Construction of Recombinant Vaccinia Virus for Oncolyitc Therapy

Homologous recombination is a mechanism conducted to exchange nucleotides between two similar sequences, which allows researchers to knock out and add genetic sequences of their interest in vaccinia virus (VACV). In this study, a Western Reserve VACV strain with inserted genes encoding Red Florescence Protein (RFP) and Green Florescence Protein (GFP), will be targeted and replaced with the Thymidine Kinase gene, J2R as well as C11 gene, respectively. This will set the foundation for continual removal of genes as well as additions to make a virus that will more effectively target cancer cells without affecting healthy cells. To conduct this experiment, the J2R gene was constructed via overlapping PCR and shared homology with the Western Reserve strain allowing for the homologous recombination to occur. HeLa cells are then infected and transfected with the VACV and the J2R overlapping PCR fragment, respectively. The transfection of the overlapping J2R PCR fragment into the infected HeLa cells will allow the virus to recombine. The infected cells are then collected and used to infect BS-C-1 cells, which allowed formation of plaques. The green (GFP) plaques without RFP are to be collected and undergo several rounds of purification. Upon purification, the DNA will be extracted using an SQ Blood Kit and enhanced via PCR. To confirm the virus recombined appropriately, the DNA will be sent out for sequencing and the procedure will be repeated for the replacement of GFP with C11 overlapping PCR fragment. Upon successful recombinant virus construction, the virus will undergo additional knockouts as well as additions that will allow the virus to target cancerous cells more effectively

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors

5to. Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad. Memoria académica

El V Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad, CITIS 2019, realizado del 6 al 8 de febrero de 2019 y organizado por la Universidad Politécnica Salesiana, ofreció a la comunidad académica nacional e internacional una plataforma de comunicación unificada, dirigida a cubrir los problemas teóricos y prácticos de mayor impacto en la sociedad moderna desde la ingeniería. En esta edición, dedicada a los 25 años de vida de la UPS, los ejes temáticos estuvieron relacionados con la aplicación de la ciencia, el desarrollo tecnológico y la innovación en cinco pilares fundamentales de nuestra sociedad: la industria, la movilidad, la sostenibilidad ambiental, la información y las telecomunicaciones. El comité científico estuvo conformado formado por 48 investigadores procedentes de diez países: España, Reino Unido, Italia, Bélgica, México, Venezuela, Colombia, Brasil, Estados Unidos y Ecuador. Fueron recibidas un centenar de contribuciones, de las cuales 39 fueron aprobadas en forma de ponencias y 15 en formato poster. Estas contribuciones fueron presentadas de forma oral ante toda la comunidad académica que se dio cita en el Congreso, quienes desde el aula magna, el auditorio y la sala de usos múltiples de la Universidad Politécnica Salesiana, cumplieron respetuosamente la responsabilidad de representar a toda la sociedad en la revisión, aceptación y validación del conocimiento nuevo que fue presentado en cada exposición por los investigadores. Paralelo a las sesiones técnicas, el Congreso contó con espacios de presentación de posters científicos y cinco workshops en temáticas de vanguardia que cautivaron la atención de nuestros docentes y estudiantes. También en el marco del evento se impartieron un total de ocho conferencias magistrales en temas tan actuales como la gestión del conocimiento en la universidad-ecosistema, los retos y oportunidades de la industria 4.0, los avances de la investigación básica y aplicada en mecatrónica para el estudio de robots de nueva generación, la optimización en ingeniería con técnicas multi-objetivo, el desarrollo de las redes avanzadas en Latinoamérica y los mundos, la contaminación del aire debido al tránsito vehicular, el radón y los riesgos que representa este gas radiactivo para la salud humana, entre otros

Identification of Vaccinia Virus Inhibitors and Cellular Functions Necessary for Efficient Viral Replication by Screening Bioactives and FDA-Approved Drugs

