Silica-coated magnetic particles for efficient RNA extraction for SARS-CoV-2 detection.

Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

PluriBAC: A Versatile Baculovirus-Based Modular System to Express Heterologous Genes in Different Biotechnological Platforms.

Baculoviruses are insect-specific pathogens widely used in biotechnology. In particular, the Autographa californica nucleopolyhedrovirus (AcMNPV) has been exploited as a platform for bio-inputs production. This is why the improvement of the technologies used for the production of recombinant baculoviruses takes on particular relevance. To achieve this goal, we developed a highly versatile baculoviral transfer vector generation system called PluriBAC. The PluriBAC system consists of three insert entry levels using Golden Gate assembly technology. The wide availability of vectors and sticky ends allows enough versatility to combine more than four different promoters, genes of interest, and terminator sequences. Here, we report not only the rational design of the PluriBAC system but also its use for the generation of baculoviral reporter vectors applied to different fields of biotechnology. We demonstrated that recombinant AcMNPV baculoviruses generated with the PluriBAC system were capable of infecting Spodoptera frugiperda larvae. On the other hand, we found that the recombinant budded virions (BV) generated using our system were capable of transducing different types of tumor and normal cells both in vitro and in vivo. Our findings suggest that the PluriBAC system could constitute a versatile tool for the generation of insecticide and gene therapy vectors.

Mitochondrial Peptide Humanin Facilitates Chemoresistance in Glioblastoma Cells.

Humanin (HN) is a mitochondrial-derived peptide with robust cytoprotective effects in many cell types. Although the administration of HN analogs has been proposed to treat degenerative diseases, its role in the pathogenesis of cancer is poorly understood. Here, we evaluated whether HN affects the chemosensitivity of glioblastoma (GBM) cells. We found that chemotherapy upregulated HN expression in GBM cell lines and primary cultures derived from GBM biopsies. An HN analog (HNGF6A) boosted chemoresistance, increased the migration of GBM cells and improved their capacity to induce endothelial cell migration and proliferation. Chemotherapy also upregulated FPR2 expression, an HN membrane-bound receptor, and the HNGF6A cytoprotective effects were inhibited by an FPR2 receptor antagonist (WRW4). These effects were observed in glioma cells with heterogeneous genetic backgrounds, i.e., glioma cells with wild-type (wtIDH) and mutated (mIDH) isocitrate dehydrogenase. HN silencing using a baculoviral vector that encodes for a specific shRNA for HN (BV.shHN) reduced chemoresistance, and impaired the migration and proangiogenic capacity of GBM cells. Taken together, our findings suggest that HN boosts the hallmark characteristics of GBM, i.e., chemoresistance, migration and endothelial cell proliferation. Thus, strategies that inhibit the HN/FPR2 pathway may improve the response of GBM to standard therapy.

Smart manufacturing inspired approach to research, development, and scale-up of electrified chemical manufacturing systems.

As renewable electricity becomes cost competitive with fossil fuel energy sources and environmental concerns increase, the transition to electrified chemical and fuel synthesis pathways becomes increasingly desirable. However, electrochemical systems have traditionally taken many decades to reach commercial scales. Difficulty in scaling up electrochemical synthesis processes comes primarily from difficulty in decoupling and controlling simultaneously the effects of intrinsic kinetics and charge, heat, and mass transport within electrochemical reactors. Tackling this issue efficiently requires a shift in research from an approach based on small datasets, to one where digitalization enables rapid collection and interpretation of large, well-parameterized datasets, using artificial intelligence (AI) and multi-scale modeling. In this perspective, we present an emerging research approach that is inspired by smart manufacturing (SM), to accelerate research, development, and scale-up of electrified chemical manufacturing processes. The value of this approach is demonstrated by its application toward the development of CO2 electrolyzers.

Evaluation of Baculoviruses as Gene Therapy Vectors for Brain Cancer.

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Exploring the Role of the Inhibitor of Apoptosis BIRC6 in Breast Cancer: A Database Analysis.

PURPOSE: The aim of the present work was to investigate the role of apoptosis inhibitor BIRC6 (baculoviral IAP repeat-containing protein 6) in breast cancer (BC), focusing particularly on its involvement in the metastatic cascade. METHODS: We analyzed BIRC6 mRNA expression levels and copy number variations in three BC databases from The Cancer Genome Atlas comparing clinical and molecular attributes. Genomic analysis was performed using the cBioPortal platform, whereas transcriptomic studies (mRNA expression levels, correlation heatmaps, survival plots, and gene ontology) were performed using USC Xena and R. Statistical significance was set at P < .05. RESULTS: Our bioinformatic analyses showed that there was a differential expression of BIRC6 in cancer samples when compared with normal samples. Copy number variations that involve amplification and gain of BIRC6 gene were correlated with negative hormone receptor tumors, higher prognostic indexes, younger age at diagnosis, and both chemotherapy and radiotherapy administration. Transcriptomic and gene ontology analyses showed that, under conditions of high BIRC6 mRNA levels, there are differential expression patterns in apoptotic, proliferation, and metastatic pathways. CONCLUSION: In summary, our in silico data suggest that BIRC6 plays an antiapoptotic, pro-proliferative, and apparent prometastatic role and could be a relevant molecular target for treatment of BC tumors.

