Transcription factors YY1, Sp1 and Sp3 modulate dystrophin Dp71 gene expression in hepatic cells.

Dystrophin Dp71, the smallest product encoded by the Duchenne muscular dystrophy gene, is ubiquitously expressed in all non-muscle cells. Although Dp71 is involved in various cellular processes, the mechanisms underlying its expression have been little studied. In hepatic cells, Dp71 expression is down-regulated by the xenobiotic ß-naphthoflavone. However, the effectors of this regulation remain unknown. In the present study we aimed at identifying DNA elements and transcription factors involved in Dp71 expression in hepatic cells. Relevant DNA elements on the Dp71 promoter were identified by comparing Dp71 5'-end flanking regions between species. The functionality of these elements was demonstrated by site-directed mutagenesis. Using EMSAs and ChIP, we showed that the Sp1 (specificity protein 1), Sp3 (specificity protein 3) and YY1 (Yin and Yang 1) transcription factors bind to the Dp71 promoter region. Knockdown of Sp1, Sp3 and YY1 in hepatic cells increased endogenous Dp71 expression, but reduced Dp71 promoter activity. In summary, Dp71 expression in hepatic cells is carried out, in part, by YY1-, Sp1- and Sp3-mediated transcription from the Dp71 promoter.

Paxlovid™ reduces the incidence of pneumonia, hospitalization, and death in a cohort of COVID-19 patients from northeast Mexico.

BACKGROUND: Information on Paxlovid™ effectiveness must be monitored and updated in real world scenarios. Our research question was what is the effectiveness of Paxlovid™ in adult patients with COVID-19? Therefore, we investigated the effectiveness of Paxlovid™ on reducing the incidence of pneumonia, hospitalization, and mortality in a cohort of COVID-19 positive adult patients from northeast Mexico. METHODS: A retrospective cohort study of COVID-19 positive adult patients from Nuevo Leon, Mexico from December 2020 to May 2023 (after Omicron BA-5 circulation) was performed. Paxlovid™ use was authorized in September 2022. Therefore, we analyzed effectiveness in patients with confirmed diagnosis who met selection criteria between September 2022 and May 2023 (n = 20,799; 5,673 with and 15,126 without Paxlovid™). RESULTS: The pneumonia (0.1% vs. 0.4%, p < 0.0001), hospitalization (0.1% vs. 1.2%, p < 0.0001), and death rates (0.04% vs. 0.2%, p < 0.0001) were lower in patients with Paxlovid™ treatment independently of age, sex, comorbidity, and COVID-19 and pneumococcal vaccination history. Effectiveness was 88.2%, 95.9% y 91.9% for pneumonia, hospitalization, and death, respectively. CONCLUSIONS: Paxlovid™ reduces the presentation of pneumonia, hospitalization, and death secondary to COVID-19. It is recommended to continue monitoring Paxlovid™ effectiveness, as other SARS-CoV-2 variants continue to emerge.

Measuring of Mycobacterium tuberculosis growth. A correlation of the optical measurements with colony forming units.

The quantification of colony forming units (cfu), turbidity, and optical density at 600 nm (OD600) measurements were used to evaluate Mycobacterium tuberculosis growth. Turbidity and OD600 measurements displayed similar growth curves, while cfu quantification showed a continuous growth curve. We determined the cfu equivalents to McFarland and OD600 units.

Side Effects of COVID-19 Vaccines in Pregnant and Lactating Mexican Women and Breastfed Infants: A Survey-Based Study.

COVID-19 vaccines' safety has been extensively studied; however, further analysis is required in pregnant women, nursing mothers, and breastfed infants. Our aim was to compare the extension and severity of self-reported COVID-19 vaccine side effects in pregnant and breastfeeding women, and breastfed infants. In this cross-sectional study, COVID-19-vaccinated subjects were enrolled using an online survey in Mexico. Women were classified by pregnancy and breastfeeding status at the time of vaccination (n = 3167). After the first or only dose, there was a trend toward fewer systemic effects in pregnant women (p = 0.06). BNT162b2 (Pfizer-BioNTech) had a higher frequency of local symptoms in pregnancy. Lactating women experienced fewer local symptoms after the first or single dose (p = 0.04) and the opposite occurred after the second dose (p = 0.001). ChAdOx1 (AstraZeneca) increased the chances of developing both local and systemic symptoms after the first dose but decreased them after the second dose. The severity was similar across groups, although the result of lack of association in pregnancy requires studies with a larger sample size. Irritability was the most reported symptom in breastfed infants. This study contributes to the knowledge about the side effects in pregnant and lactating women, and breastfed babies.

