La educación a distancia en la Enseñanza Técnica Superior de Prótesis Estomatológica: experiencias de 2 cursos

Introducción: La asignatura Introducción a la Metodología de la Investigación, para la Enseñanza Técnica Superior de Prótesis Estomatológica, de la Facultad de Estomatología de La Habana, se incorporó a la educación a distancia, durante el curso 2021-2022. Objetivo: Describir la experiencia en la ejecución de la asignatura con el uso del aula virtual de la Institución. Material y Métodos: Se realizó una investigación de tipo descriptiva, retrospectiva, acerca de las experiencias en el proceso de enseñanza-aprendizaje, durante los cursos 2021-2022 y 2022-2023. Resultados: Se cumplieron los objetivos metodológicos de la asignatura, se debatieron los temas propuestos en los foros, se aclararon las dudas utilizando las herramientas interactivas de la plataforma y la atención al alumno fue personalizada. Conclusiones: Se logró impartir por primera vez con éxito, la asignatura, se digitalizaron los objetos de aprendizaje por temas, se diseñaron actividades para la evaluación del aprendizaje, y se obtuvieron calificaciones satisfactorias.

Introduction: The subject Introduction to Research Methodology, for the Higher Technical Education of Stomatological Prosthetics, of the Faculty of Stomatology of Havana, was incorporated into distance education, during the 2021-2022 academic year. Objective: Describe the experience in the execution of the subject with the use of the Institution's virtual classroom. Material and Methods: A descriptive, retrospective research was carried out on the experiences in the teaching-learning process, during the 2021-2022 and 2022-2023 academic years. Results: The methodological objectives of the subject were met, the topics proposed in the forums were debated, doubts were clarified using the platform's interactive tools, and student attention was personalized. Conclusions: The subject was successfully taught for the first time, the learning objects were digitized by topic, activities were designed for the evaluation of learning, and satisfactory grades were obtained.

Role of non-coding RNAs in tuberculosis and their potential for clinical applications.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of mortality due to infectious diseases, only surpassed in 2020 by COVID-19. Despite the development in diagnostics, therapeutics, and evaluation of new vaccines for TB, this infectious disease remains uncontrollable due to the emergence of multidrug-resistant (MDR) and extremely drug-resistant (XDR) TB, among other factors. The development in transcriptomics (RNomics) has enabled the study of gene expression in TB. It is considered that non-coding RNAs (ncRNAs) from host [microRNAs (miRNAs)] and Mtb [small RNAs (sRNAs)] are important elements in TB pathogenesis, immune resistance, and susceptibility. Many studies have shown the importance of host miRNAs in regulating immune response against Mtb via in vitro and in vivo mice models. The bacterial sRNAs play a major role in survival, adaptation, and virulence. Here, we review the characterization and function of host and bacteria ncRNAs in TB and their potential use in clinical applications as diagnostic, prognostic, and therapeutic biomarkers.

Challenges and the Way forward in Diagnosis and Treatment of Tuberculosis Infection.

Globally, it is estimated that one-quarter of the world's population is latently infected with Mycobacterium tuberculosis (Mtb), also known as latent tuberculosis infection (LTBI). Recently, this condition has been referred to as tuberculosis infection (TBI), considering the dynamic spectrum of the infection, as 5-10% of the latently infected population will develop active TB (ATB). The chances of TBI development increase due to close contact with index TB patients. The emergence of multidrug-resistant TB (MDR-TB) and the risk of development of latent MDR-TB has further complicated the situation. Detection of TBI is challenging as the infected individual does not present symptoms. Currently, there is no gold standard for TBI diagnosis, and the only screening tests are tuberculin skin test (TST) and interferon gamma release assays (IGRAs). However, these tests have several limitations, including the inability to differentiate between ATB and TBI, false-positive results in BCG-vaccinated individuals (only for TST), false-negative results in children, elderly, and immunocompromised patients, and the inability to predict the progression to ATB, among others. Thus, new host markers and Mtb-specific antigens are being tested to develop new diagnostic methods. Besides screening, TBI therapy is a key intervention for TB control. However, the long-course treatment and associated side effects result in non-adherence to the treatment. Additionally, the latent MDR strains are not susceptible to the current TBI treatments, which add an additional challenge. This review discusses the current situation of TBI, as well as the challenges and efforts involved in its control.

