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OBJECTIVE: In pediatric patients, middle cranial fossa (MCF) arachnoid cysts are often discovered incidentally on imaging in asymptomatic patients during workup for other indications. This study aims to describe current management gestalt and threshold for surgical intervention by surveying an international cohort of neurosurgeons. METHODS: A web-based survey was circulated via email list of attendants of the 2019 Canadian Pediatric Neurosurgery Study Group (CPNSG) and International Society of Pediatric Neurosurgery (ISPN) mailing list. The survey consisted of 8 clinical scenarios involving patients with MCF arachnoid cysts. Demographic variables of respondents and their decisions regarding management for each scenario were analyzed using R computing software. RESULTS: A total of 107 respondents were included. Cysts in asymptomatic patients (92%), younger age at diagnosis (81%), and presence of a mild learning delay were predominantly managed non-surgically (80.7 ± 9.4%). Patients with cyst enlargement, headaches, new seizures, or hemorrhage were divided between non-surgical (55.8 ± 3.3%) and surgical (44.2 ± 2.9%) management. Patients with contralateral hemiparesis were treated predominantly surgically (67%). For both Galassi I and II, papilledema was favored as the primary indication for surgical intervention in 54% of patients. Those inclined to surgery (n = 17) were more likely to practice and train outside North America compared to those not pro-surgical (adjusted P = 0.092). CONCLUSION: Incidental MCF arachnoid cysts in asymptomatic patients and younger age of diagnosis are predominantly managed non-surgically. Mild learning delay was not considered an indication to intervene. In contrast, radiological progression, hemorrhagic evolution, or non-focal neurological deficits lead to uncertainty in management, while focal neurological deficits and papilledema with MCF cysts were favored to be intervened surgically. Among the provider level factors, only location of training and practice trended towards a pro-surgery approach.
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Quistes Aracnoideos , Papiledema , Niño , Humanos , Quistes Aracnoideos/diagnóstico por imagen , Quistes Aracnoideos/cirugía , Canadá , Procedimientos Neuroquirúrgicos/métodos , Craneotomía/métodos , Estudios RetrospectivosRESUMEN
We chose to evaluate Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) as a possible biomarker for prostate cancer due to its involvement in nucleotide synthesis and cell cycle progression. We utilized two prostate cancer cell lines (PC3 and DU145) along with patient tissue and knockdowns to evaluate overall HPRT expression. The surface localization of HPRT was determined utilizing flow cytometry, confocal microscopy, and scanning electron microscopy followed by ADCC to evaluate targeting potential. We found significant upregulation of HPRT within malignant samples with approximately 47% of patients had elevated levels of HPRT compared to normal controls. We also observed a significant association between HPRT and the plasma membrane of DU145 cells (p = 0.0004), but found no presence on PC3 cells (p = 0.14). This was confirmed with scanning electron microscopy and confocal microscopy. ADCC experiments were performed to determine whether HPRT could be used as a target antigen for selective cell-mediated killing. We found that DU145 cells treated with HPRT antibodies had a significantly higher incidence of cell death than both isotype treated samples and PC3 cells treated with the same concentrations of HPRT antibody. Finally, we determined that p53 had a significant impact on HPRT expression both internally and on the surface of cancer cells. These results suggest HPRT as a possible biomarker target for the treatment of patients with prostate cancer.
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División Celular/fisiología , Citotoxicidad Inmunológica/inmunología , Hipoxantina Fosforribosiltransferasa/metabolismo , Neoplasias de la Próstata/metabolismo , Línea Celular , Membrana Celular/metabolismo , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/inmunología , Masculino , Neoplasias de la Próstata/inmunología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Human pathogens belonging to the Alphavirus genus, in the Togaviridae family, are transmitted primarily by mosquitoes. The signs and symptoms associated with these viruses include fever and polyarthralgia, defined as joint pain and inflammation, as well as encephalitis. In the last decade, our understanding of the interactions between members of the alphavirus genus and the human host has increased due to the re-appearance of the chikungunya virus (CHIKV) in Asia and Europe, as well as its emergence in the Americas. Alphaviruses affect host immunity through cytokines and the interferon response. Understanding alphavirus interactions with both the innate immune system as well as the various cells in the adaptive immune systems is critical to developing effective therapeutics. In this review, we summarize the latest research on alphavirus-host cell interactions, underlying infection mechanisms, and possible treatments.