The Magic Staff: A Comprehensive Overview of Baculovirus-Based Technologies Applied to Human and Animal Health.

Baculoviruses are enveloped, insect-specific viruses with large double-stranded DNA genomes. Among all the baculovirus species, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied. Due to its characteristics regarding biosafety, narrow host range and the availability of different platforms for modifying its genome, AcMNPV has become a powerful biotechnological tool. In this review, we will address the most widespread technological applications of baculoviruses. We will begin by summarizing their natural cycle both in larvae and in cell culture and how it can be exploited. Secondly, we will explore the different baculovirus-based protein expression systems (BEVS) and their multiple applications in the pharmaceutical and biotechnological industry. We will focus particularly on the production of vaccines, many of which are either currently commercialized or in advanced stages of development (e.g., Novavax, COVID-19 vaccine). In addition, recombinant baculoviruses can be used as efficient gene transduction and protein expression vectors in vertebrate cells (e.g., BacMam). Finally, we will extensively describe various gene therapy strategies based on baculoviruses applied to the treatment of different diseases. The main objective of this work is to provide an extensive up-to-date summary of the different biotechnological applications of baculoviruses, emphasizing the genetic modification strategies used in each field.

Platinum Complexes from C-H Activation of Sterically Hindered [C

Primary amines and benzothiophene-3-carboxaldehyde were reacted to give four large, bulky imine ligands. These imine ligands were reacted with a tetramethyl platinum dimer and by heteroatom-assisted C-H activation, both monometalated compounds and bismetalated compounds were synthesized. In all cases, five-membered platinacycles were formed. The compounds were characterized by NMR spectroscopy, and one bismetalated compound was characterized by single-crystal X-ray diffraction. The UV-vis absorption and emission spectra and the excited-state lifetimes were recorded for these complexes. Density functional theory (DFT) and time-dependent-DFT calculations were performed to aid in the assignment of the absorption and emission spectra of the newly synthesized complexes.

Development and perception of a multiple-site nontraditional postgraduate year 1 residency program.

PURPOSE: To describe the development and perception of a multiple-site nontraditional postgraduate year 1 (PGY1) residency program from the resident and preceptor perspectives. SUMMARY: A multiple-site nontraditional residency program was developed within a Florida health system to increase the education level and clinical responsibilities of hospital staff pharmacists. The program provided pharmacists interested in residency training an opportunity to pursue postgraduate credentials while maintaining their current position. The nontraditional residency program was implemented at 1 site and subsequently expanded across multiple affiliated hospital sites due to its success. Pharmacists currently working in the health system's network of hospitals for at least 2 years were eligible to enter into the 24-month program after successfully completing the application, interview, and matching process. The number of nontraditional resident positions available has varied by residency year and site. Offering this opportunity has increased the clinical knowledge of pharmacists, exposed them to a variety of practice areas, and increased their departmental contributions. In response to a request for feedback regarding the multiple-site nontraditional program, both residents and preceptors have reported benefits and challenges. CONCLUSION: Adequate resources are needed and a number of factors must be considered in developing a multiple-site nontraditional PGY1 residency program. Although there are potential challenges, it is perceived that the benefits justify continuation of the program.

Obtención de un diagnosticador rápido de cólera, basado en partículas de látex acopladas a un anticuerpo monoclonal contra lipopolisacárido de Vibrio cholerae O1 / Obtaining of a rapid diagnostic test for Cholera, based on latex particles coupled with a monoclonal antibody against Vibrio cholerae O1 lipopolysaccharide