Four decades after the eradication of smallpox, poxviruses continue to threaten the health of humans and other animals. Vaccinia virus (VACV) was used as the vaccine that successfully eradicated smallpox and is a prototypic member of the poxvirus family. Many cellular pathways play critical roles in productive poxvirus replication. These pathways provide opportunities to expand the arsenal of poxvirus antiviral development by targeting the cellular functions required for efficient poxvirus replication. In this study, we developed and optimized a secreted Gaussia luciferase-based, simplified assay procedure suitable for high throughput screening. Using this procedure, we screened a customized compound library that contained over 3200 bioactives and FDA (Food and Drug Administration)-approved chemicals, most having known cellular targets, for their inhibitory effects on VACV replication. We identified over 140 compounds that suppressed VACV replication. Many of these hits target cellular pathways previously reported to be required for efficient VACV replication, validating the effectiveness of our screening. Importantly, we also identified hits that target cellular functions with previously unknown roles in the VACV replication cycle. Among those in the latter category, we verified the antiviral role of several compounds targeting the janus kinase/signal transducer and activator of transcription-3 (JAK/STAT3) signaling pathway by showing that STAT3 inhibitors reduced VACV replication. Our findings identify pathways that are candidates for use in the prevention and treatment of poxvirus infections and additionally provide a foundation to investigate diverse cellular pathways for their roles in poxvirus replications

Isoniazid Mono-Resistant Tuberculosis: Impact on Treatment Outcome and Survival of Pulmonary Tuberculosis Patients in Southern Mexico 1995-2010

<div><p>Background</p><p>Isoniazid mono-resistance (IMR) is the most common form of mono-resistance; its world prevalence is estimated to range between 0.0 to 9.5% globally. There is no consensus on how these patients should be treated.</p><p>Objective</p><p>To describe the impact of IMR tuberculosis (TB) on treatment outcome and survival among pulmonary TB patients treated under programmatic conditions in Orizaba, Veracruz, Mexico.</p><p>Materials and Methods</p><p>We conducted a prospective cohort study of pulmonary TB patients in Southern Mexico. From 1995 to 2010 patients with acid-fast bacilli or culture proven <i>Mycobacterium tuberculosis</i> in sputum samples underwent epidemiological, clinical and microbiological evaluation. We included patients who harbored isoniazid mono-resistant (IMR) strains and patients with strains susceptible to isoniazid, rifampicin, ethambutol and streptomycin. All patients were treated following Mexican TB Program guidelines. We performed annual follow-up to ascertain treatment outcome, recurrence, relapse and mortality.</p><p>Results</p><p>Between 1995 and 2010 1,243 patients with pulmonary TB were recruited; 902/1,243 (72.57%) had drug susceptibility testing; 716 (79.38%) harbored pan-susceptible and 88 (9.75%) IMR strains. Having any contact with a person with TB (adjusted odds ratio (aOR)) 1.85, 95% Confidence interval (CI) 1.15–2.96) and homelessness (adjusted odds ratio (aOR) 2.76, 95% CI 1.08–6.99) were associated with IMR. IMR patients had a higher probability of failure (adjusted hazard ratio (HR) 12.35, 95% CI 3.38–45.15) and death due to TB among HIV negative patients (aHR 3.30. 95% CI 1.00–10.84). All the models were adjusted for socio-demographic and clinical variables.</p><p>Conclusions</p><p>The results from our study provide evidence that the standardized treatment schedule with first line drugs in new and previously treated cases with pulmonary TB and IMR produces a high frequency of treatment failure and death due to tuberculosis. We recommend re-evaluating the optimal schedule for patients harboring IMR. It is necessary to strengthen scientific research for the evaluation of alternative treatment schedules in similar settings.</p></div

Treatment Outcomes Among Pulmonary Tuberculosis Patients According to Drug Susceptibility.

<p>Orizaba, Veracruz, 1995–2010</p

Definition of Treatment Outcomes.

<p>Definition of Treatment Outcomes.</p

Socio-demographic, Clinical, and Radiological Characteristics of Patients with Pulmonary Tuberculosis, According to Type of Resistance.

<p>Orizaba, Veracruz 1995–2010.</p