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Infecciones por Alphavirus , Alphavirus , Alphavirus/inmunología , Alphavirus/patogenicidad , Infecciones por Alphavirus/epidemiología , Infecciones por Alphavirus/prevención & control , Infecciones por Alphavirus/virología , Animales , Humanos , Vacunas Virales/inmunologíaRESUMEN
BACKGROUND: Thymidine kinase 1 (TK1) is a pyrimidine salvage pathway enzyme that is up-regulated in malignant tissues and elevated in the serum of cancer patients. While TK1 has been well established as a tumor biomarker, little has been done to explore its potential as a tumor target. Recently, we reported the membrane expression of TK1 on malignant cells, but not on normal cells. This study explores the possible use of monoclonal antibodies for the targeting of membrane associated TK1 in lung, breast, colon and prostate cancer cells. METHODS: We generated and evaluated a panel of monoclonal antibodies against six different epitopes exposed in the tetrameric form of TK1. Antibodies were developed with hybridoma technology and validated with Western blot, siRNA TK1 knockdown, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The therapeutic potential of the antibodies was evaluated in vitro in antibody-dependent cell-mediated-cytotoxicity (ADCC) experiments. RESULTS: Binding of the antibodies to TK1 was confirmed by Western blot in purified recombinant protein, cancer serum, and cell lysate. After a TK1 knockdown was performed, a reduction of TK1 expression was observed with five antibodies. Using indirect ELISA, we identified 3B2E11, 9C10, 7H2, 3B4, 8G2 among the most sensitive antibodies (LOD = 10.73-66.9 pg/ml). Surface expression of TK1 on the membrane of various cancer cell lines was analyzed with flow cytometry. Antibodies 8G2, 3B4, 7HD and 5F7G11 detected TK1 on the membrane of various cancer cell lines, including lung, prostate, colon and breast. No significant binding was detected on normal lymphocytes. Increased cytolysis of lung (~ 70%. p = 0.0001), breast (~ 70%, p = 0.0461) and colon (~ 50% p = 0.0216) cancer cells by effector cells was observed when anti-TK1 antibodies were added during ADCC experiments. CONCLUSIONS: The antibodies developed showed potential to be used to detect and target TK1 on the membrane of various tumor cells. The targeting of TK1 in malignant cells using monoclonal antibodies may be a feasible approach for the elimination of high TK1 expressing tumor cells.
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BACKGROUND: The aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies. With 4.3% of all new cancers diagnosed as Non-Hodgkin lymphoma, finding new biomarkers for the treatment of B cell cancers is an ongoing pursuit. HPRT is a nucleotide salvage pathway enzyme responsible for the synthesis of guanine and inosine throughout the cell cycle. METHODS: Raji cells were used for this analysis due to their high HPRT internal expression. Internal expression was evaluated utilizing western blotting and RNA sequencing. Surface localization was analyzed using flow cytometry, confocal microscopy, and membrane biotinylation. To determine the source of HPRT surface expression, a CRISPR knockdown of HPRT was generated and confirmed using western blotting. To determine clinical significance, patient blood samples were collected and analyzed for HPRT surface localization. RESULTS: We found surface localization of HPRT on both Raji cancer cells and in 77% of the malignant ALL samples analyzed and observed no significant expression in healthy cells. Surface expression was confirmed in Raji cells with confocal microscopy, where a direct overlap between HPRT specific antibodies and a membrane-specific dye was observed. HPRT was also detected in biotinylated membranes of Raji cells. Upon HPRT knockdown in Raji cells, we found a significant reduction in surface expression, which shows that the HPRT found on the surface originates from the cells themselves. Finally, we found that cells that had elevated levels of HPRT had a direct correlation to XRCC2, BRCA1, PIK3CA, MSH2, MSH6, WDYHV1, AK7, and BLMH expression and an inverse correlation to PRKD2, PTGS2, TCF7L2, CDH1, IL6R, MC1R, AMPD1, TLR6, and BAK1 expression. Of the 17 genes with significant correlation, 9 are involved in cellular proliferation and DNA synthesis, regulation, and repair. CONCLUSIONS: As a surface biomarker that is found on malignant cells and not on healthy cells, HPRT could be used as a surface antigen for targeted immunotherapy. In addition, the gene correlations show that HPRT may have an additional role in regulation of cancer proliferation that has not been previously discovered.