Small RNA datasets of drug-susceptible Mycobacterium tuberculosis strains from Sabah, Malaysia.

These datasets present a list of small RNAs from three drug-susceptible Mycobacterium tuberculosis strains isolated from Sabah, Malaysia. Sputum samples were obtained from three tuberculosis patients belonging to different districts. The bacteria were detected using GeneXpert MTB/RIF, isolated and cultured in BACTECTM MGITTM 320, and tested for their drug susceptibility. Total RNAs were extracted, sequenced, and analyzed using bioinformatic tools to filter out small RNA present in the Mycobacterium tuberculosis strains. Small RNA sequencing generated total raw reads of 63,252,209, 63,636,812, and 61,148,224 and total trimmed reads (15-30 nucleotides) of 51,533,188, 53,520,197, and 51,363,772 for Mycobacterium tuberculosis strain SBH49, SBH149, and SBH372, respectively. The raw data were submitted to the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI) under the accession numbers of SRX16744291 (SBH49), SRX16744292 (SBH149), and SRX16744293 (SBH372). Small RNAs play important roles in cellular processes such as cell differentiation, cell signaling, development of resistance to antibiotics and immune response, and metabolism regulation. The small RNAs determined here could provide further insights into various cellular processes crucial for Mycobacterium tuberculosis survivability and a better understanding of their gene regulation which ultimately opens a new pathway for combating tuberculosis infection.

Transcriptomic Signature of Horseshoe Crab Carcinoscorpius rotundicauda Hemocytes' Response to Lipopolysaccharides.

Carcinoscorpius rotundicauda (C. rotundicauda) is one of the four species of horseshoe crabs (HSCs). The HSC hemocytes store defense molecules that are released upon encountering invading pathogens. The HSCs rely on this innate immunity to continue its existence as a living fossil for more than 480 million years. To gain insight into the innate mechanisms involved, transcriptomic analysis was performed on isolated C. rotundicauda hemocytes challenged with lipopolysaccharides (LPS), the main components of the outer cell membrane of gram-negative bacteria. RNA-sequencing with Illumina HiSeq platform resulted in 232,628,086 and 245,448,176 raw reads corresponding to 190,326,253 and 201,180,020 high-quality mappable reads from control and LPS-stimulated hemocytes, respectively. Following LPS-stimulation, 79 genes were significantly upregulated and 265 genes were downregulated. The differentially expressed genes (DEGs) were related to multiple immune functional categories and pathways such as those of the cytoskeleton, Toll and Imd, apoptosis, MAP kinase (MAPK), inositol phosphate metabolism, phagosome, leucocyte endothelial migration, and gram-negative bacterial infection, among others. This study provides important information about the mechanisms of response to LPS, which is relevant for the understanding the HSCs' immune response.

Proteomic analysis of Malaysian Horseshoe crab (Tachypleus gigas) hemocytes gives insights into its innate immunity host defence system and other biological processes.

Horseshoe crabs are one of the most studied invertebrates due to their remarkable innate immunity mechanism and biological processes. In this work, the proteins of the lipopolysaccharides (LPS)-stimulated and non-stimulated hemocytes of Malaysian Tachypleus gigas were profiled using LC-MS/MS. A total of 154 proteins were identified in both types of samples. Additionally, seventy-seven proteins were commonly found in both conditions, while 52 and 25 proteins were uniquely found in the LPS-stimulated and non-stimulated hemocytes, respectively. ATP-dependent energy-generating proteins such as actins and BLTX actin-related proteins were detected in both stimulated and non-stimulated T. gigas hemocytes, but more of such proteins were found in the former type. Proteins such as tachylectin-2, coagulogen, c-reactive proteins, histones, hemocyanin, and DNA polymerase, which play key roles in the organism's innate immunity, were differentially expressed in the hemocytes following LPS challenge. In conclusion, the proteins identified in the hemolymph of T. gigas are vital for the organism's molecular functions, biological processes, and activation of innate immunity.

