Osteogenic potential of apical papilla stem cells mediated by platelet-rich fibrin and low-level laser.

To evaluate the osteogenic potential of platelet-rich fibrin (PRF) and low-level laser therapy (LLLT) on human stem cells from the apical papilla (SCAP) we isolated, characterized, and then cultured in an osteogenic medium cells with PRF and/or LLLT (660 nm, 6 J/m2-irradiation). Osteogenic differentiation was assessed by bone nodule formation and expression of bone morphogenetic proteins (BMP-2 and BMP-4), whereas the molecular mechanisms were achieved by qRT-PCR and RNA-seq analysis. Statistical analysis was performed by ANOVA and Tukey's post hoc tests (p < 0.05* and p < 0.01**). Although PRF and LLLT increased bone nodule formation after 7 days and peaked at 21 days, the combination of PRF + LLLT led to the uppermost nodule formation. This was supported by increased levels of BMP-2 and -4 osteogenic proteins (p < 0.005). Furthermore, the PRF + LLLT relative expression of specific genes involved in osteogenesis, such as osteocalcin, was 2.4- (p = 0.03) and 28.3- (p = 0.001) fold higher compared to the PRF and LLLT groups, and osteopontin was 22.9- and 1.23-fold higher, respectively (p < 0.05), after 7 days of interaction. The transcriptomic profile revealed that the combination of PRF + LLLT induces MSX1, TGFB1, and SMAD1 expression, after 21 days of osteogenic differentiation conditions exposition. More studies are required to understand the complete cellular and molecular mechanisms of PRF plus LLLT on stem cells. Overall, we demonstrated for the first time that the combination of PRF and LLLT would be an excellent therapeutic tool that can be employed for dental, oral, and craniofacial repair and other tissue engineering applications.

Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates.

Chikungunya (CHIKV), Zika (ZIKV), and dengue viruses (DENV) are vector-borne pathogens that cause emerging and re-emerging epidemics throughout tropical and subtropical countries. The symptomatology is similar among these viruses and frequently co-circulates in the same areas, making the diagnosis arduous. Although there are different methods for detecting and quantifying pathogens, real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for detecting viruses. However, the currently developed assays frequently involve probes and high-cost reagents, limiting access in low-income countries. Therefore, this study aims to design and evaluate a quantitative one-step RT-qPCR assay to detect CHIKV, ZIKV, and DENV with high specificity, reproducibility, and low cost in multiple cell substrates. We established a DNA intercalating green dye-based RT-qPCR test that targets nsP1 of CHIKV, and NS5 gene of ZIKV, and DENV for the amplification reaction. The assay exhibited a high specificity confirmed by the melting curve analysis. No cross-reactivity was observed between the three viruses or unspecific amplification of host RNA. The sensitivity of the reaction was evaluated for each virus assay, getting a limit of detection of one RNA copy per virus. Standard curves were constructed, obtaining a reaction efficiency of ~ 100%, a correlation coefficient (R2) of ~ 0.97, and a slope of -3.3. The coefficient of variation (CV) ranged from 0.02 to 1.43. In addition, the method was optimized for viral quantification and tested in Vero, BHK-21, C6/36, LULO, and the Aedes cell lines. Thus, the DNA intercalating green dye-based RT-qPCR assay was a highly specific, sensitive, reproducible, and effective method for detecting and quantifying CHIKV, ZIKV, and DENV in different cell substrates that could also be applied in clinical samples.

Antiviral activity of myricetin glycosylated compounds isolated from Marcetia taxifolia against chikungunya virus.