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BACKGROUND: Incidence of endometrial cancer are rising both in the United States and worldwide. As endometrial cancer becomes more prominent, the need to develop and characterize biomarkers for early stage diagnosis and the treatment of endometrial cancer has become an important priority. Several biomarkers currently used to diagnose endometrial cancer are directly related to obesity. Although epigenetic and mutational biomarkers have been identified and have resulted in treatment options for patients with specific aberrations, many tumors do not harbor those specific aberrations. A promising alternative is to determine biomarkers based on differential gene expression, which can be used to estimate prognosis. METHODS: We evaluated 589 patients to determine differential expression between normal and malignant patient samples. We then supplemented these evaluations with immunohistochemistry staining of endometrial tumors and normal tissues. Additionally, we used the Library of Integrated Network-based Cellular Signatures to evaluate the effects of 1826 chemotherapy drugs on 26 cell lines to determine the effects of each drug on HPRT1 and AURKA expression. RESULTS: Expression of HPRT1, Jag2, AURKA, and PGK1 were elevated when compared to normal samples, and HPRT1 and PGK1 showed a stepwise elevation in expression that was significantly related to cancer grade. To determine the prognostic potential of these genes, we evaluated patient outcome and found that levels of both HPRT1 and AURKA were significantly correlated with overall patient survival. When evaluating drugs that had the most significant effect on lowering the expression of HPRT1 and AURKA, we found that Topo I and MEK inhibitors were most effective at reducing HPRT1 expression. Meanwhile, drugs that were effective at reducing AURKA expression were more diverse (MEK, Topo I, MELK, HDAC, etc.). The effects of these drugs on the expression of HPRT1 and AURKA provides insight into their role within cellular maintenance. CONCLUSIONS: Collectively, these data show that JAG2, AURKA, PGK1, and HRPT1 have the potential to be used independently as diagnostic, prognostic, or treatment biomarkers in endometrial cancer. Expression levels of these genes may provide physicians with insight into tumor aggressiveness and chemotherapy drugs that are well suited to individual patients.
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[This corrects the article DOI: 10.1186/s12935-018-0633-9.].
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Carbapenem-resistant bacteria have quickly become a worldwide concern in nosocomial infections. Of the seven known carbapenemases, four have been shown to be particularly problematic: KPC, NDM, IMP, and VIM. To date, many local and species- or carbapenemase-specific epidemiological studies have been performed, which often focus on the organism itself. This report attempts to perform an inclusive (encompass both species and carbapenemase) epidemiologic study using publicly available plasmid sequences from NCBI. In this report, the gene content of these various plasmids has been characterized, replicon types of the plasmids identified, and the global spread and species promiscuity of the plasmids analyzed. Additionally, support to several groups targeting plasmid maintenance and transfer mechanisms to slow the spread of resistance plasmids is given.
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Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Plásmidos/genética , beta-Lactamasas/genética , Antibacterianos , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Carbapenémicos , China , Bases de Datos de Ácidos Nucleicos , Plásmidos/clasificación , Replicón , Estados UnidosRESUMEN
Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.
