Development and Optimization of a Multiplex Real-Time RT-PCR to Detect SARS-CoV-2 in Human Samples.

PCR and its variants (RT-PCR and qRT-PCR) are valuable and innovative molecular techniques for studying nucleic acids. qPCR has proven to be highly sensitive, efficient, and reproducible, generating reliable results that are easy to analyze. During the COVID-19 pandemic, qPCR became the gold standard technique for detecting the SARS-CoV-2 virus that allowed to confirm the infection event, and those asymptomatic ones, and thus save millions of lives. In-house multiplex qPCR tests were developed worldwide to detect different viral targets and ensure results, follow the infections, and favor the containment of a pandemic. Here, we present the detailed fundamentals of the qPCR technique based on fluorogenic probes and processes to develop and optimize a successful multiplex RT-qPCR test for detecting SARS-CoV-2 that could be used to diagnose COVID-19 accurately.

Modulation of TRPV1 on Odontoblast-like Cells Using Capsazepine-Loaded Nanogels.

The modulation of TRPV1 emerges as a promising strategy for dental pain management. This study aimed to assess TRPV1 modulation in a human odontoblast-like cell model using Capsazepine (CZP) loaded in a nanogel delivery system. Gelatin nanogels, synthesized via the emulsification-gelation technique, were characterized and loaded with the TRPV1 antagonist, CZP. HPLC determined a remarkable 67.5 ± 0.04% CZP loading efficiency, with 71.7% of nanogels falling within the 300-950 nm size range, as evidenced by light microscopy. Moreover, CZP-loaded nanogels had a low cytotoxicity. An FTIR analysis showed no adverse chemical interactions, ensuring stability and active release. When examining biological responses, TRPV1 expression and channel activity were assessed in odontoblast-like cells. On the fifth day post-treatment, cells treated with CZP-loaded nanogels exhibited an increased TRPV1 expression and a reduction in calcium fluxes after agonist stimulus (F/F0 ratio 1.18 ± 0.18), resembling the response in free CZP-treated cells (1.28 ± 0.15). A two-way analysis of variance and the Tukey's test were used to determine statistical significance (p < 0.05). This delivery system, proven to be economical and straightforward, holds promise for dental pain management and potential local use. Local administration minimizes systemic adverse effects, making it a practical solution for releasing molecules in the oral cavity.

Association of Root Biofilm Bacteriome with Root Caries Lesion Severity and Activity.

INTRODUCTION: This research aimed to assess the association of root biofilm bacteriome with root caries lesion severity and activity in institutionalised Colombian elderlies and was conducted to gather data on the root caries bacteriome in this population. METHODS: A bacteriome evaluation of biofilm samples from sound and carious root surfaces was performed. Root caries was categorised (ICDAS Root criteria) based on severity (sound surfaces, initial: non-cavitated, moderate/extensive combined: cavitated) and activity status (active and inactive). DNA was extracted and the V4 region of the 16S rRNA gene was sequenced; afterwards the classification of features was conducted employing amplicon sequence variants and taxonomic assignment via the Human Oral Microbiome Database (HOMD). Bacterial richness, diversity (Simpson's and Shannon's indices), and relative abundance estimation were assessed and compared based on root caries severity and activity status (including Sound surfaces). RESULTS: A total of 130 biofilm samples were examined: sound (n = 45) and with root caries lesions (n = 85; by severity: initial: n = 41; moderate/extensive: n = 44; by activity: active: n = 60; inactive: n = 25). Species richness was significantly lower in biofilms from moderate/extensive and active groups compared to sound sites. There was a higher relative abundance of species like Lechtotricia wadei, Capnocytophaga granulosa, Cardiobacterium valvarum, Porphyromonas pasteri - in sound sites; Dialister invisus, Streptococcus mutans, Pseudoramibacter alactolyticus and Bacteroidetes (G-5) bacterium 511 - in moderate/extensive lesions, and Fusobacterium nucleatum subsp. animalis, Prevotella denticola, Lactobacillus fermentum, Saccharibacteria (TM7) (G-5)bacterium HMT 356 - in active lesions. CONCLUSION: Root caries bacteriome exhibited differences in species proportions between the compared groups. Specifically, cavitated caries lesions and active caries lesions showed higher relative abundance of acidogenic bacteria.

