RESUMEN
Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.
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Infecciones del Sistema Respiratorio , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/microbiología , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Dispositivos Laboratorio en un Chip , Técnicas de Amplificación de Ácido Nucleico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Bacterias/aislamiento & purificación , Bacterias/genética , Recombinasas/metabolismo , Automatización , Infecciones Bacterianas/diagnósticoRESUMEN
AIMS: This study aimed to characterize the first complete genome of Corynebacterium parakroppenstedtii and clarify the evolutionary relationship in the Corynebacterium kroppenstedtii complex (CKC) by using comparative genomics analysis. METHODS AND RESULTS: The genome of isolate yu01 from a breast specimen was sequenced, and 35 CKC genomes were collected. Analysis of 16S rRNA, rpoB, and fusA suggested ambiguous identification, whereas ANI analysis assigned isolate yu01 as Coryne. parakroppenstedtii. The fourth genospecies "Corynebacterium aliikroppenstedtii" was identified in CKC. Comparative genomics analysis suggested that the genomic arrangement in CKC was highly conserved. A total of 43 potential virulence genes and 79 species-specific genes were detected. Most genome-based phylogenetic analysis were incapable of resolving the interspecific evolutionary relationships among CKCs. A total of 20 core genes were found to be distinguishable in CKC. CONCLUSIONS: This study suggested the limited divergence and unavailability of normal single gene-based identification in CKC and questioned the precise species of strains associated with mastitis, identified as Coryne. kroppenstedtii in previous studies. The 20 genes showed potential to enhance the methods for the identification and epidemiological investigation of CKC.
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Infecciones por Corynebacterium , Mastitis , Femenino , Humanos , Infecciones por Corynebacterium/complicaciones , Infecciones por Corynebacterium/microbiología , Filogenia , ARN Ribosómico 16S/genética , Corynebacterium/genética , Mastitis/complicaciones , GenómicaRESUMEN
During our studies on the microorganism diversity from air of manufacturing shop in a pharmaceutical factory in Shandong province, China, a Gram-stain-positive, aerobic, cocci-shaped bacterium, designated LY-0111T, was isolated from a settling dish. Strain LY-0111T grew at temperature of 10-42 °C (optimum 35 °C), pH of 5.0-10.0 (optimum pH 7.0) and NaCl concentration of 1-12% (optimum 0.5-3%, w/v). Based on the 16S rRNA gene sequence analysis, the strain shared the highest sequence similarities to Nesterenkonia halophila YIM 70179T (96.2%), and was placed within the radiation of Nesterenkonia species in the phylogenetic trees. The genome of the isolate was sequenced, which comprised 2,931,270 bp with G + C content of 66.5%. A supermatrix tree based on the gene set bac120 indicated that LY-0111T was close related to Nesterenkonia xinjiangensis YIM 70097T (16S rRNA gene sequence similarity 95.3%). Chemotaxonomic analysis indicated that the main respiratory quinones were MK-7, MK-8, and MK-9, the predominant cellular fatty acids were anteiso-C15:0 and iso-C15:0, and the major polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. According to the phenotypic, chemotaxonomic and phylogenetic features, strain LY-0111T is considered to represent a novel species, for which the name Nesterenkonia aerolata sp. nov. is proposed. The type strain is LY-0111T (= JCM 36375T = GDMCC 1.3945T). In addition, Nesterenkonia jeotgali was proposed as a later synonym of Nesterenkonia sandarakina, according to the ANI (96.8%) and dDDH (72.9%) analysis between them.