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Proteínas Bacterianas/metabolismo , Bacteriocinas/metabolismo , Péptidos/metabolismo , Staphylococcaceae/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriocinas/química , Bacteriocinas/genética , Vías Biosintéticas/genética , Modelos Moleculares , Estructura Molecular , Péptidos/química , Péptidos/genética , Conformación Proteica , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Staphylococcaceae/enzimología , Staphylococcaceae/genéticaRESUMEN
BACKGROUND: Lung, breast, and colorectal malignancies are the leading cause of cancer-related deaths in the world causing over 2.8 million cancer-related deaths yearly. Despite efforts to improve prevention methods, early detection, and treatments, survival rates for advanced stage lung, breast, and colon cancer remain low, indicating a critical need to identify cancer-specific biomarkers for early detection and treatment. Thymidine kinase 1 (TK1) is a nucleotide salvage pathway enzyme involved in cellular proliferation and considered an important tumor proliferation biomarker in the serum. In this study, we further characterized TK1's potential as a tumor biomarker and immunotherapeutic target and clinical relevance. METHODS: We assessed TK1 surface localization by flow cytometry and confocal microscopy in lung (NCI-H460, A549), breast (MDA-MB-231, MCF7), and colorectal (HT-29, SW620) cancer cell lines. We also isolated cell surface proteins from HT-29 cells and performed a western blot confirming the presence of TK1 on cell membrane protein fractions. To evaluate TK1's clinical relevance, we compared TK1 expression levels in normal and malignant tissue through flow cytometry and immunohistochemistry. We also analyzed RNA-Seq data from The Cancer Genome Atlas (TCGA) to assess differential expression of the TK1 gene in lung, breast, and colorectal cancer patients. RESULTS: We found significant expression of TK1 on the surface of NCI-H460, A549, MDA-MB-231, MCF7, and HT-29 cell lines and a strong association between TK1's localization with the membrane through confocal microscopy and Western blot. We found negligible TK1 surface expression in normal healthy tissue and significantly higher TK1 expression in malignant tissues. Patient data from TCGA revealed that the TK1 gene expression is upregulated in cancer patients compared to normal healthy patients. CONCLUSIONS: Our results show that TK1 localizes on the surface of lung, breast, and colorectal cell lines and is upregulated in malignant tissues and patients compared to healthy tissues and patients. We conclude that TK1 is a potential clinical biomarker for the treatment of lung, breast, and colorectal cancer.
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UNLABELLED: Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in Bacillus subtilis Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2. IMPORTANCE: The emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism B. subtilis was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics.
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Bacillus subtilis/metabolismo , Bacteriocinas/biosíntesis , Regulación Bacteriana de la Expresión Génica/fisiología , Bacillus subtilis/genética , Bacteriocinas/química , Bacteriocinas/genética , Regulación Enzimológica de la Expresión Génica , Estructura Molecular , Familia de Multigenes , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Péptidos/química , Péptidos/genéticaRESUMEN
Francisella tularensis has been the focus of much research over the last two decades mainly because of its potential use as an agent of bioterrorism. F. tularensis is the causative agent of zoonotic tularemia and has a worldwide distribution. The different subspecies of F. tularensis vary in their biogeography and virulence, making early detection and diagnosis important in both the biodefense and public health sectors. Recent genome sequencing efforts reveal aspects of genetic diversity, evolution and phylogeography previously unknown for this relatively small organism, and highlight a role for detection by various PCR assays. This review explores the advances made in understanding the evolution and genetic diversity of F. tularensis and how these advances have led to better PCR assays for detection and identification of the subspecies.
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Francisella tularensis/genética , Tularemia/microbiología , Animales , ADN Bacteriano/genética , Evolución Molecular , Variación Genética , Genoma Bacteriano , Humanos , Técnicas de Diagnóstico Molecular , Tipificación Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Tularemia/diagnósticoRESUMEN
A fundamental tenet of evolution is that alleles that are under negative selection are often deleterious and confer no evolutionary advantage. Negatively selected alleles are removed from the gene pool and are eventually extinguished from the population. Conversely, alleles under positive selection do confer an evolutionary advantage and lead to an increase in the overall fitness of the organism. These alleles increase in frequency until they eventually become fixed in the population. Francisella tularensis is a zoonotic pathogen and a potential biothreat agent. The most virulent type of F. tularensis, Type A, is distributed across North America with Type A.I occurring mainly in the east and Type A.II appearing mainly in the west. F. tularensis is thought to be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. To test this hypothesis, we sequenced and compared whole genomes of Type A.I and A.II isolates. We analyzed a subset of virulence and housekeeping genes from several F. tularensis subspecies genomes to ascertain the presence and extent of positive selection. Eleven previously identified virulence genes were screened for positive selection along with 10 housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection at loci implicated in cell surface structures and membrane proteins, metabolism and biosynthesis, transcription, translation and cell separation, and substrate binding and transport. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution driven by complex interactions between host, pathogen, and thier environment, as evidenced by several of its virulence genes which are undergoing strong, positive selection.
