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Streptococcus pneumoniae is a significant pathogen causing infectious diseases, including pneumonia, otitis media, septicemia, and meningitis. The introduction of multivalent vaccines has coincided with a remarkable decrease in the number of pneumococcal-related deaths. Despite this, pneumococcal infection remains a significant cause of death among children under 5 years old and adults aged 65 or older at a global level. Therefore, early detection of S. pneumoniae infection is crucial for prognosis of pneumococcal infection patients. In this study, we evaluated the utility of a real-time multienzyme isothermal rapid amplification (MIRA) assay for detecting S. pneumoniae and other non-S. pneumoniae bacterial species. A primer-probe set targeting the S. pneumoniae lytA gene was designed, followed by optimization of parameters for the MIRA assay. At the same time, we validated the real-time MIRA assay for detecting S. pneumoniae using 79 clinical isolates identified by VITEK MS. The results showed a detection sensitivity and specificity of 100%. These results demonstrate that the designed real-time MIRA assay is a promising, rapid, simple, and reliable method for detecting S. pneumoniae infection in resource-limited areas. It has great potential for application in detecting not only S. pneumoniae but also other non-S. pneumoniae bacterial species.
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Técnicas de Amplificação de Ácido Nucleico , Infecções Pneumocócicas , Sensibilidade e Especificidade , Streptococcus pneumoniae , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Humanos , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodosRESUMO
The prevention and treatment strategies for traumatic infection often focus on the use of antibiotics, while eschew the combined treatment of the bacteria, their toxins, and inflammatory mediators. This might be a main reason the prognosis of wound victims has not improved. Although our previous work found that the combination of indomethacin (IND) and ciprofloxacin (CIP) could promote skin wound repair and enhance the immune function, the efficacy and safety of this strategy for severe traumatic infection-mediated complications remain unknown. Additionally, there is no study on the relevant target cells and molecular mechanisms. In this study, C57BL/6 adult male mice were modeled for severe traumatic infection, and the optimal doses of IND and CIP alone were determined. After that, the efficacy and safety of IND plus CIP in traumatic infection mice were explored. Then the differentially expressed genes of activated macrophages in this process were analysed and verified by transcriptomic methods and conventional experimental techniques. The role of a candidate signalling pathway (PI3K/Akt) in regulating macrophage function and drug combination therapy was evaluated. The results showed that IND plus CIP increased the survival rate, reduced the degree of inflammatory response, and enhanced the bacteriostatic effect in mice under traumatic infection. This combined therapy did not cause significant damage to the functions of important organs (liver, kidney, heart). In addition, IND combined with CIP induced macrophages to significantly change their expression levels of several cytokines, including interleukin (IL) -1ß, IL-6, IL-10, IL-22, IL-23A, IL-17A, IL-17F, cluster of differentiation (CD) 11b and other genes/encode proteins. Further study showed that intervention with the PI3K inhibitor LY294002 modulated the secretion function of the above-mentioned macrophages and Akt activation (phosphorylation at serine 473). IND plus CIP can regulate macrophage function through the PI3K/Akt signalling pathway and improve the prognosis of severe traumatic infected mice. This may be a new therapeutic strategy for the prevention and treatment of severe traumatic infection.
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Corrosion-resistant steel plays a vital role in marine steel structures. This study developed an SS304/HRB400 stainless-steel-clad rebar for application in a cross-sea bridge in Zhejiang Province. CO2 gas shielded welding was employed in the prefabricated steel structure, with SS304 steel as the welding wire. This study investigated the welding on the corrosion resistance of clad rebars and explored corrosion protection measures for welded joints.The results indicated that refined grains appeared in both stainless steel and carbon steel due to distinct dynamic recrystallization (DRX) during welding. The corrosion resistance, as determined by potentiodynamic polarization curve analysis of the material's interaction with the solution ranked as follows: clad rebar (polished) > clad rebar welding (CRW) > painting the clad rebar after welding (PCRW) > clad rebar (unpolished) > carbon-steel welding (CSW) > carbon-steel bar > cold spraying zinc after clad rebar welding (ZCRW). However, an accelerated corrosion test with four samples for 600 s with a corrosion current of 0.8 A revealed minimal corrosion damage on zinc-coated surfaces. Hence, welding joints for clad steel structures are considered feasible and must be subject to cold zinc spraying after polishing to enhance their corrosion resistance.