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Ácidos Grasos , Fosfolípidos , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Hibridación de Ácido Nucleico , Ácidos Grasos/análisis , Preparaciones Farmacéuticas , China , ADN Bacteriano/genética , Técnicas de Tipificación Bacteriana , Fosfolípidos/análisisRESUMEN
To achieve high-throughput ultrasensitive detection of mycotoxins in food, a functional DNA-guided transition-state CRISPR/Cas12a microfluidic biosensor (named FTMB) was successfully constructed. The signal transduction CRISPR/Cas12a strategy in FTMB has utilized DNA sequences with a specific recognition function and activators to form trigger switches. Meanwhile, the transition-state CRISPR/Cas12a system was constructed by adjusting the composition ratio of crRNA and activator to achieve a high response for low concentrations of target mycotoxins. On the other hand, the signal enhancement of FTMB has efficiently integrated the signal output of quantum dots (QDs) with the fluorescence enhancement effect of photonic crystals (PCs). The construction of universal QDs for the CRISPR/Cas12a system and PC films matching the photonic bandgap produced a significant signal enhancement by a factor of 45.6. Overall, FTMB exhibited a wide analytic range (10-5-101 ng·mL-1), low detection of limit (fg·mL-1), short detection period (â¼40 min), high specificity, good precision (coefficients of variation <5%), and satisfactory practical sample analysis capacity (the consistency with HPLC at 88.76%-109.99%). It would provide a new and reliable solution for the rapid detection of multiple small molecules in the fields of clinical diagnosis and food safety.
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Técnicas Biosensibles , Micotoxinas , Microfluídica , Bioensayo , Cromatografía Líquida de Alta Presión , Transducción de Señal , Sistemas CRISPR-CasRESUMEN
Palmarumycin P3 (PP3) reduces fluconazole-induced MDR1 transcription to reverse azole resistance in clinical Candida strains. Here, we demonstrated that PP3 restores the susceptibility to several antifungal drugs for Candida albicans strains with gain-of-function mutations in the transcription factor Mrr1. In addition, PP3 inhibits the efflux of Mdr1 substrates by C. albicans strains harboring hyperactive MRR1 alleles. Molecular docking revealed that PP3 is a potential Mdr1 blocker that binds to the substrate binding pocket of Mdr1.
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Azoles , Regulación Fúngica de la Expresión Génica , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antifúngicos/metabolismo , Antifúngicos/farmacología , Azoles/metabolismo , Azoles/farmacología , Candida albicans/genética , Candida albicans/metabolismo , Farmacorresistencia Fúngica/genética , Fluconazol/metabolismo , Fluconazol/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Simulación del Acoplamiento MolecularRESUMEN
The role of complement system in heart diseases is controversial. Besides, the mechanisms by which complement components participate in cardiac remodeling (CR) and heart failure during uremia are unclear. In this study, 5/6 nephrectomy was performed to adult mice to establish the uremic model and CR deteriorated over the course of uremia. Although complement pathways were not further activated over the course of the disease, soluble complement factor B (CFB) was upregulated at post-nephrectomy day 90 (PNx90) compared with PNx30. Further, CFB notably deteriorated CR in uremic mice but this effect was reversed by depletion of macrophages with liposomal clodronate. In vivo and in vitro CFB upregulated arginase 1 (ARG1) expression, increased ARG1 enzymatic activity, and stimulated the syntheses of ornithine, leading to polyamine overproduction in macrophages. Putrescine, an important polyamine, promoted cardiac fibroblast proliferation and collagen production, resulting in progressive CR. In vivo the inhibition of ARG1 activity with Nω -hydroxyl-l-arginine remarkably improved the general survival rates, inhibited the infiltration of cardiac fibroblasts, and alleviated progression of CR in uremic mice. Taken together, the CFB-ARG1-putrescine axis is related to progression of CR and ARG1 hyperactivity in macrophages may provide a novel therapeutic target against the heart injury in uremia.
