Exploring the secrets of virus entry: the first respiratory syncytial virus carrying beta lactamase.

Background: Respiratory Syncytial Virus (RSV) presents a significant health threat, especially to young children. In-depth understanding of RSV entry mechanisms is essential for effective antiviral development. This study introduces an innovative RSV variant, featuring the fusion of the beta-lactamase (BlaM) enzyme with the RSV-P phosphoprotein, providing a versatile tool for dissecting viral entry dynamics. Methods: Using the AlphaFold2 algorithm, we modeled the tertiary structure of the P-BlaM chimera, revealing structural similarities with both RSV-P and BlaM. Functional assessments, utilizing flow cytometry, quantified beta-lactamase activity and GFP expression in infected bronchial epithelial cells. Western blot analysis confirmed the integrity of P-BlaM within virions. Results: The modeled P-BlaM chimera exhibited structural parallels with RSV-P and BlaM. Functional assays demonstrated robust beta-lactamase activity in recombinant virions, confirming successful P-BlaM incorporation as a structural protein. Quercetin, known for its antiviral properties, impeded viral entry by affecting virion fusion. Additionally, Ulixertinib, an ERK-1/2 inhibitor, significantly curtailed viral entry, implicating ERK-1/2 pathway signaling. Conclusions: Our engineered RSV-P-BlaM chimera emerges as a valuable tool, illuminating RSV entry mechanisms. Structural and functional analyses unveil potential therapeutic targets. Quercetin and Ulixertinib, identified as distinct stage inhibitors, show promise for targeted antiviral strategies. Time-of-addition assays pinpoint quercetin's specific interference stage, advancing our comprehension of RSV entry and guiding future antiviral developments.

Frequency of high-risk genotypes of human papilloma virus in oral lesions / Frecuencia de genotipos alto riesgo del virus papiloma humano en lesiones bucales

Some genotypes of the human papilloma virus (HPV) in the oral cavity cause genetic instability that may lead to cancer. Clinical and histological diagnoses are key tools; however, molecular techniques allow predicting, detecting and monitoring the disease. Objective: To identify the frequency of four high-risk HPV genotypes and their association with lesions in the oral cavity. Materials and Methods: Descriptive cross-sectional study with a sample of 48 patients diagnosed with hyperplastic lesions and others currently classified as potentially malignant disorders (PMDs) of the oral cavity, who underwent biopsies, histopathological analysis, and HPV16, 18, 31, and 45 detection and genotyping by polymerase chain reaction (PCR). Results: Epithelial hyperplasia was the most frequent lesion found in 45.8% (n=22) of patients. Nicotine palatinus and leukoplakia were found in 8.3% and 6.2%, respectively; oral cancer in 6.2%. The total frequency of HPV was 12.5% (6/48). Oral papilloma was found in 6.1% (3/48), and nicotine palatinus and oral cancer in 2.0% each (1/48). HPV16, HPV31, and HPV45 were detected, while HPV18 was not observed. HPV16 was the most frequent genotype found (4 out of 6 patients), while HPV31 and HPV45 were found in one patient each. Only one genotype per lesion was found. The presence of HPV was associated with lesions (χ2=11.810; p=0.0375). No significant association with age and gender was found. Conclusion: High-risk HPV continues to be present in oral lesions. The HPV16 viral genotype was the most frequent in the studied lesions.

Algunos genotipos del virus del papiloma (VPH) en boca, producen inestabilidad genética dando lugar al cáncer. El diagnóstico clínico e histológico son herramientas claves, sin embargo, técnicas moleculares permiten predecir, detectar y dar seguimiento a la enfermedad. Objetivo: Identificar la frecuencia de cuatro genotipos del VPH de alto riesgo y su asociación con lesiones en cavidad bucal. Material y Métodos: Estudio descriptivo de corte transversal con una muestra de 48 pacientes diagnosticados con lesiones hiperplásicas y otros clasificados actualmente como desordenes potencialmente malignos (DPM) de la cavidad bucal, a quienes se les realizó biopsias, análisis histopatológico y detección y genotipificación VPH16, 18, 31, y 45 mediante reacción en cadena a la polimerasa (PCR). Resultado: La hiperplasia epitelial fue la lesión más frecuente en 45,8% (n=22). La palatinitis nicotínica y la leucoplasia, se encontraron 8,3% y 6,2% respectivamente, cáncer oral, en 6,2%. La frecuencia total de VPH fue 12,5% (6/48). El papiloma oral estuvo en un 6,1% (3/48), palatinitis nicotínica y cáncer oral en 2,0% (1/48).Se detectó VPH16, VPH31 y VPH45, mientras que VPH18 estuvo ausente. ElVPH16 fue el de mayor frecuencia con 66,7% (4/6), el VPH31 y VPH45 se encontraron en 16,7% (1/6). No se evidenció más de un genotipo por lesión. La presencia de VPH estuvo asociado con las lesiones (χ2=11,810; p=0,0375). No se encontró asociación significativa con edad y género. Conclusión: El VPH de alto riesgo sigue estando presente en lesiones bucales. El genotipo viral VPH16 se encontró con mayor frecuencia en las lesiones estudiadas.

