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Introduction: Cholesteatoma usually harbors a poly-microbial infection. As the diversity of bacterial pathogens in the Indian COM is unknown, we set out to identify the bacteria associated with cholesteatoma disease in different patients of North India using targeted metagenomic analysis of the 16 S rRNA gene. Methods: We recruited 15 patients of cholesteatomatous chronic otitis media (COM), who underwent surgical disease clearance. We divided these patients into four groups based on the four clinic-radiological stages categorized as per the EAONO/JOS joint consensus statement classification. Representative samples were extracted during the surgery and sent for bacterial culture and sensitivity and 16 S rRNA gene metagenomic analysis. Results: While 12 (80%) of the patients belonged to clinical Stage I/II; one patient had an extracranial complication (stage III) and two patients had an intracranial complication (stage IV). Our detailed bacterial metagenomics analyses showed that while phylum Proteobacteria was most abundant (reads up to ⼠95%) in specimens from nine patients, phylum Firmicutes was most abundant (up to ⼠80%) in specimens from four patients. Gamma (γ) Proteobacteria and Epsilon (ε) Proteobacteria were the most abundant class amongst Proteobacteria. Class Tissierellia stood out as the most abundant Firmicutes (40-60%), followed by Clostridia (20%) and Bacilli (10%). There was negligible difference in the bacterial profiles across all four clinical stages. Conclusion: Cholesteatoma is primarily associated with Proteobacteria and Firmicutes phyla, even in complicated disease. Further studies with a larger sample size are required to validate our findings. Supplementary Information: The online version contains supplementary material available at 10.1007/s12070-024-04678-9.
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This publication describes a method for the determination of total bisphenol A (BPA and conjugated BPA) following enzyme hydrolysis and is intended as a companion to our previously developed analytical method for the determination of free BPA (the aglycone) in human blood and urine using high-performance liquid chromatography-tandem mass spectrometry ( 1). That free BPA method provided a means to account for and/or eliminate background contamination and demonstrated accuracy and reproducibility in both matrices fortified with BPA or a surrogate analyte ((13)C BPA) at a low method quantitation limit (MQL) of 0.1-0.2 ng/mL. In contrast to the free BPA method results and based on stringent accuracy, precision and confirmation criteria set for the MQLs of the method developed for total BPA, the MQL achieved in blood was 1.020-2.550 and 0.510-1.020 ng/mL in urine. These data showed higher MQLs than the desired MQLs of 0.5 ng/mL (blood) and 0.2 ng/mL (urine) with increased variability between analyses which demonstrates the importance of generating method validation data with each analysis. In contrast, the MQL achieved for (13)C BPA-G (monoglucuronide as a surrogate analyte in blood was 0.2-0.5 and 0.2 ng/mL in urine illustrating that the method is capable of meeting lower MQL requirements if the contribution from exogenous BPA can be well controlled. This method for the determination total BPA in human blood and urine is intended to be used in conjunction with the free BPA method ( 1) to obtain accurate and complete BPA biomonitoring data to support human exposure assessments.
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Compuestos de Bencidrilo , Cromatografía Líquida de Alta Presión/métodos , Monitoreo del Ambiente/métodos , Contaminantes Ambientales , Fenoles , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Compuestos de Bencidrilo/sangre , Compuestos de Bencidrilo/orina , Calibración , Cromatografía Líquida de Alta Presión/instrumentación , Monitoreo del Ambiente/instrumentación , Contaminantes Ambientales/sangre , Contaminantes Ambientales/orina , Humanos , Límite de Detección , Fenoles/sangre , Fenoles/orina , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
AIM: To evaluate the efficacy and safety of fixed drug combination (FDC) halometasone 0.05% and fusidic acid 2% (group A) vs FDC betamethasone 0.12% and neomycin sulfate 0.5% cream (group B) in acute or chronic infected eczematous dermatosis, through a randomized open-label, comparative, multicentric study. MATERIALS AND METHODS: A total of 152 patients were randomized to either Group A or Group B. EASI (Eczema Area and Severity Index), IGA (Investigator's global assessment), scale for severity of eczema, pruritus, and safety parameters were assessed at baseline, Day 5/Day 10, Day 10/20, and Day 20/Day 30 for acute/chronic cases. Skin swabs were tested at screening, Day 10, and end of the study. RESULTS: Staphylococcus aureus was the frequently encountered causative agent. There was a significant reduction within the study groups in EASI, IGA scales for severity of eczema, pruritus at various visits, compared to baseline. At the end of study, 83.87% in group A and 65.71% in group B were culture negative. Cure rate was 54.28% and 50% in group A and B, respectively. Five adverse events were reported in five patients, of which three patients withdrew from the study. CONCLUSION: Halometasone 0.05% and Fusidic acid 2% cream is effective, safe, well tolerated with comparable efficacy to the comparator in the treatment of acute and chronic infected eczematous dermatosis.