Seroprevalence, infection, and personal protective equipment use among Colombian healthcare workers during the COVID-19 pandemic.

Introduction: Healthcare workers (HCWs) are at the forefront of the COVID-19 response and frequently come into close contact with patients and their virus-contaminated body fluids. Recent studies have identified differential risks of infection and the use of personal protective equipment (PPE) among HCWs. However, available data might be interpreted with caution because of differences in the national health systems, local implementation issues, and adherence limitations to guidelines. A comprehensive description of infection, exposure at work, and biosafety habits during the COVID-19 pandemic has not been conducted among the HCW groups in Latin American populations. Objective: To describe SARS-CoV-2 seroprevalence, infections, and extent of PPE use during the COVID-19 pandemic among HCWs at three different times, including dental practitioners (DP), nursing assistants (NA), physicians (P), and respiratory therapists (RT), from Bogotá, Colombia. Methods: After IRB approval, this cross-sectional study included 307 HCWs. Participants provided nasopharyngeal swabs and blood samples to detect viral RNA (RT-qPCR) and IgM/IgG anti-SARS-CoV-2 (ELFA-ELISA) at baseline (BL) and two follow-ups. Infection prevalence was defined as the number of positive-tested participants (RT-qPCR and/or IgM). Data on clinical status and biosafety habits were collected each time. Results: Differential infection prevalence was found among HCWs through the study timeline (BL: RT-qPCR = 2.6%, IgM = 1.6%; follow-up 1 (45 days after BL): RT-qPCR = 4.5%, IgM = 3.9%; follow-up 2 (60 days after BL): RT-qPCR = 3.58%, IgM = 1.3%. Dental practitioners showed a higher infection frequency in BL and follow-up 1. IgG-positive tested HCWs percentage progressively increased from BL to follow-ups among the whole sample while index values decreased. Limitations in N95 availability and a high perception of occupational risk were reported. Conclusion: A low prevalence of active SARS-CoV-2 infections among HCWs groups was found. Over time, there was an increase in participants showing IgG antibodies, although the levels of these antibodies in the blood decreased. Additionally, HCWs reported limitations in the availability of PPE as well as a variation in their safety practices.

Relaxin treatment stimulates the differentiation of mesenchymal stem cells into osteoblasts.

Background/purpose: Several studies have determined that relaxin stimulates differentiation and regulates the activity of mature osteoclasts, but little is known about its effect on the differentiation of mesenchymal cells towards the osteogenic lineage. Therefore, this study aimed to determine the effect of relaxin on the proliferation and differentiation of the osteoblastic lineage of mesenchymal cells derived from human dental pulp (hDPSC). Materials and methods: In this in vitro study, hDPSC were characterized and treated with relaxin at different doses (10-80 ng/ml) and times (1-21 days). Morphology was assessed by microscopy, and proliferation was assessed using a resazurin assay. Osteoblastic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase (ALP) labeling, and changes in the expression of the osteoblastic differentiation genes RUNX2 and BMP2. Results: Relaxin treatment did not induce changes in the proliferation or viability of hDPSCs; however, larger cells and increased cytoplasmic prolongation were observed. Relaxin treatment (20 and 80 ng/ml) significantly increased calcified nodule formation on days 14 and 21. The cytochemical signals for ALP, RUNX2, and BMP2 gene expression were significantly (P < 0.05) increased by the relaxin treatment. Conclusion: Relaxin treatment does not induce changes in hDPSC proliferation but induces morphological changes, increases ALP detection, calcified nodule formation, and increases expression of RUNX2 and BMP2, suggesting the induction of osteoblastic differentiation of hDPSC.

A strategy for entomo-virological surveillance of yellow fever, dengue, zika, and chikungunya viruses in field-collected mosquitoes.