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Francisella tularensis/genética , Francisella tularensis/patogenicidad , Alelos , Proteínas Bacterianas/genética , Frecuencia de los Genes , Genoma Bacteriano , Selección Genética , Virulencia/genéticaRESUMEN
Thiopeptides are small (12- to 17-amino-acid), heavily modified peptides of bacterial origin. This antibiotic family, with more than 100 known members, is characterized by the presence of sulfur-containing heterocyclic rings and dehydrated residues within a macrocyclic peptide structure. Thiopeptides, including micrococcin P1, have garnered significant attention in recent years for their potent antimicrobial activity against bacteria, fungi, and even protozoa. Micrococcin P1 is known to target the ribosome; however, like those of other thiopeptides, its biosynthesis and mechanisms of self-immunity are poorly characterized. We have discovered an isolate of Staphylococcus epidermidis harboring the genes for thiopeptide production and self-protection on a 24-kb plasmid. Here we report the characterization of this plasmid, identify the antimicrobial peptide that it encodes, and provide evidence of a target replacement-mediated mechanism of self-immunity.
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Bacteriocinas/genética , Familia de Multigenes , Péptidos Cíclicos/genética , Plásmidos , Staphylococcus epidermidis/genética , Antiinfecciosos/farmacología , Bacteriocinas/farmacología , Productos Biológicos/farmacología , Péptidos Cíclicos/farmacologíaRESUMEN
Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. The high mortality rates of glanders and melioidosis, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. The development of PCR technologies has revolutionized diagnostic testing and these detection methods have become popular due to their speed, sensitivity, and accuracy. The purpose of this review is to provide a comprehensive overview and evaluation of the advancements in PCR-based detection and differentiation methodologies for the B. pseudomallei complex, and examine their potential uses in diagnostic and environmental testing.
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Armas Biológicas , Burkholderia mallei/aislamiento & purificación , Burkholderia pseudomallei/aislamiento & purificación , Burkholderia/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , Animales , Técnicas de Tipificación Bacteriana , Burkholderia/genética , Burkholderia mallei/genética , Burkholderia pseudomallei/genética , Muermo/microbiología , Muermo/patología , Caballos , Humanos , Melioidosis/microbiología , Melioidosis/patología , Reacción en Cadena de la Polimerasa/normas , Polimorfismo de Nucleótido Simple , Sensibilidad y EspecificidadRESUMEN
Bacillus anthracis, classified as a Tier 1 Select Agent by the Centers for Disease Control and Prevention (CDC), is the causative agent of anthrax in both humans and livestock. Herein, we report the full genome sequences of 13 bacteriophages that infect B. anthracis Sterne. These phages are grouped into four clusters and are similar to previously described Bacillus phages.
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Background: Yersinia pestis, a Gram-negative bacterium, is the causative agent of plague. Y. pestis is a zoonotic pathogen that occasionally infects humans and became endemic in the western United States after spreading from California in 1899. Methods: To better understand evolutionary patterns in Y. pestis from the southwestern United States, we sequenced and analyzed 22 novel genomes from New Mexico. Analytical methods included, assembly, multiple sequences alignment, phylogenetic tree reconstruction, genotype-phenotype correlation, and selection pressure. Results: We identified four genes, including Yscp and locus tag YPO3944, which contained codons undergoing negative selection. We also observed 42 nucleotide sites displaying a statistically significant skew in the observed residue distribution based on the year of isolation. Overall, the three genes with the most statistically significant variations that associated with metadata for these isolates were sapA, fliC, and argD. Phylogenetic analyses point to a single introduction of Y. pestis into the United States with two subsequent, independent movements into New Mexico. Taken together, these analyses shed light on the evolutionary history of this pathogen in the southwestern US over a focused time range and confirm a single origin and introduction into North America.
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Peste , Yersinia pestis , Humanos , Yersinia pestis/genética , Filogenia , New Mexico/epidemiología , Peste/epidemiología , Análisis de SecuenciaRESUMEN
A simple method was developed for detection of Bacillus anthracis (BA) endospores and for differentiation of them from other species in the Bacillus cereus group. Chemical profiles that include lipids (i.e., fatty acids), carbohydrates (i.e., sugars), and the spore-specific biomarker, dipicolinic acid, were generated by one-step thermochemolysis (TCM) at 140 °C in 5 min to provide specific biomarker signatures. Anthrose, which is a biomarker characteristic of the B. cereus group of bacteria, was determined from a fragment produced by TCM. Surprisingly, several virulent BA strains contained very low levels of anthrose, which confounded their detection. A statistical discrimination algorithm was constructed using a combination of biomarkers, which was robust against different growth conditions (medium and temperature). Fifteen endospore-forming Bacillus species were confirmed in a statistically designed test (~90%) using the algorithm, including six BA strains (four virulent isolates), five B. thuringiensis (BT) isolates, and one isolate each for B. cereus (BC), B. mycoides (BM), B. atrophaeus (BG), and B. subtilis (BS). The detection limit for B. anthracis was found to be 50,000 endospores, on the basis of the GC/MS detection limits for 3-methyl-2-butenoic acid methyl ester, which is the biomarker derived from TCM of anthrose.
