação do ...vírus. Embora o Sequenciamento de Genoma Total (Whole Genome Sequencing - WGS) seja considerado o padrão ouro para a identificação de variantes, apresenta alto custo, tempo de entrega dos resultados prolongado e necessidade de equipe especializada. Por outro lado, técnicas baseadas na amplificação de ácidos nucleicos, como a transcrição reversa seguida de reação de polimerase em cadeia (RT-qPCR), têm a capacidade de identificar variantes por meio de mutações específicas de maneira mais rápida e econômica. Objetivo: Avaliar a concordância entre um kit comercial de genotipagem por RT-qPCR e o WGS na identificação de variantes do SARS-CoV-2. Métodos: Foram selecionadas 349 amostras positivas para SARS-CoV-2 de laboratórios públicos e privados da 4ª Coordenadoria Regional de Saúde do estado do RS, coletadas nas semanas epidemiológicas 13 a 27 de 2022. Essas amostras foram submetidas a RT-qPCR utilizando o kit 4Plex para a detecção de variantes de preocupação desenvolvido pelo Biomanguinhos. Além disso, as amostras foram sequenciadas nas plataformas MinION MK1C ou Illumina iSeq100. As sequências consenso foram geradas utilizando os protocolos de bioinformática ARTIC nCoV-2019 (MinION) ou Dragen COVID (Illumina). Os clados e linhagens foram atribuídos utilizando as ferramentas Nextclade e Pangolin, respectivamente. Resultados: No sequenciamento, 316 amostras foram classificadas como Ômicron, sendo a maioria pertencente à subvariante BA.2 (238 amostras). 33 amostras foram identificadas como variantes recombinantes, sendo a maioria da subvariante XAG (31 amostras). Na genotipagem por RT-qPCR, todas as variantes Ômicron foram identificadas corretamente, no entanto, não foi possível a identificação das variantes recombinantes. O Kappa de Cohen indicou 90,54% de concordância da RT-qPCR com o WGS. No entanto, não foi possível diferenciar as subvariantes utilizando a RT-qPCR. Conclusão: O RT-qPCR é uma metodologia rápida e econômica. No entanto, possui baixo poder discriminatório, sendo incapaz de identificar subvariantes e variantes recombinantes. Portanto, é necessário realizar o sequenciamento para obter essas informações. Assim, o RT-qPCR pode ser utilizado como uma metodologia complementar ao WGS para um rastreamento abrangente e mais rápido das variantes em circulação.
Multiple sclerosis (MS) is an autoimmune disease of the central
nervous system (CNS) caused by auto-reactive T cells against
myelin antigens. T-cell immunoglobulin mucin -3 (TIM-3) is a
negative ...regulator glycoprotein expressed by a range of immune
cells. A defect in TIM-3 regulation has been shown in multiple
sclerosis patients. This study was planned to investigate the
correlation between serum TIM-3 and TIM-3 gene expression in
a sample of Multiple Sclerosis Iraqi patients. Three ml of blood
samples were collected from fifty Iraqi patients who have
Multiple Sclerosis (men and women) with ages ranging between
20-57 years, and 50 healthy volunteers as a control group;
0.25ml of blood was put in Trizol tube for RNA extraction,
subsequently to estimate TIM-3 gene expression by one step
RT-qPCR, and 2.75 ml of blood placed into gel tube for
determination TIM-3 serum level by enzyme-linked
immunosorbent assay (ELISA), the Statistical analysis was done
by using program of Statistical Analysis System (SAS). There
was a significant increase (P ≤ 0.05) in TIM-3 gene expression
for patients (5.30-fold) when compared with control (7.86-fold).
Moreover, the result demonstrated a high significant elevated (P
≤ 0.01) in TIM-3 serum level of patients (0.398 pg/ml) as
compared to control (3.17 pg/ml. Furthermore, the findings
showed a strong positive association between TIM-3 serum
level and TIM-3 mRNA expression with significant differences.