Arboviruses transmitted by Culicidae insects are significant threats to human health, presenting dynamic transmission cycles and involving different vectors and hosts. The surveillance and characterization of the vectors involved in these cycles are crucial for understanding and preventing potential outbreaks. Therefore, we propose a strategy that we used for entomological surveillance of urban, rural, and sylvatic mosquitoes and to characterize natural infection by four major arboviruses.•Immature and adult mosquitoes were collected intra, peri and extradomicilie of urban and rural households, using different collection methodologies.•Mosquitoes were pooled or separated in head-thorax and abdomen, according to the species.•A multiplex nested RT-PCR (Reverse transcription polymerase chain reaction) method was used for the simultaneous detection of dengue virus (DENV), zika virus (ZIKV), chikungunya virus (CHIKV), and yellow fever virus (YFV).Overall, this strategy proved helpful for vectors surveillance at different ecosystems, as well as for implementing a low-cost molecular surveillance system that allows the early detection of potential outbreaks, and identify other potential vectors involved in viral transmission.

The Interaction Effect of Anti-RgpA and Anti-PPAD Antibody Titers: An Indicator for Rheumatoid Arthritis Diagnosis.

Porphyromonas gingivalis secretes virulence factors like Arg-gingipains and peptidyl arginine deiminase (PPAD), that are associated with rheumatoid arthritis (RA) pathogenesis. However, there is no information regarding the antibody titers for these bacterial enzymes as systemic indicators or biomarkers in RA. In this cross-sectional study, 255 individuals were evaluated: 143 were diagnosed with RA, and 112 were without RA. Logistic regression models adjusted for age, sex, basal metabolic index, smoking, and periodontitis severity were used to evaluate the association of RA with rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), erythrocyte sedimentation rate, high sensitivity C-reactive protein, anti-RgpA, anti-PPAD, and double positive anti-RgpA/anti-PPAD. It was found that RF (odds ratio [OR] 10.6; 95% confidence interval [CI] 4.4-25), ACPAs (OR 13.7; 95% CI 5.1-35), and anti-RgpA/anti-PPAD double positivity (OR 6.63; 95% CI 1.61-27) were associated with RA diagnoses. Anti-RgpA was also associated with RA (OR 4.09; 95% CI 1.2-13.9). The combination of anti-RgpA/anti-PPAD showed a high specificity of 93.7% and 82.5% PPV in identifying individuals with RA. RgpA antibodies were associated with the periodontal inflammatory index in RA individuals (p < 0.05). The double positivity of the anti-RgpA/anti-PPAD antibodies enhanced the diagnosis of RA. Therefore, RgpA antibodies and anti-RgpA/anti-PPAD may be biomarkers for RA.

Tracing ΦX174 bacteriophage spreading during aerosol-generating procedures in a dental clinic.

OBJECTIVE: The aim of this study was to test the plausibility of using the ΦX174 bacteriophage as a tracer of viral aerosols spreading in a dental aerosol-generating procedure (AGP) model. METHODS: ΦX174 bacteriophage (~ 108 plaque-forming units (PFU)/mL) was added into instrument irrigation reservoirs and aerosolized during class-IV cavity preparations followed by composite fillings on natural upper-anterior teeth (n = 3) in a phantom head. Droplets/aerosols were sampled through a passive approach that consisted of Escherichia coli strain C600 cultures immersed in a LB top agar layer in Petri dishes (PDs) in a double-layer technique. In addition, an active approach consisted of E coli C600 on PDs sets mounted in a six-stage cascade Andersen impactor (AI) (simulating human inhalation). The AI was located at 30 cm from the mannequin during AGP and afterwards at 1.5 m. After collection PDs were incubated overnight (18 h at 37 °C) and bacterial lysis was quantified. RESULTS: The passive approach disclosed PFUs mainly concentrated over the dental practitioner, on the mannequin's chest and shoulder and up to 90 cm apart, facing the opposite side of the AGP's source (around the spittoon). The maximum aerosol spreading distance was 1.5 m in front of the mannequin's mouth. The active approach disclosed collection of PFUs corresponding to stages (and aerodynamic diameters) 5 (1.1-2.1 µm) and 6 (0.65-1.1 µm), mimicking access to the lower respiratory airways. CONCLUSION: The ΦX174 bacteriophage can be used as a traceable viral surrogate in simulated studies contributing to understand dental bioaerosol's behavior, its spreading, and its potential threat for upper and lower respiratory tract. CLINICAL RELEVANCE: The probability to find infectious virus during AGPs is high. This suggests the need to continue characterizing the spreading viral agents in different clinical settings through combination of passive and active approaches. In addition, subsequent identification and implementation of virus-related mitigation strategies is relevant to avoid occupational virus infections.