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Bacillus anthracis/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Algoritmos , Bacillus/metabolismo , Biomarcadores/análisis , Carbohidratos/análisis , Ácidos Grasos/análisis , Ácidos Picolínicos/análisis , Esporas Bacterianas/metabolismoRESUMEN
Background: Chikungunya virus (CHIKV) is a mosquito-borne pathogen, within the Alphavirus genus of the Togaviridae family, that causes ~1.1 million human infections annually. CHIKV uses Aedes albopictus and Aedes aegypti mosquitoes as insect vectors. Human infections can develop arthralgia and myalgia, which results in debilitating pain for weeks, months, and even years after acute infection. No therapeutic treatments or vaccines currently exist for many alphaviruses, including CHIKV. Targeting the phagocytosis of CHIKV by macrophages after mosquito transmission plays an important role in early productive viral infection in humans, and could reduce viral replication and/or symptoms. Methods: To better characterize the transcriptional response of macrophages during early infection, we generated RNA-sequencing data from a CHIKV-infected human macrophage cell line at eight or 24 hours post-infection (hpi), together with mock-infected controls. We then calculated differential gene expression, enriched functional annotations, modulated intracellular signaling pathways, and predicted therapeutic drugs from these sequencing data. Results: We observed 234 pathways were significantly affected 24 hpi, resulting in six potential pharmaceutical treatments to modulate the affected pathways. A subset of significant pathways at 24 hpi includes AGE-RAGE, Fc epsilon RI, Chronic myeloid leukemia, Fc gamma R-mediated phagocytosis, and Ras signaling. We found that the MAPK1 and MAPK3 proteins are shared among this subset of pathways and that Telmisartan and Dasatinib are strong candidates for repurposed small molecule therapeutics that target human processes. The results of our analysis can be further characterized in the wet lab to contribute to the development of host-based prophylactics and therapeutics.
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Aedes , Fiebre Chikungunya , Virus Chikungunya , Animales , Humanos , Virus Chikungunya/genética , Mosquitos Vectores , Fiebre Chikungunya/tratamiento farmacológico , Línea Celular , MacrófagosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China in December 2019 and caused a global pandemic resulting in millions of deaths and tens of millions of patients positive tests. While studies have shown a D614G mutation in the viral spike protein are more transmissible, the effects of this and other mutations on the host response, especially at the cellular level, are yet to be fully elucidated. In this experiment we infected normal human bronchial epithelial (NHBE) cells with the Washington (D614) strain or the New York (G614) strains of SARS-CoV-2. We generated RNA sequencing data at 6, 12, and 24 hours post-infection (hpi) to improve our understanding of how the intracellular host response differs between infections with these two strains. We analyzed these data with a bioinformatics pipeline that identifies differentially expressed genes (DEGs), enriched Gene Ontology (GO) terms and dysregulated signaling pathways. We detected over 2,000 DEGs, over 600 GO terms, and 29 affected pathways between the two infections. Many of these entities play a role in immune signaling and response. A comparison between strains and time points showed a higher similarity between matched time points than across different time points with the same strain in DEGs and affected pathways, but found more similarity between strains across different time points when looking at GO terms. A comparison of the affected pathways showed that the 24hpi samples of the New York strain were more similar to the 12hpi samples of the Washington strain, with a large number of pathways related to translation being inhibited in both strains. These results suggest that the various mutations contained in the genome of these two viral isolates may cause distinct effects on the host transcriptional response in infected host cells, especially relating to how quickly translation is dysregulated after infection. This comparison of the intracellular host response to infection with these two SARS-CoV-2 isolates suggest that some of the mechanisms associated with more severe disease from these viruses could include virus replication, metal ion usage, host translation shutoff, host transcript stability, and immune inhibition.