El cólera es una enfermedad infecto-contagiosa intestinal aguda, causada por la ingestión de alimentos o agua contaminada con los serotipos O1 y O139 de la bacteria Vibrio cholerae. Se caracteriza por diarrea secretora abundante que llevan rápidamente a la deshidratación. Sin tratamiento adecuado produce la muerte en horas, por lo que el diagnostico precoz es muy importante sobre todo porque al inicio es difícil diferenciarla de otras enfermedades diarreicas agudas. La prueba diagnóstico de oro es el coprocultivo; sin embargo, no garantiza una detección rápida de la enfermedad. Recientemente se desarrollaron ensayos rápidos, se basan en tiras reactivas y aglutinación con partículas de látex, muy efectivos, pero difíciles de adquirir por sus altos precios. El objetivo de este trabajo fue obtener un diagnosticador rápido basado en partículas de látex acopladas a un anticuerpo monoclonal (AcM) contra el lipopolisacárido de V. cholerae O1, el cual se obtuvo en el Instituto Finlay. Se usaron partículas de látex de 0,8 µm en una suspensión al 10 por ciento a las que se acopló durante 2 h a 37°C el AcM a 0,25 mg/mL. Se evaluó la sensibilidad, especificidad y el desempeño en 84 muestras de heces de pacientes con diagnóstico presuntivo de cólera. El diagnosticador que se obtuvo no evidenció reactividad cruzada frente a cepas no-O1, ni contra otros enteropatógenos. El diagnosticador látex presentó valores de sensibilidad, especificidad y eficacia de 97,87; 97,29 y 97,6 por ciento respectivamente, muy similares al diagnosticador comercial CTK-Biotech. El diagnosticador látex que se obtuvo se puede utilizar en el diagnóstico rápido de la enfermedad(AU)

Cholera is an acute contagious intestinal disease caused by ingestion of food or water contaminated with O1 and O139 serotypes of the bacterium Vibrio cholerae . Cholera is characterized by abundant secretory diarrhea leading to dehydration. Death occurs within hours without treatment, so early diagnosis is very important, especially at the beginning of the disease, because it is difficult to differentiate from other acute diarrheal diseases. The diagnostic golden test is the stool culture; however, it does not guarantee a rapid detection of the disease. Rapid tests have been recently developed; they are based on test strips and agglutination with latex particles, which are very effective, but difficult to acquire for their high prices. The objective of this research was to obtain a quick assay based on latex particles coupled with a monoclonal antibody (mAb) against V. cholerae O1 lipopolysaccharide obtained in Finlay Institute. Latex particles of 0.8 µm were used in a 10 percent suspension, and they were coupled to the mAb (0.25 mg/ml) for 2 hours at 37°C. The sensitivity, specificity and performance were evaluated in 84 stool samples from patients with presumptive diagnosis of cholera. The diagnostic test obtained showed no cross-reactivity against no-O1 strains and other enteropathogens. Latex diagnostic test showed values of sensitivity, specificity and efficacy of 97.87; 97.29 and 97.6 percent respectively, very similar to the commercial diagnostic test CTK-Biotech. The latex reagent obtained can be used in the rapid diagnosis of the disease(AU)

Comportamiento y distribución espacial del virus rábico en la provincia La Habana, Cuba, 1998-2010 / Behavior and spatial distribution of rabic virus in Havana province, Cuba, 1998-2010

Objetivo: Analizar el comportamiento y distribución espacial de los focos rábicos en la provincia La Habana, de 1998 hasta 2010. Métodos: Se realizó un estudio observacional descriptivo retrospectivo de los casos de especie animal positivos a rabia, examinados en el Departamento de Rabia del Laboratorio Provincial de forma pasiva, de 1998 hasta 2010. Se incluyó 1 196 muestras de cerebros de carnívoros, quirópteros y herbívoros estudiados en la provincia. Se empleó la técnica de inmunofluorescencia directa para el diagnóstico de rabia. La descripción de la distribución espacial se realizó a partir de los sistemas de información geográficos digitalizados. Resultados: Los municipios periféricos tienen mayor índice de casos positivos a rabia en mangostas (mal llamado hurón en Cuba), perros y gatos; con 85 casos positivos de un total 1 196 muestras (7,11%) en un periodo de 13 años. Conclusiones: La utilización de mapas nos brinda gran utilidad ubicando geográficamente los focos rábicos. El mayor reporte de casos se notificó en cuatro municipios periféricos (Boyeros, Arroyo Naranjo, Cotorro y Guanabacoa), ubicados geográficamente en el centro y sur de la provincia La Habana.

Objective: To analyze the behavior and spatial distribution of rabies foci in the province of Havana, 1998-2010. Methods: We conducted a retrospective observational study of cases of animal species positive for rabies, examined passively in the Department of Rabies of the Provincial Laboratory, from 1998-2010. 1196 brain samples of carnivores and herbivores bats studied in the province were included. We used the direct immunofluorescence technique for the diagnosis of rabies. The description of the spatial distribution was made from the digitized geographical information systems. Results: The peripheral municipalities have larger number of cases positive for rabies in mongooses (so-called ferret in Cuba), dogs and cats, with 85 positive cases out of a total 1196 samples (7.11%) over a period of 13 years. Conclusions: The use of maps is useful in the geographic location of rabies foci. The greater reporting of cases occurred in four peripheral municipalities (Boyeros, Arroyo Naranjo, Cotorro and Guanabacoa), geographically located in the center and south of the province of Havana.