Overlapping of Pulmonary Fibrosis of Postacute COVID-19 Syndrome and Tuberculosis in the Helminth Coinfection Setting in Sub-Saharan Africa.

There is an increasing attention to the emerging health problem represented by the clinical and functional long-term consequences of SARS-CoV-2 infection, referred to as postacute COVID-19 syndrome. Clinical, radiographic, and autopsy findings have shown that a high rate of fibrosis and restriction of lung function are present in patients who have recovered from COVID-19. Patients with active TB, or those who have recovered from it, have fibrotic scarred lungs and, consequently, some degree of impaired respiratory function. Helminth infections trigger predominantly type 2 immune responses and the release of regulatory and fibrogenic cytokines, such as TGF-ß. Here, we analyze the possible consequences of the overlapping of pulmonary fibrosis secondary to COVID-19 and tuberculosis in the setting of sub-Saharan Africa, the region of the world with the highest prevalence of helminth infection.

Genetic determination and JARID2 over-expression in a thermal incubation experiment in Casque-Headed Lizard.

Non-avian reptiles, unlike mammals and birds, have undergone numerous sex determination changes. Casque-Headed Lizards have replaced the ancestral XY system shared across pleurodonts with a new pair of XY chromosomes. However, the evolutionary forces that triggered this transition have remained unclear. An interesting hypothesis suggests that species with intermediate states, with sex chromosomes but also thermal-induced sex reversal at specific incubation temperatures, could be more susceptible to sex determination turnovers. We contrasted genotypic data (presence/absence of the Y chromosome) against the histology of gonads of embryos from stages 35-37 incubated at various temperatures, including typical male-producing (26°C) and female-producing (32°C) temperatures. Our work apparently reports for the first time the histology of gonads, including morphological changes, from stages 35-37 of development in the family Corytophanidae. We also observed that all embryos developed hemipenes, suggesting sex-linked developmental heterochrony. We observed perfect concordance between genotype and phenotype at all temperatures. However, analysis of transcriptomic data from embryos incubated at 26°C and 32°C identified transcript variants of the chromatin modifiers JARID2 and KDM6B that have been linked to temperature-dependent sex determination in other reptiles. Our work tested the validity of a mixed sex determination system in the family Corytophanidae. We found that XY chromosomes are dominant; however, our work supports the hypothesis of a conserved transcriptional response to incubation temperatures across non-avian reptiles that could be a reminiscence of an ancestral sex determination system.

Application of liposomes in the treatment of infectious diseases.

The advances in the development of drugs and vaccines for major infectious diseases of tuberculosis (TB), malaria and HIV represent some of the most significant milestones in their therapeutic strategies. Yet, current drugs and vaccines display limitations such as drug resistance and low efficacy level. In recent years, new emerging and advanced nano-technology carrier liposomes have been widely studied towards producing drugs and vaccines capable of targeting infectious diseases. Liposomes portrayed biocompatible and biodegradable properties with versatile flexibility, characteristics that are advantageous for a good targeting at the site of action. The success of liposomes has renewed interest in the research and development of liposomal drugs and vaccines shifting the paradigm in infectious diseases treatment. This review focuses on the limitations of current therapeutic drugs and vaccines, the knowledge of liposomes in terms of their classifications and advantages, and a review of the application of liposomes in the treatment of TB, malaria, and HIV infection.

Comparative transcriptome profiling of horseshoe crab Tachypleus gigas hemocytes in response to lipopolysaccharides.