Deubiquitinating Enzyme Inhibitors Block Chikungunya Virus Replication.

Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection.

Capsazepine antagonizes TRPV1 activation induced by thermal and osmotic stimuli in human odontoblast-like cells.

Objectives: Dental pain, which is the main reason for patients consulting dentists, is classified as a public health concern. The study of cellular and molecular mechanisms contributing to pain is a fundamental element for developing new analgesics. By using a selective antagonist in an in vitro model, this study aimed to establish the role of TRPV-1 in human odontoblast-like cells (OLCs) as a therapeutic target for dental pain mediated by noxious thermal and osmotic stimuli. Methods: OLCs were differentiated from dental pulp mesenchymal cells and TRPV1 expression was evaluated. Activation of TRPV-1 was determined by evaluating changes in calcium concentration after stimulation with mannitol and xylitol hyperosmotic solutions or DMEM heated at 45 °C, using the fluorescent calcium probe Fluo-4 AM. In addition, changes in fluorescence (F/F0) due to calcium flux were evaluated using fluorometry and flow cytometry. Simultaneously, the cells were co-stimulated with the selective antagonist capsazepine (CZP). Results: OLCs expressed DSPP and DMP-1, confirming their cellular phenotype. TRPV1 was expressed, and its activation by different stimuli produced an increase in cytosolic Ca2+ which was reduced by the antagonist. Both methods used to evaluate TRPV1 activation through the measurement of calcium probe fluorescence showed similar patterns. Conclusions: These results suggest that TRPV-1 modulation using an antagonist can be implemented as a pharmacological strategy for managing dental pain mediated by hyperosmotic and thermal stimuli.

Effect of chitosan irrigant solutions on the release of bioactive proteins from root dentin.

OBJECTIVE: To identify the effect of two chitosan solutions on the release of root dentin matrix proteins and to describe the chemical changes observed following conditioning with chelating agents. MATERIALS AND METHODS: The release of dentin sialoprotein (DSP), transforming growth factor-beta 1 (TGF-ß1), vascular endothelial growth factor (VEGF), and platelet-derived growth factor-BB (PDGF-BB) with different chelating agents, including ethylenediaminetetraacetic acid (EDTA), chitosan solution (CS), and nanoparticulate chitosan (CSnp), was investigated. DSP was quantified using an enzyme-linked immunosorbent assay (ELISA). TGF-ß1, VEGF, and PDGF-BB were quantified using a cytokine bead panel (CBA). Raman spectroscopy was performed to identify surface chemical changes. Statistical analysis was performed using Kruskal-Wallis test with Mann-Whitney-Wilcoxon rank-sum test (p < 0.05). RESULTS: TGF-ß1, VEGF, and DSP solubilized in all irrigants tested. CSnp showed the highest concentration of DSP. PDGF-BB did not exceed the detection limits. Raman spectroscopy revealed a decrease in the phosphate and carbonate peaks, representing the chelating effect of EDTA, CS, and CSnp. Additionally, CSnp showed the greatest preservation of the amide I and III content. CONCLUSION: Proteins can be released from dentin via EDTA, CS, and CSnp conditioning. Raman spectroscopic revealed changes in the inorganic content of the root dentin after chelation. Furthermore, use of CSnp facilitated a preservation of the organic content. CLINICAL RELEVANCE: Chelation allows the release of proteins, justifying the use of chelating agents in regenerative endodontics. The chitosan-dentin matrix interaction also promotes the protection of the organic content as an additional benefit to its protein releasing effect.