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Arginasa/metabolismo , Factor B del Complemento/metabolismo , Uremia/metabolismo , Remodelación Ventricular/fisiología , Animales , Arginina/metabolismo , Macrófagos/metabolismo , Ratones , Óxido Nítrico/metabolismo , Ornitina/metabolismo , Poliaminas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Bacterial infections cause considerable morbidity and expensive healthcare costs. The prescription of broad-spectrum antimicrobial drugs results in failure of treatment or overtreatment and exacerbates the spread of multidrug-resistant pathogens. There is an emergent demand for rapid and accurate methods to identify pathogens and conduct personalized therapy. Here, we develop a herringbone microfluidic chip integrated with vancomycin modified magnetic beads (herringbone-VMB microchip) to enrich pathogens. The enriched pathogens are identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The herringbone-VMB microchip applies passive mixing of bacterial samples by generating microvortices, which significantly enhances the interaction between bacteria and vancomycin modified magnetic beads and leads to more efficient enrichment compared to in-tube extraction. Four common pathogens in urinary tract infections are utilized to validate the method, and the capture efficiency of the bacteria from urine is up to 90%. The whole procedure takes 1.5 hours from enrichment to identification. This method shows potential in shortening the turnaround time in the clinical diagnosis of bacterial infections.
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Infecciones Bacterianas , Infecciones Urinarias , Bacterias , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/tratamiento farmacológico , Humanos , Microfluídica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Heart failure is the leading cause of mortality in patients with end-stage renal disease, and progressive cardiac remodeling is the key pathological basis of heart failure. However, the mechanism by which uremia-induced cardiac remodeling occurs is not well understood. Here, we showed that platelets were significantly activated in 5/6 nephrectomy-operated mice, and cardiac remodeling in the uremic mice was significantly improved when platelets were effectively depleted. A cardiac fibrosis-related gene expression profile revealed that Mmp7, encoding matrix metalloproteinase-7 (MMP-7), exhibited the greatest degree of downregulation in the hearts of uremic mice with platelets depleted. Using fluorescence-activated cell sorting, we discovered that MMP-7 was mainly expressed in M1 and M4 cardiac macrophages, although it was also extensively expressed in heart tissues. For the upstream therapeutic target, neutralization of platelet factor 4 (PF4) with monoclonal antibody not only significantly suppressed M4 macrophages in vivo, but also notably prevented collagen destruction in heart tissues. For the downstream therapeutic target, the pharmacological inhibition of MMP-7 with selective inhibitor failed to notably affect the platelet status, but significantly reduced heart collagen destruction in mice, a further indication that MMP-7 is a crucial downstream molecular target of platelet activation. In vitro, platelets interacted with macrophages and drove them to upregulate MMP-7 expression via free molecules, especially PF4. Taken together, the data suggest that MMP-7 is a key downstream target of platelet activation during uremia. Thus, MMP-7 is a likely and novel therapeutic target for intervention of cardiac remodeling during uremia.
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Plaquetas/metabolismo , Macrófagos/enzimología , Metaloproteinasa 7 de la Matriz/metabolismo , Activación Plaquetaria/fisiología , Remodelación Ventricular/fisiología , Animales , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Fallo Renal Crónico/complicaciones , Ratones , Uremia/complicacionesRESUMEN
Increasing clinical significance of coagulase-negative staphylococci requires effective methods for species identification and genotyping. In this study, six housekeeping genes (femA, ftsZ, gap, pyrH, rpoB, and tuf) with extensive allelic polymorphisms were identified and evaluated to develop a comprehensive multilocus sequence typing (MLST) scheme. Selected primers were capable of amplification of the six loci from all of the 180 Staphylococcus strains belonging to 18 different species. Sequence analysis of each locus (44-63 alleles) revealed higher nucleotide diversity than 16S rRNA (28 alleles). Phylogenetic analysis of the concatenated sequences (3054 bp) of the six loci provided accurate species identification and highly discriminatory typing for all the strains. Multilocus allelic analysis of the 180 Staphylococcus strains generated 103 different sequence profiles, suggesting high genetic diversity of the strains. For example, 30 S. aureus, 37 S. epidermidis, 32 S. haemolyticus, and 14 S. hominis strains were typed into 15, 21, 11, and 10 sequence profiles, respectively. Compared with published MLST schemes that restrict on a few particular species, this new scheme both achieved similar discrimination for typing S. aureus, S. epidermidis, S. haemolyticus, and S. hominis and provided sufficient discriminatory power for typing additional opportunistic species, such as S. cohnii, S. capitis, and S. warneri. Importantly, the comprehensive MLST scheme for Staphylococcus strains provides a better genotyping tool for understanding the phylogeny of coagulase-positive Staphylococcus aureus strains.