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Bisphenol A (BPA) is an industrial chemical used to make polymers including some used in food contact applications. Virtually complete presystemic clearance of orally administered BPA occurs in humans by metabolism to BPA-glucuronide (BPA-G), but some biomonitoring studies report low concentrations of free (parent) BPA in human blood and urine. Trace contamination of BPA from exogenous sources or hydrolysis of BPA-G to free BPA, either during or after biomonitoring specimen collection, may have contributed to the reported concentrations of free BPA. An analytical method for the determination of free BPA in human blood and urine was developed and validated in two independent laboratories, using the latest generation of high-performance liquid chromatography-tandem mass spectrometry instrumentation to ensure the desired high sensitivity and selectivity. The method was designed to account for and/or eliminate background contamination from all sources and demonstrated that contamination could occur from devices used for specimen collection or storage, as well as other sources. The method employed an internal standard (BPA-d(8)) and demonstrated accuracy and reproducibility in both matrices fortified with BPA or a surrogate analyte ((13)C-BPA) at a low quantitation limit (0.1-0.2 ng/mL). For validation, five replicate samples were analyzed to evaluate reproducibility. Importantly, it was demonstrated that the conditions of the method did not result in the hydrolysis of BPA-G to free BPA, another possible source of error in BPA analysis. Application of the principles defined by this method will be critical to assure valid analytical results in any future biomonitoring studies.
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Monitoreo del Ambiente/métodos , Contaminantes Ambientales/metabolismo , Fenoles/metabolismo , Compuestos de Bencidrilo , Cromatografía Líquida de Alta Presión , Contaminantes Ambientales/sangre , Contaminantes Ambientales/orina , Humanos , Fenoles/sangre , Fenoles/orina , Espectrometría de Masas en TándemRESUMEN
Inorganic polyphosphate (Poly P) is a polymer of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds as in ATP. Found in abundance in all cells in nature, it is unique in its likely role in the origin and survival of species. Here, we present extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth. Beyond that, Poly P has proved in a variety of ways to be essential for growth of cells, their responses to stresses and stringencies, and the virulence of pathogens. In this review, we pay particular attention to the enzyme, polyphosphate kinase 1 (Poly P kinase 1 or PPK1), responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens. Mutants lacking PPK1 are defective in motility, quorum sensing, biofilm formation, and virulence. Structural studies are cited that reveal the conserved ATP-binding site of PPK1 at atomic resolution and reveal that the site can be blocked with minute concentrations of designed inhibitors. Another widely conserved enzyme is PPK2, which has distinctive kinetic properties and is also implicated in the virulence of some pathogens. Thus, these enzymes, absent in yeast and animals, are novel attractive targets for treatment of many microbial diseases. Still another enzyme featured in this review is one discovered in Dictyostelium discoideum that becomes an actin-like fiber concurrent with the synthesis, step by step, of a Poly P chain made from ATP. The Poly P-actin fiber complex, localized in the cell, lengthens and recedes in response to metabolic signals. Homologs of DdPPK2 are found in pathogenic protozoa and in the alga Chlamydomonas. Beyond the immediate relevance of Poly P as a target for anti-infective drugs, a large variety of cellular operations that rely on Poly P will be considered.