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We report a clinical isolate of Burkholderia thailandensis 2022DZh obtained from a patient with an infected wound in southwest China. Genomic analysis indicates that this isolate clusters with B. thailandensis BPM, a human isolate from Chongqing, China. We recommend enhancing monitoring and surveillance for B. thailandensis infection in both humans and livestock.
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Infecções por Burkholderia , Burkholderia , Filogenia , Infecção dos Ferimentos , Humanos , Masculino , Burkholderia/genética , Burkholderia/isolamento & purificação , Burkholderia/classificação , Infecções por Burkholderia/microbiologia , Infecções por Burkholderia/diagnóstico , China/epidemiologia , Genoma Bacteriano , Infecção dos Ferimentos/microbiologia , Pessoa de Meia-IdadeRESUMO
AIM: This study assessed the efficacy and safety of co-administering retagliptin and henagliflozin versus individual agents at corresponding doses in patients with type 2 diabetes mellitus who were inadequately controlled with metformin. METHODS: This multicentre, phase 3 trial consisted of a 24-week, randomized, double-blind, active-controlled period. Patients with glycated haemoglobin (HbA1c) levels between 7.5% and 10.5% were randomized to receive once-daily retagliptin 100 mg (R100; n = 155), henagliflozin 5 mg (H5; n = 156), henagliflozin 10 mg (H10; n = 156), co-administered R100/H5 (n = 155), or R100/H10 (n = 156). The primary endpoint was the change in HbA1c from baseline to week 24. RESULTS: Based on the primary estimand, the least squares mean reductions in HbA1c at week 24 were significantly greater in the R100/H5 (-1.51%) and R100/H10 (-1.54%) groups compared with those receiving the corresponding doses of individual agents (-0.98% for R100, -0.86% for H5 and -0.95% for H10, respectively; p < .0001 for all pairwise comparisons). Achievement of HbA1c <7.0% at week 24 was observed in 27.1% of patients in the R100 group, 21.2% in the H5 group, 24.4% in the H10 group, 57.4% in the R100/H5 group and 56.4% in the R100/H10 group. Reductions in fasting plasma glucose and 2-h postprandial glucose were also more pronounced in the co-administration groups compared with the individual agents at corresponding doses. Decreases in body weight and systolic blood pressure were greater in the groups containing henagliflozin than in the R100 group. The incidence rates of adverse events were similar across all treatment groups, with no reported episodes of severe hypoglycaemia. CONCLUSIONS: For patients with type 2 diabetes mellitus inadequately controlled by metformin monotherapy, the co-administration of retagliptin and henagliflozin yielded more effective glycaemic control through 24 weeks compared with the individual agents at their corresponding doses.
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Glicemia , Diabetes Mellitus Tipo 2 , Quimioterapia Combinada , Hemoglobinas Glicadas , Hipoglicemiantes , Metformina , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Masculino , Pessoa de Meia-Idade , Feminino , Método Duplo-Cego , Metformina/administração & dosagem , Metformina/uso terapêutico , Hemoglobinas Glicadas/análise , Hemoglobinas Glicadas/efeitos dos fármacos , Hemoglobinas Glicadas/metabolismo , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/uso terapêutico , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Idoso , Adulto , Resultado do TratamentoRESUMO
BACKGROUND: It is challenging to identify residual or recurrent fistulas from the surgical region, while MR imaging is feasible. The aim was to use dynamic contrast-enhanced MR imaging (DCE-MRI) technology to distinguish between active anal fistula and postoperative healing (granulation) tissue. METHODS: Thirty-six patients following idiopathic anal fistula underwent DCE-MRI. Subjects were divided into Group I (active fistula) and Group IV (postoperative healing tissue), with the latter divided into Group II (≤ 75 days) and Group III (> 75 days) according to the 75-day interval from surgery to postoperative MRI reexamination. MRI classification and quantitative analysis were performed. Correlation between postoperative time intervals and parameters was analyzed. The difference of parameters between the four groups was analyzed, and diagnostic efficiency was tested by receiver operating characteristic curve. RESULTS: Wash-in rate (WI) and peak enhancement intensity (PEI) were significantly higher in Group I than in Group II (p = 0.003, p = 0.040), while wash-out rate (WO), time to peak (TTP), and normalized signal intensity (NSI) were opposite (p = 0.031, p = 0.007, p = 0.010). Area under curves for discriminating active fistula from healing tissue within 75 days were 0.810 in WI, 0.708 in PEI, 0.719 in WO, 0.783 in TTP, 0.779 in NSI. All MRI parameters were significantly different between Group I and Group IV, but not between Group II and Group III, and not related to time intervals. CONCLUSION: In early postoperative period, DCE-MRI can be used to identify active anal fistula in the surgical area. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR2000033072.