Horseshoe crabs (HSCs) are living fossil species of marine arthropods with a long evolutionary history spanning approximately 500 million years. Their survival is helped by their innate immune system that comprises cellular and humoral immune components to protect them against invading pathogens. To help understand the genetic mechanisms involved, the present study utilised the Illumina HiSeq platform to perform transcriptomic analysis of hemocytes from the HSC, Tachypleus gigas, that were challenged with lipopolysaccharides (LPS). The high-throughput sequencing resulted in 352,077,208 and 386,749,136 raw reads corresponding to 282,490,910 and 305,709,830 high-quality mappable reads for the control and LPS-treated hemocyte samples, respectively. Based on the log-fold change of > 0.3 or < -0.3, 1338 genes were significantly upregulated and 215 genes were significantly downregulated following LPS stimulation. The differentially expressed genes (DEGs) were further identified to be associated with multiple pathways such as those related to immune defence, stress response, cytoskeleton function and signal transduction. This study provides insights into the underlying molecular and regulatory mechanisms in hemocytes exposed to LPS, which has relevance for the study of the immune response of HSCs to infection.

Mitochondrial DNA sequence of the horseshoe crab Tachypleus gigas.

This paper reports on the complete mitochondrial (mt) genome of a horseshoe crab, Tachypleus gigas (T. gigas), in Kuala Kemaman, Terengganu, Malaysia. Whole-genome sequencing of hemocyte DNA was performed with Illumina HiSeq system and the generated reads were de novo assembled with ABySS 2.1.5 and reassembled using mitoZ against Carcinoscorpius rotundicauda and Limulus polyphemus, resulting in a contig of 15 Kb. Phylogenetic analysis of the assembled mt genome suggests that the Tachypleus gigas is closely related to Tachypleus tridentatus than to Carcinoscorpius rotundicauda.

Engineered Mycobacterium tuberculosis antigen assembly into core-shell nanobeads for diagnosis of tuberculosis.

Despite recent advances in diagnosis, tuberculosis (TB) remains one of the ten leading causes of death worldwide. Here, we engineered Mycobacterium tuberculosis (Mtb) proteins (ESAT6, CFP10, and MTB7.7) to self-assemble into core-shell nanobeads for enhanced TB diagnosis. Respective purified Mtb antigen-coated polyester beads were characterized and their functionality in TB diagnosis was tested in whole blood cytokine release assays. Sensitivity and specificity were studied in 11 pulmonary TB patients (PTB) and 26 healthy individuals composed of 14 Tuberculin Skin Test negative (TSTn) and 12 TST positive (TSTp). The production of 6 cytokines was determined (IFNγ, IP10, IL2, TNFα, CCL3, and CCL11). To differentiate PTB from healthy individuals (TSTp + TSTn), the best individual cytokines were IL2 and CCL11 (>80% sensitivity and specificity) and the best combination was IP10 + IL2 (>90% sensitivity and specificity). We describe an innovative approach using full-length antigens attached to biopolyester nanobeads enabling sensitive and specific detection of human TB.

Immunomodulatory Effects of Recombinant Mycobacterium smegmatis Expressing Antigen-85B Epitopes in Infected J774A.1 Murine Macrophages.