Incidencia del virus rábico en mangostas (Herpestes auropuntactus). Cuba, enero 2004 a diciembre 2011 / Incidence of rabies virus in mongooses (Herpestes auropuntactus). Cuba, January 2004 to December 2011

Objetivo: Analizar la incidencia del virus rábico en mangosta (Herpestes auropunctatus) de vida libre en Cuba, de enero 2004 a diciembre 2011. Métodos: Se realizó un estudio descriptivo-retrospectivo en el Centro Provincial de Higiene Epidemiología y Microbiología (CPHEM), en el Laboratorio de Rabia, La Habana-Cuba. Se analizaron las muestras colectadas correspondientes a 561 cerebros de mangostas lesionadoras a humanos y de vigilancia epidemiológica perteneciente a catorce provincias del país, diagnosticadas mediante la técnica de inmunofluorescencia directa (IFD) y representadas mediante el sistemas de información Mapinfo. Resultados: El porcentaje total de positividad de las muestras estudiadas es de 47.95 por ciento; desde el punto de vista geográfico la mayor concentración de muestras positivas fue en las províncias del occidente del país y además Ciego de Ávila y Holguín. Conclusiones: El porcentaje de positividad en esta especie es de 47.95 por ciento. Siendo la mangosta el principal reservorio de la rabia en Cuba; no es suficiente el conocimiento, monitoreo, control y erradicación de esta especie; se debe realizar un mayor estudio morfomátrico para su control.

January 2004 to December 2011. Methods: We conducted a retrospective descriptive study at the rabies laboratory of the Provincial Center of Hygiene, Epidemiology and Microbiology (CPHEM), Havana, Cuba. 561 brain samples from human attacker mongooses and epidemiological surveillance were analyzed, belonging to fourteen provinces. The diagnosis was made by direct immunofluorescence (DIF). Data were represented in the information system Mapinfo. Results: Results: The overall rate of positivity of the studied samples is 47.95 percent. From the geographical point of view the greatest concentration of positive samples was in the western provinces of the country and also Ciego de Avila and Holguin. Conclusions: The percentage of positivity in this species is of 47.95 percent. Since the mongoose is the main reservoir of rabies in Cuba, besides knowledge, monitoring, control and eradication of this species, more morphometric studies are needed for control.

Variante protrombótica C677T del gen metilenotetrahidrofolato reductasa es un biomarcador molecular de fallas de implantación en mujeres chilenas sometidas a protocolos de reproducción asistida / Prothrombotic variant C677T at the methylenetetrahydrofolate reductase gene is a molecular biomarker of implantation failure in chilean women undergoing assisted reproductive protocols

Las fallas de implantación son consideradas una importante causa de infertilidad en mujeres sometidas a protocolos de Fecundación Asistida. Evidencia reciente sugiere que la presencia de variantes protrombóticas está asociada a diversos defectos obstétricos y falla reproductiva. Sin embargo, los resultados en diversas poblaciones son contradictorios. De acuerdo a esta evidencia, se evaluó la potencial asociación entre variantes protrombóticas y fallas de implantación en mujeres chilenas incluidas en protocolos de fecundación asistida. Un total de 180 mujeres, 80 pacientes sometidas a protocolos de reproducción asistida y 100 controles fueron incluidas en este estudio. La genotipificación molecular de variantes protrombóticas en genes candidatos fue realizada por PCR-RFLP. Observamos una paciente heterocigota para la variante F5 G1691A y ausencia total del polimorfismo F2 G20210A en pacientes y controles. La distribución genotípica y la distribución relativa de alelos del polimorfismo MTHFR C677T fueron significativamente diferentes entre pacientes y controles. Odds Ratio para fallas de implantación asociadas al genotipo homocigoto fue 2,78 (95 por ciento IC 1,147 - 6,755; p= 0,0199). En resumen, nuestros resultados sugieren que la variante MTHFR C677T constituye un biomarcador molecular de susceptibilidad a fallas de implantación población chilena.

Implantation failures are considered an important cause of infertility in women undergoing assisted reproductive protocols. Recent evidence suggests that the presence of prothrombotic variants is associated with obstetric defects and reproductive failure. However, results between several populations are contradictory. According this evidence, we evaluated the potential association between prothrombotic variants and implantation failure susceptibility in Chilean women undergoing Assisted Reproductive protocols. 180 women, 80 patients undergoing assisted reproductive protocols and 100 controls were included in this study. Molecular genotyping of prothrombotic variants in candidate genes was realized by PCR-RFLP. We observed one patient heterozygote for factor V G1691A variant, and total absence to prothrombin G20210A polymorphism in patients and controls. Genotype distribution and allelic frequencies of MTHFR C677T polymorphism were significantly different between patients and controls (p<0.05). Odss Ratio for implantation failure associated to homozygote genotype was 2.78 (95 percent IC 1.147 ¡ 6.755; p= 0.0199). In summary, our data suggest that the MTHFR C677T polymorphism constituted a molecular biomarker of implantation failure susceptibility in Chilean population.