The chikungunya virus (CHIKV) has produced epidemic outbreaks of significant public health impact. The clinical symptoms of this disease are fever, polyarthralgia, and skin rash, generally self-limiting, although patients may develop a chronic disabling condition or suffer lethal complications. Unfortunately, there is no specific treatment or vaccine available. Thus, the search for effective therapies to control CHIKV infection is an urgent need. This study evaluated the antiviral activity of flavonoids isolated from Marcetia taxifolia by in vitro and in silico analysis. Cytotoxicity of compounds was determined by MTT assay and viral load was assessed in cell substrates supernatants by plaque-forming and RT-qPCR assays. Selected molecules were analyzed by molecular docking assays. Myricetin 3-rhamnoside (MR) and myricetin 3-(6-rhamnosylgalactoside) (MRG) were tested for antiviral assays and analyzed by the TCID50 method and RT-qPCR. MR exhibited dose-dependent antiviral activity, reducing viral titer at concentrations of 150-18.8 µg/mL by at least 1-log. Similarly, MRG showed a significant decrease in viral titer at concentrations of 37.5, 9.4, and 2.3 µg/mL. RT-qPCR analysis also displayed a substantial reduction of CHIKV RNA for both flavonoids. Furthermore, molecular docking of the selected flavonoids proposed the nsP3 macrodomain as a possible target of action. Our study reveals that MR and MRG could be considered promising anti-CHIKV therapeutic agents. Molecular modeling studies showed MR and MRG ligands with a high affinity for the N-terminal region of the nsP3 macrodomain, postulating them as a potential target of action for the CHIKV control.

Reduced levels of convalescent neutralizing antibodies against SARS-CoV-2 B.1+L249S+E484K lineage.

The E484K mutation at the SARS-CoV-2 Spike protein emerged independently in different variants around the world and has been widely associated with immune escape from neutralizing antibodies generated during previous infection or vaccination. In this work, the B.1 + L249S+E484K lineage was isolated along with A.1, B.1.420, and B.1.111 SARS-CoV-2 lineages without the E484K mutation and the neutralizing titer of convalescent sera was compared using microneutralization assays. While no significant differences in the neutralizing antibody titers were found between A.1 and B.lineages without the E484K mutation, the neutralizing titers against B.1 + L249S+E484K were 1.5, 1.9, 2.1, and 1.3-fold lower than against A.1, B.1.420, B.1.111-I, and B.1.111-II, respectively. However, molecular epidemiological data indicate that there is no increase in the transmissibility rate associated with this new lineage. This study supports the capability of new variants with the E484K mutation to be resistant to neutralization by humoral immunity, and therefore the need to intensify surveillance programs to determine if these lineages represent a risk for public health.

Neutralizing Antibody Responses Elicited by Inactivated Whole Virus and Genetic Vaccines against Dominant SARS-CoV-2 Variants during the Four Epidemic Peaks of COVID-19 in Colombia.

Several SARS-CoV-2 variants of concern (VOC) and interest (VOI) co-circulate in Colombia, and determining the neutralizing antibody (nAb) responses is useful to improve the efficacy of COVID-19 vaccination programs. Thus, nAb responses against SARS-CoV-2 isolates from the lineages B.1.111, P.1 (Gamma), B.1.621 (Mu), AY.25.1 (Delta), and BA.1 (Omicron), were evaluated in serum samples from immunologically naïve individuals between 9 and 13 weeks after receiving complete regimens of CoronaVac, BNT162b2, ChAdOx1, or Ad26.COV2.S, using microneutralization assays. An overall reduction of the nAb responses against Mu, Delta, and Omicron, relative to B.1.111 and Gamma was observed in sera from vaccinated individuals with BNT162b2, ChAdOx1, and Ad26.COV2.S. The seropositivity rate elicited by all the vaccines against B.1.111 and Gamma was 100%, while for Mu, Delta, and Omicron ranged between 32 to 87%, 65 to 96%, and 41 to 96%, respectively, depending on the vaccine tested. The significant reductions in the nAb responses against the last three dominant SARS-CoV-2 lineages in Colombia indicate that booster doses should be administered following complete vaccination schemes to increase the nAb titers against emerging SARS-CoV-2 lineages.

Low Neutralizing Antibody Titers against the Mu Variant of SARS-CoV-2 in 31 BNT162b2 Vaccinated Individuals in Colombia.