Inflammation and focal atrophy in prostate needle biopsy cores and association to prostatic adenocarcinoma.

The possible origin of proliferative inflammatory atrophy in the regenerative proliferation of prostate epithelial cells in response to injury caused by inflammation, and their relation to prostate adenocarcinoma have not been defined. Inflammation and focal atrophy are common pathological findings in prostate biopsies, currently not routinely included in surgical pathology reports. The objective of the study was to determine the correlation between inflammation and focal atrophy with prostate adenocarcinoma. Prostate needle biopsies from 203 patients with clinical parameters suspicious for malignancy were evaluated for the presence and extent of chronic inflammation, type and grade of focal atrophy, high-grade intraepithelial neoplasia, and adenocarcinoma. Relations among them and with age were also analyzed. χ(2) tests and binary logistic regression were used to estimate associations. Chronic inflammation was observed in 77.3% of the biopsies, significantly associated to adenocarcinoma (P = .031). Moderate/severe inflammation in at least 1 biopsy core increased the risk of prostate adenocarcinoma (odds ratio, 2.94; 95% confidence interval, 1.27-6.8), whereas glandular localization of inflammation decreased the risk. Focal atrophy was present in 72.9% of the biopsies, proliferative inflammatory atrophy was the most common type, and its grade was significantly associated to inflammation (P < .0001) and inflammation intensity (P = .003). An association between prostate adenocarcinoma and inflammation was found, with higher odds in presence of moderate/severe inflammation in at least 1 biopsy core. Increasing grades of proliferative inflammatory atrophy were associated to high levels of inflammation, supporting its previously proposed inflammatory nature.

The small leucine rich proteoglycan fibromodulin is overexpressed in human prostate epithelial cancer cell lines in culture and human prostate cancer tissue.

BACKGROUND: Fibromodulin is a small leucine-rich proteoglycan important for extracellular matrix organization and essential for tissue repair in multiple organs. The main function of this proteoglycan is the regulation of collagen fibrillogenesis; however, more recently described roles for fibromodulin have expanded to include regulation of angiogenesis, reprogramming of human fibroblasts into pluripotent cells, modulation of TGF-ß activity, inflammatory processes and association with metastatic phenotypes. Additionally, fibromodulin has been identified as a novel tumor-associated antigen in leukemia, lymphoma, and leiomyoma. Knowledge about its expression in the prostate is limited. METHODS: Fibromodulin expression was analyzed in two different malignant and one non-tumorigenic prostatic cell lines in culture, and in benign and malignant human prostate tissue. Expression was analyzed by real time PCR, immunocytochemistry, and immunohistochemistry. DNA sequencing was performed on a PCR fragment amplified with primers specific for the FMOD gene from cDNA obtained from the cultured cell lines. RESULTS: Both immunostaining and real time PCR analysis of cell lines indicated that fibromodulin was differentially expressed in the cancerous cell lines compared to the non-tumorigenic cell line. Likewise, cancerous tissue expressed significantly higher levels of intracellular fibromodulin compared to matched, benign tissue from the same patients, as well as compared to tissue from patients with only benign disease. CONCLUSIONS: The expression of fibromodulin was higher in prostatic cancer cells (cell-lines and human tissue) than in normal/benign prostatic cells. Additional studies are required to determine the biological and clinical significance and whether this proteoglycan has a role in carcinogenesis of the prostate or in prostate cancer related inflammatory processes.

Gene expression profiling of prostate cancer-associated genes identifies fibromodulin as potential novel biomarker for prostate cancer.