A tangled threesome: understanding arbovirus infection in Aedes spp. and the effect of the mosquito microbiota.

Arboviral infections transmitted by Aedes spp. mosquitoes are a major threat to human health, particularly in tropical regions but are expanding to temperate regions. The ability of Aedes aegypti and Aedes albopictus to transmit multiple arboviruses involves a complex relationship between mosquitoes and the virus, with recent discoveries shedding light on it. Furthermore, this relationship is not solely between mosquitoes and arboviruses, but also involves the mosquito microbiome. Here, we aimed to construct a comprehensive review of the latest information about the arbovirus infection process in A. aegypti and A. albopictus, the source of mosquito microbiota, and its interaction with the arbovirus infection process, in terms of its implications for vectorial competence. First, we summarized studies showing a new mechanism for arbovirus infection at the cellular level, recently described innate immunological pathways, and the mechanism of adaptive response in mosquitoes. Second, we addressed the general sources of the Aedes mosquito microbiota (bacteria, fungi, and viruses) during their life cycle, and the geographical reports of the most common microbiota in adults mosquitoes. How the microbiota interacts directly or indirectly with arbovirus transmission, thereby modifying vectorial competence. We highlight the complexity of this tripartite relationship, influenced by intrinsic and extrinsic conditions at different geographical scales, with many gaps to fill and promising directions for developing strategies to control arbovirus transmission and to gain a better understanding of vectorial competence. The interactions between mosquitoes, arboviruses and their associated microbiota are yet to be investigated in depth.

Multidisciplinary approach for surveillance and risk identification of yellow fever and other arboviruses in Colombia.

Arbovirus, a critical threat to human health, have complex and dynamic life cycles. With reports of Yellow fever virus (YFV) causing spillover from sylvatic transmission cycles, and dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV) viruses expanding from urban to rural areas. We explored a multidisciplinary approach to analyze arbovirus transmission through vectors, and identify biological and sociodemographic determinants associated with their transmission risk in urban and rural areas in a Colombian municipality. We visited 178 urban and 97 rural households, registered sociodemographic characteristics and vaccination status for each of these households, collected adult and immature mosquitoes at the intra-, peri-, and extra-domicile, and surveyed forest patches in rural areas. Infections of YFV, DENV, ZIKV, and CHIKV in the mosquitoes collected in the wild were analyzed using a reverse transcriptase PCR. We identified various risk factors of transmission associated with a high Aedes aegypti infestation in urban areas and their presence in rural settlements and Haemagogus janthinomys and other sylvatic mosquitoes near urban areas. The collected Ae. aegypti females from urban areas had a high infection rate of YFV (5.8%) and CHIKV (58.8%), and those from rural settlements had a high infection rate of DENV (33%), CHIKV (16.7%), and ZIKV (16.7%). The infection rates of YFV in the thorax of the sylvatic mosquitoes H. janthinomys and Aedes serratus collected from the forest patches were 14.3 and 42.1%, respectively. We could discern the transmission determinants associated with climatic, socioeconomic, and anthropogenic factors and YFV vaccination status. This multidisciplinary approach for surveillance of arboviral diseases allowed us to independently detect and integrate factors indicating an early risk of rural transmission of DENV, CHIKV, and ZIKV and rural and urban outbreaks of YFV in the study area. This study provides a helpful tool for designing and focalizing prevention strategies.

Immune challenges upregulate the expression of cannabinoid receptors in cultured human odontoblasts and gingival fibroblasts.