Global surveillance programs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are showing the emergence of variants with mutations in the spike protein. Genomic and laboratory surveillance are important to determine if these variants may be more infectious or less susceptible to antiviral treatments and vaccine-induced antibodies. Three of the most predominant SARS-CoV-2 variants in Colombia during the epidemiological peaks of 2021 were isolated: Mu, a variant of interest; Gamma, a variant of concern; B.1.111, which lacks genetic markers associated with greater virulence. Microneutralization assays were performed by incubating 120 mean tissue culture infectious doses (TCID50) of each SARS-CoV-2 isolate with five two-fold serial dilutions of sera from 31 BNT162b2-vaccinated volunteers. The mean neutralization titer (MN50) was calculated by the Reed-Muench method. At the end of August, Mu represented 49% of coronavirus disease 2019 (COVID-19) cases in Colombia, followed by 25% of Gamma. In contrast, B.1.111 became almost undetectable. The evaluation of neutralizing antibodies suggests that patients vaccinated with BNT162b2 generate neutralizing antibody titers against the Mu variant at significantly lower concentrations relative to B.1.111 and Gamma. This study shows the importance of continuing surveillance programs of emerging variants, as well as the need to evaluate the neutralizing antibody response induced by other vaccines.

Inverse Molecular Docking Study of NS3-Helicase and NS5-RNA Polymerase of Zika Virus as Possible Therapeutic Targets of Ligands Derived from Marcetia taxifolia and Its Implications to Dengue Virus.

Dengue and Zika are two mosquito-borne diseases of great impact on public health around the world in tropical and subtropical countries. DENV and ZIKV belong to the Flaviviridae family and the Flavivirus genus. Currently, there are no effective therapeutic agents to treat or prevent these pathologies. The main objective of this work was to evaluate potential inhibitors from active compounds obtained from Marcetia taxifolia by performing inverse molecular docking on ZIKV-NS3-helicase and ZIKV-NS5-RNA polymerase as targets. This computational strategy is based on renormalizing the binding scores of the compounds to these two proteins, allowing a direct comparison of the results across the proteins. The crystallographic structures of the ZIKV-NS3-helicase and ZIKV-NS5-RNA-polymerase proteins share a great similarity with DENV homologous proteins. The P-loop active site of the crystallographic structure of ZIKV-NS3-helicase presents a high percentage of homology with the four dengue serotypes. It was found that most ligands of the active compounds (5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (5DP); 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5HH); myricetin-3-O-rhamnoside (M3OR)) from Marcetia taxifolia had a better affinity for ZIKV-NS3-helicase than for ZIKV-NS5-RNA polymerase, as indicated by the negative multiple active site correction (MASC) score, except for M3RG that showed a higher affinity for ZIKV-NS5-RNA polymerase. On the other hand, the AutoDock Vina scores showed that M3OR had the highest score value (-9.60 kcal/mol) and the highest normalized score (1.13) against ZIKV-NS3-helicase. These results in silico demonstrated that the nonstructural proteins NS3-helicase and NS5-RNA polymerase, which share similar molecular structures between the selected viruses, could become therapeutic targets for some bioactive compounds derived from Marcetia taxifolia.

Importance of clinical and laboratory characteristics in the diagnosis of dengue and chikungunya co-infection: Probable case report / Importancia de las características clínicas y de laboratorio en el diagnóstico de las infecciones concomitantes por dengue y chikungunya: reporte de un caso probable.

We report the case of a 38-year-old woman who initially consulted for an undifferentiated fever. Although her clinical condition evolved with signs and symptoms compatible with dengue with alarm signs and that the anti-dengue IgM detection in a single sample indicated it was a probable case that could have happened during the previous three months, the patient kept consulting due to little improvement. On the tenth day after the onset of symptoms, she presented with painful polyarticular symmetric edema, as well as hyperpigmented lesions in the nasolabial fold. Chikungunya diagnosis was confirmed by the presence of IgM antibodies. In endemic countries for dengue and chikungunya, the possibility of co-infection exists, but it may go unnoticed. On the other hand, the co-infection may worsen the clinical course of these diseases. Therefore, physicians should evaluate the clinical and laboratory characteristics of both infections to be able to diagnose the coinfection for adequate management and to minimize complications.