BACKGROUND: The aim of this study was to evaluate the gene expression profiles of a set of prostate cancer-associated genes in prostate cancer cell lines, to determine their association with different cancer phenotypes and identify potential novel biomarkers for this disease. METHODS: Quantitative real-time PCR was used to determine the expression profiles of 21 prostate cancer-associated genes in the human prostate cancer cell lines PC-3 and LNCaP, using the nontumorigenic cell line PWR-1E as control cell line. Genes evaluated were ESM-1, SERPINE2, CLU, BGN, A2M, PENK, FMOD, CD81, DCN, TSPAN8, KBTBD10, F2RL1, TMSB4X, SNCG, CXXC5, FOXQ1, PDPN, SPN, CAV1, CD24 and KLK3. A potential biomarker from this set of genes, the FMOD gene, encoding the small leucine-rich proteoglycan fibromodulin, was selected for further evaluation in clinical samples from patients diagnosed with benign or malignant prostatic disease. RESULTS: Several of the evaluated genes showed significantly altered expression in the prostate cancer cell lines, compared with nontumorigenic PWR-1E cells. Further evaluation of FMOD transcript in prostate clinical samples from patients diagnosed with benign or malignant prostatic disease identified a significant difference in the expression levels of this proteoglycan between benign and malignant tissue (p<0.05). CONCLUSIONS: A number of gene transcripts were differentially expressed by the cell lines assayed. Among them, FMOD was further evaluated in clinical samples and was found to be differentially expressed between benign and prostate cancer tissue. Further validation of FMOD transcript in a larger population is required to ascertain its usefulness as biomarker for prostate cancer.

Microarray analysis of the in vitro granulomatous response to Mycobacterium tuberculosis H37Ra.

BACKGROUND: The hallmark of tuberculosis is the granuloma, an organized cellular accumulation playing a key role in host defense against Mycobacterium tuberculosis. These structures sequester and contain mycobacterial cells preventing active disease, while long term maintenance of granulomas leads to latent disease. Clear understanding on mechanisms involved in granuloma formation and maintenance is lacking. OBJECTIVE: To monitor granuloma formation and to determine gene expression profiles induced during the granulomatous response to M. tuberculosis (H37Ra). METHODS: We used a previously characterized in vitro human model. Cellular aggregation was followed daily with microscopy and Wright staining for 5 days. Granulomas were collected at 24 h, RNA extracted and hybridized to Affymetrix human microarrays. RESULTS: Daily microscopic examination revealed gradual formation of granulomas in response to mycobacterial infection. Granulomatous structures persisted for 96 h, and then began to disappear. CONCLUSIONS: Microarray analysis identified genes in the innate immune response and antigen presentation pathways activated during the in vitro granulomatous response to live mycobacterial cells, revealing very early changes in gene expression of the human granulomatous response.

ANTECEDENTES: La marca histológica de la tuberculosis es el granuloma, una acumulación celular organizada que cumple funciones claves en la defensa del hospedero contra Mycobacterium tuberculosis. Estas estructuras secuestran y confinan a las micobacterias previniendo el desarrollo de enfermedad activa; el mantenimiento a largo plazo de los granulomas conlleva al establecimiento de latencia. Un mejor entendimiento de los mecanismos involucrados en la formación y mantenimiento del granuloma es necesario. OBJETIVO: Monitorear la formación del granuloma y determinar los patrones de expresión génica inducidos durante la respuesta granulomatosa a M. tuberculosis (H37Ra). MÉTODOS: En este estudio se empleó un modelo in vitro humano previamente caracterizado. La agregación celular fue examinada diariamente mediante microscopia óptica y tinción de Wright por 5 días. Para analizar la expresión génica, los granulomas fueron colectados a las 24 h, se extrajo el RNA sometiéndolo a hibridación a micromatrices de Affymetrix. RESULTADOS: Se observó la formación gradual de granulomas en respuesta a la infección. Los granulomas persistieron por 96 h, y luego se desvanecieron. CONCLUSIONES: Se identificaron genes de la respuesta inmune innata y vías de presentación antigénica activadas durante la respuesta granulomatosa in vitro a células micobacteriales vivas, lo cual reveló alteraciones tempranas de la expresión génica en el inicio de la respuesta granulomatosa humana.

Microarray analysis of the in vitro granulomatous response to Mycobacterium tuberculosis H37Ra / Análisis con Micromatrices de la respuesta granulomatosa in vitro a Mycobacterium tuberculosis H37Ra

Background: The hallmark of tuberculosis is the granuloma, an organized cellular accumulation playing a key role in host defense against Mycobacterium tuberculosis. These structures sequester and contain mycobacterial cells preventing active disease, while long term maintenance of granulomas leads to latent disease. Clear understanding on mechanisms involved in granuloma formation and maintenance is lacking. Objective: To monitor granuloma formation and to determine gene expression profiles induced during the granulomatous response to M. tuberculosis (H37Ra). Methods: We used a previously characterized in vitro human model. Cellular aggregation was followed daily with microscopy and Wright staining for 5 days. Granulomas were collected at 24h, RNA extracted and hybridized to Affymetrix human microarrays. Results: Daily microscopic examination revealed gradual formation of granulomas in response to mycobacterial infection. Granulomatous structures persisted for 96 h, and then began to disappear. Conclusions: Microarray analysis identified genes in the innate immune response and antigen presentation pathways activated during the in vitro granulomatous response to live mycobacterial cells, revealing very early changes in gene expression of the human granulomatous response.