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Técnicas de Tipificación Bacteriana/métodos , Tipificación de Secuencias Multilocus/métodos , ARN Ribosómico 16S/genética , Staphylococcus/clasificación , Proteínas Bacterianas/genética , Coagulasa/genética , Genotipo , Filogenia , Staphylococcus/aislamiento & purificación , Staphylococcus aureus/clasificación , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
The enterotoxin gene cluster (egc) has been proposed to contribute to the Staphylococcus aureus colonization, which highlights the need to evaluate genetic diversity and virulence gene profiles of the egc-positive population. Here, a total of 43 egc-positive isolates (16.2%) were identified from 266 S. aureus isolates that were obtained from various food and clinical specimens in Shanghai. Seven different egc profiles were found based on the polymerase chain reaction (PCR) result for egc genes. Then, these 43 egc-positive isolates were further typed by multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number tandem-repeat analysis (MLVA), and accessory gene regulatory (agr) typing. It showed that the 43 egc-positive isolates displayed 17 sequence types, 28 PFGE patterns, 29 MLVA types, and 4 agr types, respectively. Among them, the dominant clonal lineage was CC5-agr II (48.84%). Thirty toxin and 20 adhesion-associated genes were detected by PCR in egc-positive isolates. Notably, invasive toxin genes showed a high prevalence, such as 76.7% for Panton-Valentine leukocidin encoding genes, 27.9% for sec, and 23.3% for tsst-1. Most of the examined adhesion-associated genes were found to be conserved (76.7-100%), whereas the fnbB gene was only found in 8 (18.6%) isolates. In addition, 33 toxin gene profiles and 13 adhesion gene profiles were identified, respectively. Our results imply that isolates belonging to the same clonal lineage harbored similar adhesion gene profiles but diverse toxin gene profiles. Overall, the high prevalence of invasive virulence genes increases the potential risk of egc-positive isolates in S. aureus infection.
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Enterotoxinas/metabolismo , Microbiología de Alimentos , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , Intoxicación Alimentaria Estafilocócica/microbiología , Staphylococcus aureus/metabolismo , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , China , Electroforesis en Gel de Campo Pulsado , Enterotoxinas/genética , Exotoxinas/genética , Exotoxinas/metabolismo , Perfilación de la Expresión Génica , Humanos , Leucocidinas/genética , Leucocidinas/metabolismo , Tipificación Molecular/métodos , Tipificación de Secuencias Multilocus , Filogenia , Polimorfismo Genético , Intoxicación Alimentaria Estafilocócica/fisiopatología , Staphylococcus aureus/clasificación , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/patogenicidad , Secuencias Repetidas en Tándem , Transactivadores/genética , Transactivadores/metabolismo , Factores de Virulencia/genética , Vómitos/etiologíaRESUMEN
A 3 nm coronene coating and a 4 nm amorphous carbon coating with a uniform shell-core encapsulation structure for nanosized boron (B) powders are formed by a simple process in which coronene is directly mixed with boron particles without a solvent and heated at 520 °C for 1 h or at 630 °C for 3 h in a vacuum-sealed silica tube. Coronene has a melting point lower than its decomposition temperature, which enables liquid coronene to cover B particles by liquid diffusion and penetration without the need for a solvent. The diffusion and penetration of coronene can extend to the boundaries of particles and to inside the agglomerated nanoparticles to form a complete shell-core encapsulated structure. As the temperature is increased, thermal decomposition of coronene on the B particles results in the formation of a uniform amorphous carbon coating layer. This novel and simple nanometer-level uniform amorphous carbon coating method can possibly be applied to many other powders; thus, it has potential applications in many fields at low cost.