Se presenta el caso de una mujer de 38 años que consultó inicialmente por fiebre indiferenciada. A pesar de que el cuadro clínico evolucionó con manifestaciones clínicas de dengue con signos de alarma y de que la detección de IgM antidengue en una sola muestra indicaba que se trataba de un caso probable que había podido ocurrir durante los tres meses anteriores, la paciente consultó de forma reiterada, pues no presentaba una mejoría significativa. En el décimo día del inicio de los síntomas, se observó edema simétrico en múltiples articulaciones acompañado de dolor, así como lesiones hiperpigmentadas en el surco nasogeniano. Se confirmó el diagnóstico de chikungunya por la presencia de anticuerpos IgM. Aunque puede pasar desapercibida, en los países endémicos para dengue y chikungunya existe la posibilidad de la infección concomitante, la cual puede agravar la evolución clínica de cada una de estas enfermedades. Por ello, es necesario que el médico considere las características clínicas y de laboratorio de ambas enfermedades para diagnosticar su presencia simultánea, garantizar un manejo adecuado y minimizar las complicaciones.

Molecular characteristics and replication mechanism of dengue, zika and chikungunya arboviruses, and their treatments with natural extracts from plants: An updated review.

Viruses transmitted by arthropods (arboviruses) are the etiological agents of several human diseases with worldwide distribution; including dengue (DENV), zika (ZIKV), yellow fever (YFV), and chikungunya (CHIKV) viruses. These viruses are especially important in tropical and subtropical regions; where, ZIKV and CHIKV are involved in epidemics worldwide, while the DENV remains as the biggest problem in public health. Factors, such as, environmental conditions promote the distribution of vectors, deficiencies in health services, and lack of effective vaccines, guarantee the presence of these vector-borne diseases. Treatment against these viral diseases is only palliative since available therapies formulated lack to demonstrate specific antiviral activity and vaccine candidates fail to demonstrate enough effectiveness. The use of natural products, as therapeutic tools, is an ancestral practice in different cultures. According to WHO 80 % of the population of some countries from Africa and Asia depend on the use of traditional medicines to deal with some diseases. Molecular characteristics of these viruses are important in determining its cellular pathogenesis, emergence, and dispersion mechanisms, as well as for the development of new antivirals and vaccines to control strategies. In this review, we summarize the current knowledge of the molecular structure and replication mechanisms of selected arboviruses, as well as their mechanism of entry into host cells, and a brief overview about the potential targets accessed to inhibit these viruses in vitro and a summary about their treatment with natural extracts from plants.

Importancia de las características clínicas y de laboratorio en el diagnóstico de las infecciones concomitantes por dengue y chikungunya: reporte de un caso probable / Importance of clinical and laboratory characteristics in the diagnosis of dengue and chikungunya co-infection: Probable case report

Se presenta el caso de una mujer de 38 años que consultó inicialmente por fiebre indiferenciada. A pesar de que el cuadro clínico evolucionó con manifestaciones clínicas de dengue con signos de alarma y de que la detección de IgM antidengue en una sola muestra indicaba que se trataba de un caso probable que había podido ocurrir durante los tres meses anteriores, la paciente consultó de forma reiterada, pues no presentaba una mejoría significativa. En el décimo día del inicio de los síntomas, se observó edema simétrico en múltiples articulaciones acompañado de dolor, así como lesiones hiperpigmentadas en el surco nasogeniano. Se confirmó el diagnóstico de chikungunya por la presencia de anticuerpos IgM. Aunque puede pasar desapercibida, en los países endémicos para dengue y chikungunya existe la posibilidad de la infección concomitante, la cual puede agravar la evolución clínica de cada una de estas enfermedades. Por ello, es necesario que el médico considere las características clínicas y de laboratorio de ambas enfermedades para diagnosticar su presencia simultánea, garantizar un manejo adecuado y minimizar las complicaciones.

We report the case of a 38-year-old woman who initially consulted for an undifferentiated fever. Although her clinical condition evolved with signs and symptoms compatible with dengue with alarm signs and that the anti-dengue IgM detection in a single sample indicated it was a probable case that could have happened during the previous three months, the patient kept consulting due to little improvement. On the tenth day after the onset of symptoms, she presented with painful polyarticular symmetric edema, as well as hyperpigmented lesions in the nasolabial fold. Chikungunya diagnosis was confirmed by the presence of IgM antibodies. In endemic countries for dengue and chikungunya, the possibility of co-infection exists, but it may go unnoticed. On the other hand, the co-infection may worsen the clinical course of these diseases. Therefore, physicians should evaluate the clinical and laboratory characteristics of both infections to be able to diagnose the coinfection for adequate management and to minimize complications.