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Meios de Contraste , Fístula Retal , Humanos , Imageamento por Ressonância Magnética/métodos , Curva ROC , Fístula Retal/diagnóstico por imagem , Fístula Retal/etiologia , Fístula Retal/cirurgia , Aumento da Imagem/métodosRESUMO
BACKGROUND: Cancer stem cells (CSCs) are a small population of cells in tumor tissues that can drive tumor initiation and promote tumor progression. A small number of previous studies indirectly mentioned the role of F-box and WD repeat domain-containing 7 (FBXW7) as a tumor suppressor in Triple-negative breast cancer (TNBC). However, few studies have focused on the function of FBXW7 in cancer stemness in TNBC and the related mechanism. METHODS: We detected FBXW7 by immunohistochemistry (IHC) in 80 TNBC patients. FBXW7 knockdown and overexpression in MD-MBA-231 and HCC1937 cell models were constructed. The effect of FBXW7 on malignant phenotype and stemness was assessed by colony assays, flow cytometry, transwell assays, western blot, and sphere formation assays. Immunoprecipitation-Mass Spectrometry (IP-MS) and ubiquitination experiments were used to find and verify potential downstream substrate proteins of FBXW7. Animal experiments were constructed to examine the effect of FBXW7 on tumorigenic potential and cancer stemness of TNBC cells in vivo. RESULTS: The results showed that FBXW7 was expressed at low levels in TNBC tissues and positively correlated with prognosis of TNBC patients. In vitro, FBXW7 significantly inhibited colony formation, cell cycle progression, cell migration, EMT process, cancer stemness and promotes apoptosis. Further experiments confirmed that chromodomain-helicase-DNA-binding protein 4 (CHD4) is a novel downstream target of FBXW7 and is downregulated by FBXW7 via proteasomal degradation. Moreover, CHD4 could promote the nuclear translocation of ß-catenin and reverse the inhibitory effect of FBXW7 on ß-catenin, and ultimately activate the Wnt/ß-catenin pathway. Rescue experiments confirmed that the FBXW7-CHD4-Wnt/ß-catenin axis was involved in regulating the maintenance of CSC in TNBC cells. In animal experiments, FBXW7 reduced CSC marker expression and suppressed TNBC cell tumorigenesis in vivo. CONCLUSIONS: Taken together, these results highlight that FBXW7 degrades CHD4 protein through ubiquitination, thereby blocking the activation of the Wnt/ß-catenin pathway to inhibit the stemness of TNBC cells. Thus, targeting FBXW7 may be a promising strategy for therapeutic intervention against TNBC.