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Boro/química , Carbono/química , Nanocáscaras , Compuestos Policíclicos/química , Polvos/síntesis química , Tomografía con Microscopio Electrónico , HumanosRESUMEN
BACKGROUND: Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) have important functions in injury and repair processes of glomerular intrinsic cells. A study was conducted to analyze the urinary VEGF/creatinine (CR) and IL-6/CR levels in simple hematuria patients after excluding the interference of creatinine. We aimed to investigate the function and relationships of the above indices in the glomerular pathological injury process, and to elaborate the values of urinary VEGF and IL-6 changes in the diagnosis of asymptomatic hematuria or hematuria with proteinuria. MATERIAL/METHODS: A total of 121 renal hematuria patients diagnosed by clinical and laboratory tests were included as research subjects. The midstream fresh morning urine was collected on the day renal biopsy was performed. RESULTS: The IL-6/CR value of the group III was significantly greater than in group I (Z=-2.478, P<0.05), with a statistically significant difference between these 2 groups. The VEGF/CR value of group III was significantly greater than in group II (P<0.01). Compared with group I, the VEGF/CR of group III was significantly greater (Z=-4.65, P<0.01), with a statistically significant difference. CONCLUSIONS: The VEGF/CR and IL-6/CR values in simple hematuria patients were positively correlated with glomerular pathological injury scores. VEGF/CR and IL-6/CR might be used as biological diagnostic indicators in determining the extent of simple hematuria glomerular injury.
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Creatinina/orina , Hematuria/diagnóstico , Hematuria/orina , Interleucina-6/orina , Enfermedades Renales/orina , Glomérulos Renales/patología , Factor A de Crecimiento Endotelial Vascular/orina , Biopsia , Estudios de Casos y Controles , Receptor gp130 de Citocinas/metabolismo , Hematuria/patología , Humanos , Enfermedades Renales/patología , Proteinuria/patología , Proteinuria/orinaRESUMEN
To our knowledge, no in-depth clinicopathologic study of isolated hematuria (IH) is currently available. To address this gap, we analyzed the clinicopathologic features of IH as it manifests in child/adolescent and adult patients. The clinical data and pathological types of 543 IH patients who underwent renal pathological examinations from January 2005 to June 2010 were retrospectively analyzed. Clinical manifestations differed among the age groups: children/adolescents exhibited the highest percentage of mesangial proliferative glomerulonephritis (41.78%), whereas adults showed the highest percentage of immunoglobulin A nephropathy (IgAN) (52.39%). In addition, the percentage of IH patients who were classified according to clinical pathology differed from that of patients who were classified according to renal pathological type. Patients with IgAN who were found to have minimal proteinuria had more severe IH. For IH patients, especially those with a small amount of proteinuria, renal biopsy should be performed as early as possible in order to develop a long-term treatment plan and prognosis evaluation.
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Hematuria/patología , Riñón/patología , Adolescente , Adulto , Anciano , Biopsia , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
AIM: It is necessary to screen people at high risk for proteinuria with an economical, reliable and convenient method. The aim of this study is to establish a new approach to predict 24 h urine protein (24 h UP) by routine laboratory assays. METHODS: Five centres were included and a total of 4211 hospitalized patients were enrolled. All samples were assayed for dipstick protein (DSP), specific gravity (SG), 24 h UP and serum albumin (ALB) simultaneously. 4211 patients were randomly divided into two groups for establishing and testing the equations. Equations were built by multiple log-linear regressions. RESULTS: (i) DSP is significantly correlated to 24 h UP in a logarithmic pattern; (ii) SG interprets 24 h UP for specific DSP; (iii) Equation 1 = 0.203 × 10(dummy-variable F) × [100 (SG-1)](-0.470) ; and (iv) Equation 2 = 13.366 × 10(dummy-variable F) × [100 (SG-1)](-0.547) × [ALB (g/L)](-1.130) The dummy-variable F had a point-to-point accordance to DSP (detailed in text). CONCLUSION: Combination of DSP and SG can interpret normal-range proteinuria well, and helped by ALB, their interpretation for macro proteinuria is much improved. It is dependable and economical for routine urinalysis to evaluate pathological proteinuria by equation.