Odontoblasts and gingival fibroblasts play essential roles in the physiological and pathological processes of dental tissue. Cannabinoid receptors (CB1 and CB2) are involved in analgesia by modulating the función of calcium channels that inhibit the synthesis of some neurotransmitters. A better understanding of the physiology of these receptors would provide the possibility of using them as therapeutic targets in controlling dental pain. The aim of this study was to evaluate the presence and activity of cannabinoid receptors in human odontoblast-like cells (OLC) and human gingival fibroblasts (HGF). CB1 and CB2 transcription was analyzed by real-time PCR, proteins were detected by immunofluorescence, and functional cannabinoid receptors were evaluated by measuring intracellular calcium concentration after stimulation with cannabidiol (CBD) and pre-treatment with a CB1 antagonist, a CB2 inverse agonist and a TRPV1 antagonist. Transcripts for CB1 and CB2 were found in both odontoblasts and gingival fibroblasts. Cannabidiol induced an increase in [Ca2+]i in both cells types, but surprisingly, pre-treatment with selective cannabinoid antagonists attenuated this effect, suggesting a functional communication between specific cannabinoid receptors and other CBD target receptors. In conclusion, human odontoblasts and gingival fibroblasts express functional CB1 and CB2 cannabinoid receptors, which could be modulated to improve the treatment of pain or dental sensitivity.

Los odontoblastos y los fibroblastos gingivales desempeñan funciones esenciales en los procesos fisiológicos y patológicos de los tejidos dentales. Los receptores cannabinoides (CB1 y CB2) participan en la analgesia mediante la modulación de la función de canales de calcio que inhiben la síntesis de algunos neurotransmisores. Un mejor conocimiento de su fisiología abre la posibilidad de utilizar estos receptores como dianas terapéuticas en el control del dolor dental. Este trabajo tuvo como objetivo evaluar la presencia y la actividad de los receptores cannabinoides en células humanas similares a los odontoblastos (OLC) y en fibroblastos gingivales humanos (HGF). Se analizó la transcripción de CB1 y CB2 por PCR en tiempo real, la detección de las proteínas por inmunofluorescencia y se evaluaron los receptores cannabinoides funcionales midiendo las concentraciones de calcio intracelular, tras la estimulación con cannabidiol (CBD) y el pretratamiento con un antagonista de CB1, un agonista inverso de CB2 y un antagonista de TRPV1. Se encontraron mensajeros para CB1 y CB2 tanto en odontoblastos como en fibroblastos gingivales. El cannabidiol indujo un aumento de la [Ca2+]i en ambos tipos de células, pero sorprendentemente el pretratamiento con antagonistas cannabinoides selectivos atenuó este efecto, lo que sugiere una comunicación funcional entre receptores cannabinoides específicos y otros receptores diana del CBD. En conclusión, los odontoblastos humanos y los fibroblastos gingivales expresan receptores cannabinoides CB1 y CB2 funcionales, que podrían ser modulados para mejorar el tratamiento del dolor o la sensibilidad dental.

Purification of RgpA from external outer membrane vesicles of Porphyromonas gingivalis.

INTRODUCTION: Purification of native gingipains is challenging because these proteases are frequently associated with the cell surface, which affects yield. This study aimed to purify native Arg-gingipain (RgpA) from Porphyromonas gingivalis Outer Membrane Vesicles (OMV). METHODS: Native RgpA was purified from P. gingivalis strain ATCC33277 OMV using a strategy including ultracentrifugation, sonication, and successive anionic and cationic fast protein liquid chromatography (FPLC). The presence and purity of the protease were confirmed by SDS-PAGE and detection of protease activity using fluorogenic substrates. Rat antibodies produced against the unique adhesin hemagglutinin (H1) domain of RgpA (amino acids 719-865) were titrated by ELISA at a 1:100 dilution using whole P. gingivalis lysate as an antigen and western blotting to detect a 75 kDa band corresponding to RgpA. RESULTS: Double anionic-cationic FLPC yielded prominent peaks with evident amidolytic gingipain activity of the appropriate molecular weight, as confirmed by western blotting. The final RgpA yield from 1 L of bacterial culture with colony forming unit (CFU) (Log10) 7.4 ± 0.08/mL was of 12.6% (2 mg/mL), with 3.2 FU/µg of amidolytic activity. CONCLUSIONS: This protocol allows purification of native RgpA from OMV that retains protease activity.

Reference gene validation for the relative quantification of cannabinoid receptor expression in human odontoblasts via quantitative polymerase chain reaction.