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Fenómenos Fisiológicos Bacterianos , Fosfatos/metabolismo , Animales , Bacterias/enzimología , Bacterias/metabolismo , Dictyostelium/enzimología , Dictyostelium/fisiología , Humanos , Fosfatos/químicaRESUMEN
Polyphosphate kinase 1 (PPK1), the principal enzyme responsible for reversible synthesis of polyphosphate (poly P) from the terminal phosphate of ATP, is highly conserved in bacteria and archaea. Dictyostelium discoideum, a social slime mold, is one of a few eukaryotes known to possess a PPK1 homolog (DdPPK1). Compared with PPK1 of Escherichia coli, DdPPK1 contains the conserved residues for ATP binding and autophosphorylation, but has an N-terminal extension of 370 aa, lacking homology with any known protein. Polyphosphate or ATP promote oligomerization of the enzyme in vitro. The DdPPK1 products are heterogeneous in chain length and shorter than those of E. coli. The unique DdPPK1 N-terminal domain was shown to be necessary for its enzymatic activity, cellular localization, and physiological functions. Mutants of DdPPK1, as previously reported, are defective in development, sporulation, and predation, and as shown here, in late stages of cytokinesis and cell division.
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Citocinesis , Dictyostelium/enzimología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Proteínas Protozoarias/metabolismo , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Citocinesis/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genéticaRESUMEN
Transduction frequency with phage P1 had been observed to be very low in Escherichia coli K-12 mutants lacking the operon (ppk1-ppx) responsible for the synthesis of inorganic polyphosphate (poly P). We now find that these mutants, for lack of poly P, are lysogenic for P1 and when infected with phage P1 produce only approximately 1% the number of infective centers compared with the WT host. Both phage adsorption and release were unaffected. The host-encoded P1 late-gene transcriptional activator, SspA, failed to show the transcriptional increase in the mutant, observed in the WT. UV induction of a P1-infected mutant resulted in a 200-fold increase in the production of infectious phage particles. The lysogenized P1 (P1mut) and P1 progeny from the mutant host (Deltappk1-ppx) produced plaques of differing morphologies, whereas P1 progeny from the WT yielded only small, clear plaques. Two discernable variants, one producing small and clear plaques (P1small) and the other large plaques with turbid rims (P1large), had broader host range and produced larger burst sizes in WT compared with P1. Transmission electron microscopy showed P1mut had contractile sheath defects. Thus, the lack of poly P/PPK1 in the mutant host resulted in the formation of defective P1 particles during intracellular growth. A filamentous phage, fd, also failed to produce plaques on a mutant lawn. Although fd adsorbed to the F-pilus, its DNA failed to enter the mutant host.
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Bacteriófago M13/crecimiento & desarrollo , Bacteriófago M13/metabolismo , Bacteriófago P1/crecimiento & desarrollo , Bacteriófago P1/metabolismo , Lisogenia , Polifosfatos/metabolismo , Bacteriófago M13/genética , Bacteriófago P1/genética , Escherichia coli/virología , Lisogenia/genética , Mutación , Transducción Genética , Replicación Viral/genéticaRESUMEN
OBJECTIVE: Computed tomography (CT) is traditionally used for evaluation and staging of gallbladder carcinoma (GC). However, in the subgroup of patients with obstructive jaundice, magnetic resonance cholangiography (MRC) is generally required to assess the level of biliary obstruction. The present study was undertaken to evaluate the diagnostic potential of three-dimensional helical CT cholangiography (3-D CTC) with minimum intensity projection (minIP), to determine the presence and level of biliary obstruction. MATERIALS AND METHODS: Twenty-five consecutive patients with proven GC, presenting with clinical and biochemical features of obstructive jaundice, over a 1-year period were included in the study. Dual phase helical CT data was obtained in the arterial and venous phases, respectively, after intravenous contrast injection using a pressure injector. Axial CT data (both arterial and venous phase) was studied for staging and resectability of tumor. Three-dimensional helical CT cholangiography using minIP obtained from the venous phase data set, was used to assess the level of biliary obstruction and isolation of hepatic segmental ducts. Three-dimensional helical CT cholangiography findings were compared with MRC and percutaneous transhepatic cholangiography (PTC) (gold standard). None of the patients were operated on as they were all considered inoperable on axial CT images due to extensive local disease or distant metastasis. RESULTS: In all patients, 3-D CTC demonstrated dilated intrahepatic ducts up to tertiary branch level. The 3-D CTC correctly diagnosed the level of biliary obstruction and demonstrated isolated segmental ducts in all patients and correlated well in all cases with MRC and PTC findings in this regard. However, the 3-D CTC did not add any additional information over the axial source images. CONCLUSION: Three-dimensional helical CT cholangiography with minIP can correctly determine the level of biliary obstruction in patients with GC and may be a strong competitor with MRC, because it gives equivalent information with regard to the level of ductal obstruction even while being a part of an overall comprehensive CT staging study. Even though 3-D CTC did not provide additional information on top of the source images, the referring physicians found them very useful for conceptualization of the 3-D biliary anatomy.