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Proteinuria/orina , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Humanos , Masculino , Conceptos Matemáticos , Persona de Mediana Edad , Factores de Tiempo , Urinálisis/métodos , Urinálisis/estadística & datos numéricos , Adulto JovenAsunto(s)
Imagen de Difusión por Resonancia Magnética/métodos , Nódulo Tiroideo/diagnóstico por imagen , Nódulo Tiroideo/patología , Adolescente , Adulto , Anciano , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Movimiento (Física) , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Glándula Tiroides/diagnóstico por imagen , Glándula Tiroides/patología , Adulto JovenRESUMEN
Autophagy is a lysosomal degradative pathway, which regulates the homeostasis of eukaryotic cells. This pathway can degrade misfolded or aggregated proteins, clear damaged organelles, and eliminate intracellular pathogens, including viruses, bacteria, and parasites. But, not all types of viruses are eliminated by autophagy. Flaviviruses (e.g., Yellow fever, Japanese encephalitis, Hepatitis C, Dengue, Zika, and West Nile viruses) are single-stranded and enveloped RNA viruses, and transmitted to humans primarily through the bites of arthropods, leading to severe and widespread illnesses. Like the coronavirus SARS-CoV-II, flaviviruses hijack autophagy for their infection and escape from host immune clearance. Thus, it is possible to control these viral infections by inhibiting autophagy. In this review, we summarize recent research progresses on hijacking of autophagy by flaviviruses and discuss the feasibility of antiviral therapies using autophagy inhibitors.
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Autofagia , Infecciones por Flavivirus , Flavivirus , Humanos , Flavivirus/fisiología , Flavivirus/patogenicidad , Infecciones por Flavivirus/virología , Animales , Antivirales/uso terapéutico , Antivirales/farmacología , Interacciones Huésped-PatógenoRESUMEN
RATIONALE: Anthrax is a severe zoonotic infectious disease caused by Bacillus anthracis. Most reported cases were traditionally diagnosed through culture and microscopy. We reported here the second case of cutaneous anthrax diagnosed by metagenomic next-generation sequencing (mNGS). PATIENT CONCERNS: A 63-year-old man had a history of contact with an unwell sheep, developing local redness and swelling on wrist. The dorsal side of the left hand and forearm, with tension blisters on the back of the left. DIAGNOSIS: B anthracis was detected from culturing and mNGS of tension blisters. INTERVENTIONS: On the second day of admission, the patient was administered 3.2 million units of penicillin every 6 hours, and isolated and closely observed. OUTCOMES: The patient improves and is discharged. LESSONS: Traditional bacterial cultures are time-consuming, while mNGS offers the advantage of accurate, quick, high-throughput, unbiased sequencing of all genetic material in a sample, which is a good technical tool for assisting in the diagnosis of rare pathogen infections.
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Carbunco , Bacillus anthracis , Enfermedades Cutáneas Bacterianas , Masculino , Humanos , Animales , Ovinos , Persona de Mediana Edad , Carbunco/diagnóstico , Carbunco/microbiología , Vesícula , Enfermedades Cutáneas Bacterianas/diagnóstico , Bacillus anthracis/genética , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Objective: A comprehensive strategy for microbial identification and contamination investigation during sterile drug manufacturing was innovatively established in this study, mainly based on MALDI-TOF MS for the identification and complemented by sequencing technology on strain typing. Methods: It was implemented to monitor the bacterial contamination of a sterile drug manufacturing facility, including its bacterial distribution features and patterns. In three months, two hundred ninety-two samples were collected covering multiple critical components of raw materials, personnel, environment, and production water. Results: Based on our strategy, the bacterial profile across the production process was determined: 241/292 bacterial identities were obtained, and Staphylococcus spp. (40.25%), Micrococcus spp.(11.20%), Bacillus spp. (8.30%), Actinobacteria (5.81%), and Paenibacillus spp. (4.56%) are shown to be the most dominant microbial contaminants. With 75.8% species-level and 95.4% genus-level identification capability, MALDI-TOF MS was promising to be a first-line tool for environmental monitoring routine. Furthermore, to determine the source of the most frequently occurring Staphylococcus cohnii, which evidenced a widespread presence in the entire process, a more discriminating S. cohnii whole-genome SNP typing method was developed to track the transmission routes. Phylogenetic analysis based on SNP results indicated critical environment contamination is highly relevant to personnel flow in this case. The strain typing results provide robust and accurate information for the following risk assessment step and support effective preventive and corrective measures. Conclusion: In general, the strategy presented in this research will facilitate the development of improved production and environmental control processes for the pharmaceutical industry, and give insights about how to provide more sound and reliable evidence for the optimization of its control program.