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Neoplasias de Mama Triplo Negativas , Animais , Humanos , beta Catenina , Carcinogênese , Transformação Celular Neoplásica , Proteína 7 com Repetições F-Box-WD/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
AIM: To evaluate the effect of noiiglutide as an adjunct to lifestyle intervention on the reduction in body weight and tolerability in obese Chinese adults without diabetes. MATERIALS AND METHODS: In this 24-week, randomized, double-blind, placebo-controlled phase 2 trial, 254 obese adults with a body mass index of 28.0-40.0 kg/m2 and without diabetes were enrolled. Participants were initially randomized in a 1:1:1 ratio to one of three dose levels: 0.12, 0.24, or 0.36 mg of the study treatment. Within each dose level, participants were further randomized in a 3:1 ratio to receive either subcutaneous injection of noiiglutide or a matching placebo. The primary endpoint was the change in body weight from baseline to week 24. RESULTS: Across all noiiglutide dosage levels, least squares mean reductions in body weight from baseline to week 24 ranged from 8.03 to 8.50 kg, compared with 3.65 kg in the placebo group (all p-values <.0001). In the noiiglutide groups (0.12, 0.24, 0.36 mg/day), a significantly higher proportion of participants achieved a weight loss ≥5% (68.8%, 60.0%, 73.0%) and ≥10% (37.5%, 36.9%, 39.7%), compared with the pooled placebo group (≥5%: 29.0%; ≥10%: 8.1%). Gastrointestinal adverse events, such as nausea, diarrhoea and vomiting, were more common in all noiiglutide groups (15.4%-30.2%, 18.8%-22.2%, 15.6%-18.5%) than in the pooled placebo group (8.1%, 6.5%, 0%). CONCLUSIONS: In obese Chinese adults without diabetes, once-daily subcutaneous noiiglutide significantly reduced body week at week 24 compared with placebo, and had a manageable safety profile, primarily involving gastrointestinal disorders.
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Diabetes Mellitus Tipo 2 , Hipoglicemiantes , Adulto , Humanos , Hipoglicemiantes/uso terapêutico , Peso Corporal , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/induzido quimicamente , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/induzido quimicamente , Injeções Subcutâneas , China/epidemiologia , Método Duplo-Cego , Resultado do TratamentoRESUMO
Background: This prespecified subgroup analysis of the FIDELIO-DKD trial aimed to evaluate the efficacy and safety of finerenone in patients with chronic kidney disease (CKD) and type 2 diabetes mellitus (T2DM) in China. Methods: 372 participants were recruited from 67 centers in China and randomized 1:1 to oral finerenone or placebo with standard therapy for T2DM. The primary composite outcome included kidney failure, sustained decrease of estimated glomerular filtration rate ≥40% from baseline over at least 4 weeks, or renal death. The key secondary composite outcome included death from cardiovascular causes, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Results: After a median follow-up of 30 months, the finerenone group showed a relative risk reduction (RRR) of 41% (hazard ratio [HR] = 0.59, 95% confidence interval [CI], 0.39-0.88; p = 0.009) for the primary composite outcome compared with placebo, consistent across its components with treatment benefits with finerenone. Based on an absolute between-group difference of 12.2% after 30 months, the number of patients who needed to be treated with finerenone to prevent one primary outcome event was eight (95% CI: 4-84). For the key secondary composite outcome, the finerenone group showed a RRR of 25% (HR = 0.75, 95% CI, 0.38-1.48; p = 0.408). Adverse events were similar between the two groups. The effects of finerenone on blood pressure were modest. No gynecomastia events were reported in the study. Hyperkalemia leading to discontinuation occurred in eight (4.3%) and two (1.1%) participants in the finerenone and control groups, respectively. The incidence of acute kidney injury was comparable between the two groups (1.6% vs. 1.6%). Conclusions: Finerenone resulted in lower risks of CKD progression than placebo and a balanced safety profile in Chinese patients with CKD and T2DM.
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Bloodstream infection (BSI) caused by bacteria is highly pathogenic and lethal, and easily develops whole-body inflammatory state. Immediate identification of disease-causing bacteria can improve patient prognosis. Traditional testing methods are not only time-consuming, but such tests are limited to laboratories. Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) holds great promise for rapid nucleic acid detection, but the uncapping operation after amplification easily contaminates laboratories. Therefore, the establishment of a more effective integrated isothermal amplification system has become an urgent problem to be solved. In this study, we designed and fabricated a hermetically sealed integrated isothermal amplification system. Combining with this system, a set of RPA-LFD assays for detecting S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI were established and evaluated. The whole process could be completed in less than 15 min and the results can be visualized by the naked eye. The developed RPA-LFD assays displayed a good sensitivity, and no cross-reactivity was observed in seven similar bacterial genera. The results obtained with 60 clinical samples indicated that the developed RPA-LFD assays had high specifcity and sensitivity for identifying S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI. In conclusion, our results showed that the developed RPA-LFD assay is an alternative to existing PCR-based methods for detection of S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI in primary hospitals.