Objective: The aim of this study was to identify and validate the reference genes in cultured human odontoblasts to quantify their cannabinoid receptor transcripts. Methods: The most stably transcribed genes in cultured human odontoblast cells were identified using the RefGenes tool and were selected for real-time polymerase chain reaction (PCR) amplification. Human odontoblast cells were differentiated from mesenchymal stem cells using a transforming growth factor-ß-supplemented differentiation medium, and total RNA was purified. Reverse transcription-quantitative PCR and relative quantification analyses were performed using the Schefe's method. The relative expression dataset was analyzed to select the most stable genes. Results: The analysis showed that the transcripts of cholinergic receptor nicotinic beta 2 subunit, LIM homeobox transcription factor 1 beta, and family with sequence similarity 223 member B presented the lowest standard deviation (SD) in expression (SD: 0.2, 0.17, and 0.16, respectively). These genes showed similar expression levels as the target genes (cannabinoid receptors). Significant differences were found in the relative expression levels of cannabinoid receptors using the selected genes compared to those calculated using beta actin transcripts as references (p < 0.05). Conclusions: The strategy reported here for searching and verifying new reference genes will aid in the accurate and reliable expression of cannabinoid receptors in human odontoblast cells.

Immune challenges upregulate the expression of cannabinoid receptors in cultured human odontoblasts and gingival fibroblasts / Los desafíos inmunológicos regulan la expresión de los receptores cannabinoides en cultivos de odontoblastos y fibroblastos gingivales humanos

ABSTRACT Odontoblasts and gingival fibroblasts play essential roles in the physiological and pathological processes of dental tissue. Cannabinoid receptors (CB1 and CB2) are involved in analgesia by modulating the función of calcium channels that inhibit the synthesis of some neurotransmitters. A better understanding of the physiology of these receptors would provide the possibility of using them as therapeutic targets in controlling dental pain. The aim of this study was to evaluate the presence and activity of cannabinoid receptors in human odontoblast-like cells (OLC) and human gingival fibroblasts (HGF). CB1 and CB2 transcription was analyzed by real-time PCR, proteins were detected by immunofluorescence, and functional cannabinoid receptors were evaluated by measuring intracellular calcium concentration after stimulation with cannabidiol (CBD) and pre-treatment with a CB1 antagonist, a CB2 inverse agonist and a TRPV1 antagonist. Transcripts for CB1 and CB2 were found in both odontoblasts and gingival fibroblasts. Cannabidiol induced an increase in [Ca2+]i in both cells types, but surprisingly, pre-treatment with selective cannabinoid antagonists attenuated this effect, suggesting a functional communication between specific cannabinoid receptors and other CBD target receptors. In conclusion, human odontoblasts and gingival fibroblasts express functional CB1 and CB2 cannabinoid receptors, which could be modulated to improve the treatment of pain or dental sensitivity.

RESUMEN Los odontoblastos y los fibroblastos gingivales desempeñan funciones esenciales en los procesos fisiológicos y patológicos de los tejidos dentales. Los receptores cannabinoides (CB1 y CB2) participan en la analgesia mediante la modulación de la función de canales de calcio que inhiben la síntesis de algunos neurotransmisores. Un mejor conocimiento de su fisiología abre la posibilidad de utilizar estos receptores como dianas terapéuticas en el control del dolor dental. Este trabajo tuvo como objetivo evaluar la presencia y la actividad de los receptores cannabinoides en células humanas similares a los odontoblastos (OLC) y en fibroblastos gingivales humanos (HGF). Se analizó la transcripción de CB1 y CB2 por PCR en tiempo real, la detección de las proteínas por inmunofluorescencia y se evaluaron los receptores cannabinoides funcionales midiendo las concentraciones de calcio intracelular, tras la estimulación con cannabidiol (CBD) y el pretratamiento con un antagonista de CB1, un agonista inverso de CB2 y un antagonista de TRPV1. Se encontraron mensajeros para CB1 y CB2 tanto en odontoblastos como en fibroblastos gingivales. El cannabidiol indujo un aumento de la [Ca2+]i en ambos tipos de células, pero sorprendentemente el pretratamiento con antagonistas cannabinoides selectivos atenuó este efecto, lo que sugiere una comunicación funcional entre receptores cannabinoides específicos y otros receptores diana del CBD. En conclusión, los odontoblastos humanos y los fibroblastos gingivales expresan receptores cannabinoides CB1 y CB2 funcionales, que podrían ser modulados para mejorar el tratamiento del dolor o la sensibilidad dental.