Tuberculosis (TB) causes more than 1.5 million deaths each year, remaining a significant global health problem. Mycobacterium smegmatis (M. smegmatis) and Mycobacterium tuberculosis (M. tuberculosis) share features, which support the use of the former use in new generation TB vaccine development. In a previous study, the specific humoral and cellular immunogenicity of a recombinant M. smegmatis strain expressing epitopes from M. tuberculosis Ag85B protein (rMs064), was demonstrated in mice. In the current study, the immunomodulatory capacity of rMs064 was determined in a J774A.1 murine macrophage cell line. To determine the immunomodulatory effect of rMs064 in J774A.1 macrophages, the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) was evaluated. The expression of activation surface markers (MHC-II, CD40, CD80 and CD86) and the production of cytokines (IL-1ß, TNF-α, IL-12p70 and IL-6) was also determined in rMs064 infected J774A.1 macrophages. Our findings showed the ability of rMs064 to induce substantial increases in macrophage activation markers expression; MHC class II and CD40, compared with M. smegmatis transformed with the empty vector (rMs012) and uninfected cells. rMs064 induced significant increases in IL-12p70 compared to uninfected cells. The expression of iNOS and CD86, and the production of IL-1ß, and TNF-α were increased in rMs064 and rMs012, compared to uninfected cells. rMs064 demonstrated its immunomodulatory ability by stimulating the innate immune response, which supports its further evaluation as a TB vaccine candidate.

A Direct Role for the CD1b Endogenous Spacer in the Recognition of a Mycobacterium tuberculosis Antigen by T-Cell Receptors.

Lipids, glycolipids and lipopeptides derived from Mycobacterium tuberculosis (Mtb) are presented to T cells by monomorphic molecules known as CD1. This is the case of the Mtb-specific sulfoglycolipid Ac2SGL, which is presented by CD1b molecules and is recognized by T cells found in tuberculosis (TB) patients and in individuals with latent infections. Our group, using filamentous phage display technology, obtained two specific ligands against the CD1b-Ac2SGL complex: (i) a single chain T cell receptor (scTCR) from a human T cell clone recognizing the CD1b-AcSGL complex; and (ii) a light chain domain antibody (dAbκ11). Both ligands showed lower reactivity to a synthetic analog of Ac2SGL (SGL12), having a shorter acyl chain as compared to the natural antigen. Here we put forward the hypothesis that the CD1b endogenous spacer lipid (EnSpacer) plays an important role in the recognition of the CD1b-Ac2SGL complex by specific T cells. To support this hypothesis we combined: (a) molecular binding assays for both the scTCR and the dAbκ11 antibody domain against a small panel of synthetic Ac2SGL analogs having different acyl chains, (b) molecular modeling of the CD1b-Ac2SGL/EnSpacer complex, and (c) modeling of the interactions of this complex with the scTCR. Our results contribute to understand the mechanisms of lipid presentation by CD1b molecules and their interactions with T-cell receptors and other specific ligands, which may help to develop specific tools targeting Mtb infected cells for therapeutic and diagnostic applications.

Identification of a Mycobacterium tuberculosis-specific gene marker for diagnosis of tuberculosis using semi-nested melt-MAMA qPCR (lprM-MAMA).

Tuberculosis (TB) is the deadliest of infectious diseases. TB diagnosis, based on sputum microscopy, culture, and nucleic acid amplification tests (NAATs) to identify its main causative agent, Mycobacterium tuberculosis (MTB), remains challenging. The current available NAATs, endorsed by World Health Organization (WHO), can differentiate MTB from some MTB complex (MTBC) members. Using bioinformatics, we identified a single nucleotide polymorphism (SNP) in lprM (Rv1970) gene that differentiate MTB from other MTBC members. A forward mismatch amplification mutation assay (MAMA) primer was designed for the targeted mutation and was used in a semi-nested melt-MAMA qPCR (lprM-MAMA). Using the optimized protocol, lprM-MAMA was positive with all MTB reference and clinical strains, and negative with other MTBC members, non-tuberculous mycobacteria (NTM) and other non-mycobacterial (NM) reference strains. The limit of detection (LOD) of lprM-MAMA was 76.29 fg. Xpert® MTB/RIF (Xpert)-positive sputum samples were also positive by lprM-MAMA, except for samples classified as having "very low" bacterial load by Xpert. Xpert-negative sputum samples were also negative by lprM-MAMA. In conclusion, lprM-MAMA demonstrated to be a useful tool for specific MTB diagnosis. Further evaluation with higher number of reference strains, including NTM and NM; and sputum samples are required to determine its potential for clinical application.