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Due to the unavailability of source domain data encountered in unsupervised domain adaptation, there has been an increasing number of studies on source-free domain adaptation (SFDA) in recent years. To better solve the SFDA problem and effectively leverage the multi-modal information in medical images, this paper presents a novel SFDA method for multi-modal stroke lesion segmentation in which evidential deep learning instead of convolutional neural network. Specifically, for multi-modal stroke images, we design a multi-modal opinion fusion module which uses Dempster-Shafer evidence theory for decision fusion of different modalities. Besides, for the SFDA problem, we use the pseudo label learning method, which obtains pseudo labels from the pre-trained source model to perform the adaptation process. To solve the unreliability of pseudo label caused by domain shift, we propose a pseudo label filtering scheme using shadowed sets theory and a pseudo label refining scheme using evidential uncertainty. These two schemes can automatically extract unreliable parts in pseudo labels and jointly improve the quality of pseudo labels with low computational costs. Experiments on two multi-modal stroke lesion datasets demonstrate the superiority of our method over other state-of-the-art SFDA methods.
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Insufficient light during the growth periods has become one of the main factors restricting maize yield with global climate change. Exogenous hormones application is a feasible measure to alleviate abiotic stresses on crop productivity. In this study, a field trial was conducted to investigate the effects of spraying exogenous hormones on yield, dry matter (DM) and nitrogen (N) accumulation, leaf carbon and N metabolism of fresh waxy maize under weak-light stress in 2021 and 2022. Five treatments including natural light (CK), weak-light after pollination (Z), spraying water (ZP1), exogenous Phytase Q9 (ZP2) and 6-benzyladenine (ZP3) under weak-light after pollination were set up using two hybrids suyunuo5 (SYN5) and jingkenuo2000 (JKN2000). Results showed that weak-light stress significantly reduced the average fresh ear yield (49.8%), fresh grain yield (47.9%), DM (53.3%) and N accumulation (59.9%), and increased grain moisture content. The net photosynthetic rate (Pn), transpiration rate (Tr) of ear leaf after pollination decreased under Z. Furthermore, weak-light decreased the activities of RuBPCase and PEPCase, nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in ear leaves, and increased malondialdehyde (MDA) accumulation. And the decrease was greater on JKN2000. While ZP2 and ZP3 treatments increased the fresh ear yield (17.8%, 25.3%), fresh grain yield (17.2%, 29.5%), DM (35.8%, 44.6%) and N (42.5%, 52.4%) accumulation, and decreased grain moisture content compared with Z. The Pn, Tr increased under ZP2 and ZP3. Moreover, the ZP2 and ZP3 treatments improved the activities of RuBPCase, PEPCase; NR, GS, GOGAT; SOD, CAT, POD in ear leaves, and decreased MDA content during grain filling stage. The results also showed the mitigative effect of ZP3 was greater than ZP2, and the improvement effect was more significant on JKN2000.