The current study concluded that the TIM-3 gene expression and
TIM-3 serum level were high in MS patients, and there was a
direct positive relationship between TIM-3 gene expression and
TIM-3 serum level.
Keywords: MS, TIM-3, RT-qPCR.,ELISA
•A total of 51 known miRNAs and 95 novel miRNAs were identified from the nine small RNA libraries.•6 down-regulated DE miRNAs responded to the hypo-salinity stress.•1 up-regulated and 5 ...down-regulated DE miRNAs responded to the hyper-salinity stress.•931 and 768 potential target genes were respectively predicted from the hypo- and hyper-salinity stress.•Genes involved in the vesicle-mediated transport and metal ion binding responded to the hypo-salinity stress.•Genes related to the signal transduction and metabolic process responded to the hyper-salinity stress.
The Hong Kong oyster, Crassostrea hongkongensis, is a significant bivalve species with economic importance. It primarily inhabits the estuarine intertidal zones in southern China, making it susceptible to salinity fluctuations. Consequently, investigating the molecular mechanisms governing salinity regulation in C. hongkongensis is essential. In this study, we conducted miRNA-seq on C. hongkongensis to compare miRNA expression differences under varying salinities (5‰, 25‰, and 35‰). The miRNA sequencing revealed 51 known miRNAs and 95 novel miRNAs across nine small RNA libraries (S5, S25, and S35). Among these miRNAs, we identified 6 down-regulated differentially expressed (DE) miRNAs in response to hypo-salinity stress (5‰), while 1 up-regulated DE miRNA and 5 down-regulated DE miRNAs were associated with hyper-salinity stress (35‰). Additionally, we predicted 931 and 768 potential target genes for hypo- and hyper-salinity stress, respectively. Functional gene annotation indicated that the target genes under hypo-salinity stress were linked to vesicle-mediated transport and metal ion binding. Conversely, those under hyper-salinity stress were primarily involved in signal transduction and metabolic processes. These findings have provided insights into the regulatory role of miRNAs, their potential target genes and associated pathways in oyster hypo- and hyper-salinity stress, which establish a foundation for future studies on the roles of miRNAs in salinity acclimation mechanisms in C. hongkongensis.
Nickel compounds in dissolved form or as nanoparticles may affect planktonic invertebrates in marine ecosystems. Here, we assessed the physiological (naupliar mortality, egg production, egg hatching ...success) and molecular (quantitative gene expression) responses of the crustacean copepods Acartia clausi (indigenous Mediterranean species) and Acartia tonsa (model organism in ecotoxicology), to nickel nanoparticles (NiNPs) and nickel chloride (NiCl2), over time. We also measured NPs size and the temporal release of Ni ions in aqueous solution, through dynamic light scattering (DLS) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively.
Nauplii of A. clausi were highly vulnerable to NiCl2 in the 48 h acute test, with an EC50 in the range of Ni concentrations measured in polluted waters. Females of both species exhibited a decreased egg production and hatching success after the 4-day exposure to NiNPs. Molecular responses in A. clausi incubated in NiNPs and NiCl2 showed a stronger up- or down-regulation, compared to A. tonsa, of genes associated with detoxification (phospholipid-hydroperoxide glutathione peroxidase, glutathione-S-transferase sigma), oxidative stress (superoxide dismutase), nervous system functioning (acetylcholinesterase), and oogenesis (vitellogenin).
In conclusion, new information was here obtained on the effects of different forms of nickel on physiological and molecular responses of A. clausi, that could help to identify biomarker genes of exposure to be used as early-warning indicators. Our results also highlighted the need of employing indigenous copepod species to better evaluate the ecotoxicological impact of pollutants in different geographical area.
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•Acartia clausi nauplii were highly sensitive to NiCl2.•Nickel nanoparticles decreased female egg production in both species.•Nanoparticles release Ni ions, decreasing copepod hatching success.•NiCl2 down-regulated oxidative stress and nervous system genes in A. clausi.