Antecedentes: La marca histológica de la tuberculosis es el granuloma, una acumulación celular organizada que cumple funciones claves en la defensa del hospedero contra Mycobacterium tuberculosis. Estas estructuras secuestran y confinan a las micobacterias previniendo el desarrollo de enfermedad activa; el mantenimiento a largo plazo de los granulomas conlleva al establecimiento de latencia. Un mejor entendimiento de los mecanismos involucrados en la formación y mantenimiento del granuloma es necesario. Objetivo: Monitorear la formación del granuloma y determinar los patrones de expresión génica inducidos durante la respuesta granulomatosa a M. tuberculosis (H37Ra). Métodos: En este estudio se empleó un modelo in vitro humano previamente caracterizado. La agregación celular fue examinada diariamente mediante microscopia óptica y tinción de Wright por 5 días. Para analizar la expresión génica, los granulomas fueron colectados a las 24 h, se extrajo el RNA sometiéndolo a hibridación a micromatrices de Affymetrix. Resultados: Se observó la formación gradual de granulomas en respuesta a la infección. Los granulomas persistieron por 96 h, y luego se desvanecieron. Conclusiones: Se identificaron genes de la respuesta inmune innata y vías de presentación antigénica activadas durante la respuesta granulomatosa in vitro a células micobacteriales vivas, lo cual reveló alteraciones tempranas de la expresión génica en el inicio de la respuesta granulomatosa humana.

Molecular identification and antimicrobial susceptibility of Staphylococcus aureus nasal isolates from medical students in Cartagena, Colombia.

Staphylococcus aureus (SA) remains a major cause of nosocomial and community-acquired infections worldwide. Nasal carriage of this bacterium among hospital personnel constitutes an important source for nosocomial infections. A cross-sectional study enrolling the whole medical student population (n=387) of the School of Medicine at the Universidad de Cartagena, Colombia, was conducted to evaluate the carriage rates of both methicillin sensitive- and methicillin resistant-SA, the frequency of Panton-Valentine leukocidin genes in the isolates, and risk factors associated with carriage in this selected population. After signing an informed consent, participants completed a survey related to possible risk factors for colonization, and nasal swabs were collected from anterior nares. Staphylococcus aureus strains isolated from carriers were subjected to DNA extraction and PCR assays to determine the presence of MecA and Panton-Valentine leukocidin genes. Typing of the staphylococcal chromosomal cassette was performed for methicillin resistant strains. Molecular analysis was performed for only one strain per carrier. Prevalence of carriage for methicillin sensitive- and methicillin resistant-SA was 25% and 1.6% respectively. Most of the methicillin resistant isolates carried the staphylococcal chromosomal cassette type IV and the genes for Panton-Valentine leukocidin. To determine carrier types among medical students, each participant was subjected to four additional swabs, each taken two weeks apart. 9.8% persistent carriers, 53.1% intermittent carriers, and 37.1% non-carriers of SA were found. There was no association between risk factors analyzed and carriage of the bacterium. The study was conducted from April to September 2009 and found a persistent carriage of methicillin resistant-SA strains bearing the genes for Panton-Valentine leukocidin among medical students, evidencing the potential contribution of this portion of healthcare personnel either to the spread or introduction of these strains into the healthcare environment.

Molecular identification and antimicrobial susceptibility of Staphylococcus aureus nasal isolates from medical students in Cartagena, Colombia

Staphylococcus aureus (SA) remains a major cause of nosocomial and community-acquired infections worldwide. Nasal carriage of this bacterium among hospital personnel constitutes an important source for nosocomial infections. A cross-sectional study enrolling the whole medical student population (n = 387) of the School of Medicine at the Universidad de Cartagena, Colombia, was conducted to evaluate the carriage rates of both methicillin sensitive-and methicillin resistant-SA, the frequency of Panton-Valentine leukocidin genes in the isolates, and risk factors associated with carriage in this selected population. After signing an informed consent, participants completed a survey related to possible risk factors for colonization, and nasal swabs were collected from anterior nares. Staphylococcus aureus strains isolated from carriers were subjected to DNA extraction and PCR assays to determine the presence of MecA and Panton-Valentine leukocidin genes. Typing of the staphylococcal chromosomal cassette was performed for methicillin resistant strains. Molecular analysis was performed for only one strain per carrier. Prevalence of carriage for methicillin sensitiveand methicillin resistant-SA was 25% and 1.6% respectively. Most of the methicillin resistant isolates carried the staphylococcal chromosomal cassette type IV and the genes for Panton-Valentine leukocidin. To determine carrier types among medical students, each participant was subjected to four additional swabs, each taken two weeks apart. 9.8% persistent carriers, 53.1% intermittent carriers, and 37.1% non-carriers of SA were found. There was no association between risk factors analyzed and carriage of the bacterium. The study was conducted from April to September 2009 and found a persistent carriage of methicillin resistant-SA strains bearing the genes for Panton-Valentine leukocidin among medical students, evidencing the potential contribution of this portion of healthcare personnel either to the spread or introduction of these strains into the healthcare environment.