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Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumors and spread into the peripheral bloodstream. Mutation detection in CTCs can reveal vital genetic information about the tumors and can be used for "liquid biopsy" to indicate cancer treatment and targeted medication. However, current methods to measure the mutations in CTCs are based on PCR or DNA sequencing which are cumbersome and time-consuming and require sophisticated equipment. These largely limited their applications especially in areas with poor healthcare infrastructure. Here we report a simple, convenient, and rapid method for mutation detection in CTCs, including an example of a deletion at exon 19 (Del19) of the epidermal growth factor receptor (EGFR). CTCs in the peripheral blood of NSCLC patients were first sorted by a double spiral microfluidic chip with high sorting efficiency and purity. The sorted cells were then lysed by proteinase K, and the E19del mutation was detected via real-time recombinase polymerase amplification (RPA). Combining the advantages of microfluidic sorting and real-time RPA, an accurate mutation determination was realized within 2 h without professional operation or complex data interpretation. The method detected as few as 3 cells and 1% target variants under a strongly interfering background, thus, indicating its great potential in the non-invasive diagnosis of E19del mutation for NSCLC patients. The method can be further extended by redesigning the primers and probes to detect other deletion mutations, insertion mutations, and fusion genes. It is expected to be a universal molecular diagnostic tool for real-time assessment of relevant mutations and precise adjustments in the care of oncology patients.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Microfluídica , Recombinases/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Mutação , Células Neoplásicas Circulantes/patologiaRESUMO
Introduction. Burkholderia thailandensis is a clinically rare opportunistic pathogen in the genus Burkholderia, and the genomic features and virulence characteristics of B. thailandensis strains that cause human infection remain unclear.Gap Statement. B. thailandensis strains with different virulence induce different host innate immune responses in vitro.Aim. This work aimed to understand the sequence diversity, phylogenetic relationship, and virulence of B. thailandensis BPM causing human infection.Methodology. The comparative molecular and genomic analyses, and mouse infection studies were applied to analyse the virulence and genomic features of B. thailandensis BPM originating from China.Results. The whole genome sequence analysis showed that the genomes of BPM and other avirulent B. thailandensis strains were broadly similar, comprising two highly syntenic chromosomes with comparable numbers of coding regions (CDs), protein family distributions, and horizontally acquired genomic islands. By examining species-specific genomic regions, we obtained molecular explanations for previously known differences in virulence and discovered the potential specific virulence-associated genes of BPM, which likely work together to confer the virulence of BPM. Significantly reduced LD50 and survival rates during mouse infection experiments were found in BPM compared to the avirulent B. thailandensis E264 (BtE264).Conclusion. Taken together, the results of this study provide basic information on the genomic features and virulence characteristics of the virulent B. thailandensis strain BPM, which is helpful for understanding its evolution as it relates to pathogenesis and environmental adaptability.
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Burkholderia , Humanos , Animais , Camundongos , Virulência , Filogenia , Burkholderia/genética , Burkholderia/metabolismo , GenômicaRESUMO
This case report describes numerous warm, red, edematous, tender, fluctuant, and purulent draining nodules on the abdomen following a catgut implant procedure.
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Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Humanos , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Pele , Surtos de Doenças , Antibacterianos/uso terapêuticoRESUMO
The COVID-19 pandemic has spread worldwide, and rapid detection of the SARS-CoV-2 virus is crucial for infection surveillance and epidemic control. This study developed a centrifugal microfluidics-based multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay for endpoint fluorescence detection of the E, N, and ORF1ab genes of SARS-CoV-2. The microscope slide-shaped microfluidic chip could simultaneously accomplish three target genes and one reference human gene (i.e., ACTB) RT-RPA reactions in 30 min, and the sensitivity was 40 RNA copies/reaction for the E gene, 20 RNA copies/reaction for the N gene, and 10 RNA copies/reaction for the ORF1ab gene. The chip demonstrated high specificity, reproducibility, and repeatability. Chip performance was also evaluated using real clinical samples. Thus, this rapid, accurate, on-site, and multiplexed nucleic acid test microfluidic chip would significantly contribute to detecting patients with COVID-19 in low-resource settings and point-of-care testing (POCT) and, in the future, could be used to detect emerging new variants of SARS-CoV-2.