The increase in incidence and geographical expansion of viruses transmitted by the Aedes mosquitoes, such as dengue (DENV) and zika (ZIKV) in the Americas, represents a burden for healthcare systems ...in tropical and subtropical regions. These and other under-detected arboviruses co-circulate in Costa Rica, adding additional complexity to their management due to their shared epidemiological behavior and similarity of symptoms in early stages. Since diagnostics of febrile illness is mostly based on clinical symptoms alone, we gathered acute-phase serum and urine from 399 samples of acute dengue-like cases from two healthcare facilities of Costa Rica, during an outbreak of arboviruses from July 2017 to May 2018, and tested them using molecular and serological methods. The analyses showed that of the clinically presumptive arbovirus cases that were reported, only 39.4% (n=153) of the samples were confirmed positive by RT-PCR to be DENV (DENV (10.3%), CHIKV (0.2%), ZIKV (27.3%), or mixed infections (1.5%). RT-PCR for other alphaviruses and flaviviruses, and PCR for Leptospira sp were negative. Furthermore, to assess flavivirus positivity in post-acute patients, the negative sera were tested against Dengue-IgM. 20% of sera were found positive, confounding even more the definitive number of cases, and emphasizing the need of several distinct diagnostic tools for accurate diagnostics. Molecular characterization of the prM and E genes from isolated viruses revealed that the American/Asian genotype of DENV-2 and the Asian lineage of ZIKV were circulating during this outbreak. Two different clades of DENV-2 American/Asian genotype were identified to co-circulate in the same region and a difference in the platelet and leukocyte count was noted between people infected with each clade, suggesting a putative distinct virulence. Our study sheds light on the necessity for healthcare strategies in managing arbovirus outbreaks, emphasizing the importance of comprehensive molecular and serological diagnostic approaches, as well as molecular characterization. This approach aids in enhancing our understanding of the clinical and epidemiological aspects of arboviral diseases during outbreaks. Our research highlights the need to strengthen training programs for health professionals and the need to increase research-based on laboratory evidence for diagnostic accuracy, guidance, development and implementation of public health interventions and epidemiological surveillance.
Background
In addition to non‐coding RNAs (lncRNAs) and microRNAs (miRNAs), circular RNAs (circRNAs) are endogenous RNAs with various functions, which have recently become a research hotspot. ...CircRNAs are a kind of closed circular RNA molecule widely existing in transcriptomes. Due to lack of free ends, they are not easily cleaved by RNase R, thus avoiding degradation. They are more stable than linear RNAs.
Methods
Data were collected through PubMed. The following search terms were used: “circular RNA,” “circRNA,” “cancer,” “mechanism,” “biogenesis,” “biomarker,” “diagnosis.” Only articles published in English were included.
Results
Most circRNAs express tissue/developmental stage specificity. Moreover, circRNAs are involved in the regulation of a variety of biological activities. In this review, we discuss the formation, classification, and biological functions of circRNAs, especially their molecular diagnostic values in common cancers, including gastric cancer (hsa_circ_002059, circ_LARP4, hsa_circ_0000190, hsa_circ_0000096, circ‐SFMBT2, and circ_PVT1), hepatocellular carcinoma (circ_104075, circRNA_100338, circ_MTO1, and circZKSCAN1), colorectal cancer (hsa_circ_0136666 and hsa_circ_0000523), lung cancer (hsa_circ_0006427, circ_100876, and circ_ABCB10), breast cancer (hsa_circ_0089105, circAGFG1, and circEPSTI1), bladder cancer (circFNDC3B and circTFRC), and esophageal squamous cell carcinoma (circ_100876 and circ‐DLG1).
Conclusion
CircRNAs not only play important roles in tumorigenesis, but also may become new diagnostic biomarkers.
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