Staphylococcus aureus en residentes de un hogar de ancianos de Cartagena / Staphylococcus aureus in residents from a nursing-home in Cartagena

Objetivos Determinar la prevalencia de colonización nasal de Staphylococcus aureus, la susceptibilidad a antibióticos de los aislamientos y su posible asociación con factores de riesgo, en los residentes del Hogar Asilo de Ancianos San Pedro Claver de la ciudad de Cartagena de Indias durante el segundo semestre del año 2007. Métodos Con el debido consentimiento informado, para cada sujeto participante se tomaron hisopados nasales por una única vez durante el estudio. Se identificaron cepas de Staphylococcus aureus usando métodos clásicos y se determinó la susceptibilidad a antibióticos de los aislamientos mediante el método de difusión por disco según los estándares del CLSI. La información recolectada de las historias clínicas y con ayuda de un cuestionario se utilizó para analizar la asociación con factores de riesgo potenciales usando el programa SPSS 13.0 para Windows. Resultados Se obtuvieron 11 aislamientos positivos para Staphylococcus aureus, de un total de 69 individuos participantes, lo que correspondió a una prevalencia de 15,9 por ciento. No se detectaron cepas resistentes a meticilina. La portación nasal de Staphylococcus aureus estuvo asociada significativamente con limitaciones en el nivel de movilidad y con la presencia de lesiones cutáneas. No se encontró asociación significativa con otros de los diferentes factores de riesgo analizados. Conclusiones. La portación nasal de S. aureus encontrada en este estudio es mucho más baja que las reportadas en estudios similares en Colombia y otros países, teniendo en cuenta que la población estudio es vulnerable a la colonización con este patógeno.

Objectives Determining Staphylococcus aureus nasal carriage, antibiotic susceptibility and association with potential risk factors in residents from the Hogar Asilo de Ancianos San Pedro Claver nursing-home in Cartagena during the second semester of 2007. Methods Nasal swabs were taken from each person participating in the study after they had signed an informed consent form. Staphylococcus aureus strains were identified by classical methods; antibiotic susceptibility was determined by disk diffusion methods, according to CLSI standards. SPSS for Windows 13.0 statistical package was used for analysing data collected from medical records and from a questionnaire for analysing association with potential risk factors. Results 11 Staphylococcus aureus isolates were obtained from 69 participants, corresponding to 15.9 percent prevalence. No methicillin-resistant strains were detected. Staphylococcus aureus nasal carriage was significantly associated with limited mobility and skin lesions. There was no significant association with the other risk factors analysed. Conclusions Staphylococcus aureus nasal carriage found in this study was lower than that reported from other similar studies in other countries, taking into account that this is a population at risk for colonisation by this pathogen.

[Staphylococcus aureus in residents from a nursing-home in Cartagena]. / Staphylococcus aureus en residentes de un hogar de ancianos de Cartagena.

OBJECTIVES: Determining Staphylococcus aureus nasal carriage, antibiotic susceptibility and association with potential risk factors in residents from the Hogar Asilo de Ancianos San Pedro Claver nursing-home in Cartagena during the second semester of 2007. METHODS: Nasal swabs were taken from each person participating in the study after they had signed an informed consent form. Staphylococcus aureus strains were identified by classical methods; antibiotic susceptibility was determined by disk diffusion methods, according to CLSI standards. SPSS for Windows 13.0 statistical package was used for analysing data collected from medical records and from a questionnaire for analysing association with potential risk factors. RESULTS: 11 Staphylococcus aureus isolates were obtained from 69 participants, corresponding to 15.9% prevalence. No methicillin-resistant strains were detected. Staphylococcus aureus nasal carriage was significantly associated with limited mobility and skin lesions. There was no significant association with the other risk factors analysed. CONCLUSIONS: Staphylococcus aureus nasal carriage found in this study was lower than that reported from other similar studies in other countries, taking into account that this is a population at risk for colonisation by this pathogen.