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BACKGROUND: Isothermal exponential amplification reaction (EXPAR) is an emerging amplification technique that is most frequently used to amplify microRNA (miRNA). However, EXPAR also exhibits non-specific background amplification in the absence of the targeted sequence, which limits the attainable assay sensitivity of EXPAR. METHODS AND RESULTS: A novel modified isothermal EXPAR based on circular amplification templates (cEXPAR) was developed in this study. The circular template consists of two same linear fragments that complement the target sequence, and these two linear fragments are separated by two nicking agent recognition sequences (NARS). Compared with the linear structure template, this circular template allows DNA or RNA fragments to be randomly paired with two repeated sequences and can be successfully amplified. This reaction system developed in this study could rapidly synthesize short oligonucleotide fragments (12-22 bp) through simultaneous nicking and displacement reactions. Highly sensitive chain reactions can be specifically triggered by as low as a single copy of target molecule, and non-specific amplification can be effectively eliminated in this optimized system. Moreover, the proposed approach applied to miRNA test can discriminate single-nucleotide variations between miRNAs. CONCLUSION: The newly developed cEXPAR assay provides a useful alternative tool for rapid, sensitive, and highly specific detection of miRNAs.
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MicroRNAs , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA/química , OligonucleotídeosRESUMO
Hepatocellular carcinoma (HCC) is one of the most common malignancies with a hallmark of aberrant metabolism. The mechanism of long noncoding RNAs (lncRNAs) underlying the aggressive behaviors and glycolysis of HCC is poorly understood. In this study, we identified, via microarray, novel lncRNA NONHSAT024276 as a potential tumor suppressor in HCC. The downregulation of NONHSAT024276 closely correlated with larger tumor volume and higher aspartate transaminase levels. Functional experiments were performed to verify the role of NONHSAT024276 in HCC progression, and the negative effects of NONHSAT024276 expression on cell proliferation and migration were identified. Mechanistically, NONHSAT024276 directly bound to polypyrimidine tract-binding protein 1 (PTBP1), downregulating it and forming a feedback loop. Furthermore, NONHSAT024276 increased the ratio of M1 and M2 isoforms of pyruvate kinase (PKM1/PKM2) and also obstructed the PTBP1/PKM-mediated glycolysis. Finally, the rescue assays confirmed that NONHSAT024276 functioned in HCC via downregulating PTBP1 to increase the PKM1/PKM2 ratio. Hence, this study supported a model in which NONHSAT024276 downregulated PTBP1 and formed a feedback loop to increase the PKM1/PKM2 ratio to inhibit glycolysis and progression of HCC, opening new prospects for preventing or treating HCC.
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Carcinoma Hepatocelular , Ribonucleoproteínas Nucleares Heterogêneas , Neoplasias Hepáticas , Proteína de Ligação a Regiões Ricas em Polipirimidinas , RNA Longo não Codificante , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Retroalimentação , Glicólise/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Piruvato Quinase/genética , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Drought stress (DS) induced by post-silking have a major impact on the yield and quality of maize. In this study, the effects of different degrees of DS after pollination on grain filling, starch and protein metabolism, and functional properties were investigated using two waxy maize cultivars as materials. The levels of DS that were investigated were 'mild water stress' (WS1), 'moderate water stressed' (WS2), and 'severe waterstressed' (WS3). RESULTS: Drought stress decreased grain fresh weight, dry weight, and moisture content in both cultivars during grain filling, and reduced kernel number, kernel weight, and grain yield at maturity. The effect on grain development and yield formation gradually increased with drought aggravation. The water stress (WS) treatment downregulated the enzymatic activities related to starch biosynthesis during grain-filling process, accompanied by a decrease in soluble sugar and starch deposition. The WS treatment increased the enzymatic activities involved in protein synthesis during grain-filling process, thereby increasing the protein content of grains. On average, WS2 and WS3 treatments reduced the pasting viscosities and increased the gelatinization temperatures of grains, with WS3 having the greatest effect. However, the changes of setback viscosity, gelatinization enthalpy, retrogradation enthalpy, and retrogradation percentage under WS treatment were inconsistent in both cultivars. Pearson correlation analysis showed that starch content was negatively correlated with gelatinization temperatures and positively correlated with pasting viscosities in both cultivars. However, grain pasting and gelatinization properties have opposite correlations with protein content and starch content. CONCLUSIONS: These findings suggest that post-silking DS regulated the grain-filling process and starch and protein biosynthesis, which influenced grain yield and quality. © 2022 Society of Chemical Industry.