Endothelial Dysfunction, HMGB1, and Dengue: An Enigma to Solve.

Dengue is a viral infection caused by dengue virus (DENV), which has a significant impact on public health worldwide. Although most infections are asymptomatic, a series of severe clinical manifestations such as hemorrhage and plasma leakage can occur during the severe presentation of the disease. This suggests that the virus or host immune response may affect the protective function of endothelial barriers, ultimately being considered the most relevant event in severe and fatal dengue pathogenesis. The mechanisms that induce these alterations are diverse. It has been suggested that the high mobility group box 1 protein (HMGB1) may be involved in endothelial dysfunction. This non-histone nuclear protein has different immunomodulatory activities and belongs to the alarmin group. High concentrations of HMGB1 have been detected in patients with several infectious diseases, including dengue, and it could be considered as a biomarker for the early diagnosis of dengue and a predictor of complications of the disease. This review summarizes the main features of dengue infection and describes the known causes associated with endothelial dysfunction, highlighting the involvement and possible relationship between HMGB1 and DENV.

Molecular and biological characterization of an Asian-American isolate of Chikungunya virus.

Chikungunya virus is an arthropod-transmitted virus that causes chikungunya fever, a disease characterized by severe muscle and joint pain. In 2013, the virus was introduced to the Americas and caused approximately 2.7 million cases of infection during the subsequent two years. The lack of knowledge regarding the biological behavior of the viral strains circulating during the outbreak motivated the characterization of an isolate from the Colombian outbreak, starting from analysis of the complete genome to the biological behavior in vitro. The full genome was retrieved using next-generation sequencing. The infective and replicative capacities were evaluated in HEK293T, Huh-7, and MRC-5 cell lines. The infection rates were determined by flow cytometry, and the cytopathic effect was assessed by a resazurin fluorescent metabolic assay. The viral yield was quantified using the virus plaque formation assay, while the viral proteins and genomic RNA kinetics were subsequently evaluated by western-blot and RT-qPCR. The COL7624 isolate clustered with other American and Caribbean sequences in the Asian American lineage. The T669A substitution in E2 protein distinguished it from other Colombian sequences reported in 2014. After 48 h post infection (hpi), the three cell lines analyzed reached infection percentages exceeding 65%, generating a high load of infectious viral progeny. The infection kinetics indicated that the replication peak of this CHIKV isolate is around 24 hpi, although gRNA is detectable in the culture supernatant from 4 hpi onwards. The infection caused the overexpression of interferon and pro-inflammatory cytokines, such as IL-1ß, TNF-α, and IL-8. The COL7624 CHIKV isolate exhibited a high infective and replicative capacity as well as activation of cellular immune responses, similar to isolates belonging to the other genotypes.

Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples.

In low-resource settings, resilience to infectious disease outbreaks can be hindered by limited access to diagnostic tests. Here we report the results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America. The tests involved a cell-free expression system relying on isothermal amplification and toehold-switch reactions, a purpose-built portable reader and onboard software for computer vision-enabled image analysis. In patients suspected of infection, the accuracies and sensitivities of the tests for the Zika and chikungunya viruses were, respectively, 98.5% (95% confidence interval, 96.2-99.6%, 268 serum samples) and 98.5% (95% confidence interval, 91.7-100%, 65 serum samples) and approximately 2 aM and 5 fM (both concentrations are within clinically relevant ranges). The analytical specificities and sensitivities of the tests for cultured samples of the viruses were equivalent to those of the real-time quantitative PCR. Cell-free synthetic biology tools and companion hardware can provide de-centralized, high-capacity and low-cost diagnostics for use in low-resource settings.