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Objective: To mine specific proteins and their protein-coding genes as suitable molecular biomarkers for the Burkholderia cepacia Complex (BCC) bacteria detection based on mega analysis of microbial proteomic and genomic data comparisons and to develop a real-time recombinase polymerase amplification (rt-RPA) assay for rapid isothermal screening for pharmaceutical and personal care products. Methods: We constructed an automatic screening framework based on Python to compare the microbial proteomes of 78 BCC strains and 263 non-BCC strains to identify BCC-specific protein sequences. In addition, the specific protein-coding gene and its core DNA sequence were validated in silico with a self-built genome database containing 158 thousand bacteria. The appropriate methodology for BCC detection using rt-RPA was evaluated by 58 strains in pure culture and 33 batches of artificially contaminated pharmaceutical and personal care products. Results: We identified the protein SecY and its protein-coding gene secY through the automatic comparison framework. The virtual evaluation of the conserved region of the secY gene showed more than 99.8% specificity from the genome database, and it can distinguish all known BCC species from other bacteria by phylogenetic analysis. Furthermore, the detection limit of the rt-RPA assay targeting the secY gene was 5.6 × 102 CFU of BCC bacteria in pure culture or 1.2 pg of BCC bacteria genomic DNA within 30 min. It was validated to detect <1 CFU/portion of BCC bacteria from artificially contaminated samples after a pre-enrichment process. The relative trueness and sensitivity of the rt-RPA assay were 100% in practice compared to the reference methods. Conclusion: The automatic comparison framework for molecular biomarker mining is straightforward, universal, applicable, and efficient. Based on recognizing the BCC-specific protein SecY and its gene, we successfully established the rt-RPA assay for rapid detection in pharmaceutical and personal care products.
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Heat-stress nephropathy (HSN) is associated with recurrent dehydration. However, the mechanisms underlying HSN remain largely unknown. In this study, we evaluated the role of dehydration in HSN and kidney injury in mice. Firstly, we found that complement was strongly activated in the mice that were exposed to dehydration; and among complement components, the interaction between C3a and its receptor, C3aR, was more closely associated with kidney injury. Then two-month-old mice were intraperitoneally injected with 2% dimethyl sulfoxide (DMSO) or the C3aR inhibitor SB290157 during dehydration. DMSO-treated mice exhibited excessive macrophage infiltration, renal cell apoptosis, and kidney fibrosis. In contrast, SB290157-treated mice had no apparent kidney injury. By fluorescence-activated cell sorting (FACS), we found that SB290157 treatment in mice remarkably inhibited macrophage infiltration and suppressed CCR2 expression in macrophages. In addition, C3a binding to C3aR promoted macrophage polarization toward the M1 phenotype and increased the production of TNF-α, which induced renal tubular epithelial cell (RTEC) apoptosis in vivo and in vitro. Interestingly, C3a treatment failed to directly induce TNF-α production and apoptosis in RTECs. However, TNF-α production in response to C3a treatment was significantly elevated when RTECs were cocultured with macrophages, suggesting that macrophages rather than RTECs are the target of C3a-C3aR interaction. At last, we proved that infusion of macrophages which highly expressed TNF-α would significantly deteriorate HSN in TNF-KO mice when they were exposed to recurrent dehydration. This study uncovers a novel mechanism underlying the pathogenesis of HSN, and a potential pathway to prevent kidney injury during dehydration.