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Colangiocarcinoma/diagnóstico por imagen , Neoplasias de la Vesícula Biliar/diagnóstico por imagen , Ictericia Obstructiva/diagnóstico por imagen , Adulto , Anciano , Sistema Biliar/diagnóstico por imagen , Sistema Biliar/patología , Colangiocarcinoma/complicaciones , Colangiografía/métodos , Femenino , Neoplasias de la Vesícula Biliar/complicaciones , Humanos , Imagenología Tridimensional/métodos , Ictericia Obstructiva/etiología , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Tomografía Computarizada Espiral/métodosRESUMEN
Chains of inorganic polyphosphate (poly-P) with hundreds of P(i) residues linked by phosphoanhydride bonds, as in ATP, are found in every bacterial, fungal, plant, and animal cell, in which they perform various functions. In the spore-forming Bacillus cereus, we have identified three principal enzymes and genes involved in the metabolism of poly-P, namely, (i) poly-P kinase (PPK), which synthesizes poly-P reversibly from ATP, (ii) exopolyphosphatase (PPX), which hydrolyzes poly-P to P(i), and (iii) poly-P/AMP phosphotransferase (PAP), which uses poly-P as a donor to convert AMP to ADP, reversibly. In the null mutant of ppk, poly-P levels are reduced to <5% of the WT; in the ppx mutant, the PPK activity is elevated 10-fold, and the accumulation of poly-P is elevated approximately 1,000-fold. All of the null mutants of ppk, ppx, and pap showed defects in motility and biofilm formation, but sporulation efficiency was impaired only in the ppx mutant. These enzymes and genes in B. cereus are nearly identical to those in the very closely related pathogen Bacillus anthracis, and, thus, they may provide attractive targets for the treatment of anthrax.
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Bacillus cereus/enzimología , Biopelículas , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/fisiología , Bacillus cereus/genética , Bacillus cereus/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Clonación Molecular , Enzimas/genética , Enzimas/fisiología , Datos de Secuencia Molecular , Movimiento , Mutagénesis Sitio-Dirigida , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Esporas Bacterianas/enzimologíaRESUMEN
To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.
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Fosfatasa Ácida/análisis , Proteínas de Escherichia coli/análisis , Escherichia coli/enzimología , N-Glicosil Hidrolasas/análisis , Nucleotidasas/análisis , Fosfatasa Ácida/antagonistas & inhibidores , Fosfatasa Ácida/metabolismo , Cationes Bivalentes/farmacología , Desoxirribonucleótidos/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , N-Glicosil Hidrolasas/antagonistas & inhibidores , N-Glicosil Hidrolasas/metabolismo , Nucleotidasas/antagonistas & inhibidores , Nucleotidasas/metabolismo , Nucleótidos/metabolismo , Nucleótidos/farmacología , Monoéster Fosfórico Hidrolasas/metabolismo , Polifosfatos/metabolismo , Ribonucleótidos/metabolismo , Especificidad por SustratoRESUMEN
The importance of inorganic polyphosphate (poly P) and poly P kinase (PPK), the enzyme principally responsible for its synthesis, has been established previously for stationary-phase survival of Escherichia coli and virulence in Pseudomonas aeruginosa. The gene (ppk) that encodes PPK is highly conserved among many bacterial pathogens, including Shigella and Salmonella spp. In view of the phylogenetic similarity of the enteropathogens and the frequency with which virulence factors are expressed in stationary phase, the ppk gene of pathogenic Shigella flexneri, Salmonella enterica serovar Dublin, and Salmonella enterica serovar typhimurium have been cloned and deleted. In some of these mutants lacking ppk, the phenotypes included features indicative of decreased virulence such as: (i) growth defects, (ii) defective responses to stress and starvation, (iii) loss of viability, (iv) polymyxin sensitivity, (v) intolerance to acid and heat, and (vi) diminished invasiveness in epithelial cells. Thus PPK may prove, as it has with P. aeruginosa, to be an attractive target for antibiotics, with low toxicity because PPK is not found in higher eukaryotes.