Suspension culture improves iPSC expansion and pluripotency phenotype.

BACKGROUND: Induced pluripotent stem cells (iPSCs) offer potential to revolutionize regenerative medicine as a renewable source for islets, dopaminergic neurons, retinal cells, and cardiomyocytes. However, translation of these regenerative cell therapies requires cost-efficient mass manufacturing of high-quality human iPSCs. This study presents an improved three-dimensional Vertical-Wheel® bioreactor (3D suspension) cell expansion protocol with comparison to a two-dimensional (2D planar) protocol. METHODS: Sendai virus transfection of human peripheral blood mononuclear cells was used to establish mycoplasma and virus free iPSC lines without common genetic duplications or deletions. iPSCs were then expanded under 2D planar and 3D suspension culture conditions. We comparatively evaluated cell expansion capacity, genetic integrity, pluripotency phenotype, and in vitro and in vivo pluripotency potential of iPSCs. RESULTS: Expansion of iPSCs using Vertical-Wheel® bioreactors achieved 93.8-fold (IQR 30.2) growth compared to 19.1 (IQR 4.0) in 2D (p < 0.0022), the largest expansion potential reported to date over 5 days. 0.5 L Vertical-Wheel® bioreactors achieved similar expansion and further reduced iPSC production cost. 3D suspension expanded cells had increased proliferation, measured as Ki67+ expression using flow cytometry (3D: 69.4% [IQR 5.5%] vs. 2D: 57.4% [IQR 10.9%], p = 0.0022), and had a higher frequency of pluripotency marker (Oct4+Nanog+Sox2+) expression (3D: 94.3 [IQR 1.4] vs. 2D: 52.5% [IQR 5.6], p = 0.0079). q-PCR genetic analysis demonstrated a lack of duplications or deletions at the 8 most commonly mutated regions within iPSC lines after long-term passaging (> 25). 2D-cultured cells displayed a primed pluripotency phenotype, which transitioned to naïve after 3D-culture. Both 2D and 3D cells were capable of trilineage differentiation and following teratoma, 2D-expanded cells generated predominantly solid teratomas, while 3D-expanded cells produced more mature and predominantly cystic teratomas with lower Ki67+ expression within teratomas (3D: 16.7% [IQR 3.2%] vs.. 2D: 45.3% [IQR 3.0%], p = 0.002) in keeping with a naïve phenotype. CONCLUSION: This study demonstrates nearly 100-fold iPSC expansion over 5-days using our 3D suspension culture protocol in Vertical-Wheel® bioreactors, the largest cell growth reported to date. 3D expanded cells showed enhanced in vitro and in vivo pluripotency phenotype that may support more efficient scale-up strategies and safer clinical implementation.

Characterization of an Importin alpha/beta-recognized nuclear localization signal in beta-dystroglycan.

Beta-dystroglycan (beta-DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of beta-DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of beta-DG, characterizing a functional nuclear localization signal (NLS) in the beta-DG cytoplasmic domain, within amino acids 776-782. The NLS either alone or in the context of the whole beta-DG protein was able to target the heterologous GFP protein to the nucleus, with site-directed mutagenesis indicating that amino acids R(779) and K(780) are critical for NLS functionality. The nuclear transport molecules Importin (Imp)alpha and Impbeta bound with high affinity to the NLS of beta-DG and were found to be essential for NLS-dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of beta-DG may result in cytoplasmic retention, with Y(892) playing a key role. beta-DG thus follows a conventional Impalpha/beta-dependent nuclear import pathway, with important implications for its potential function in the nucleus.

Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2alpha in Dp71 promoter activity.

In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E-115 cell line. We demonstrated that Dp71 expression is up-regulated in response to cAMP-mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5'-flanking 159-bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA-less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2alpha bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over-expression of Sp1 and AP2alpha, as well as knock-down experiments on Sp1 and AP2alpha gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2alpha, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2alpha binding, which ultimately releases the promoter from repression.

High Prevalence of Avian Influenza Virus Among Wild Waterbirds and Land Birds of Mexico.

Aquatic wild birds, especially waterfowl, have been long considered the main reservoirs of the avian influenza A virus; however, recent surveys have found an important prevalence of these viruses among land birds as well. Migration has been suggested as an important factor in the avian influenza virus dissemination. We aimed to estimate the prevalence of influenza A viruses in wild birds (waterbirds and land birds; resident and migratory) in eastern Mexico, where the three main North American migration flyways converge and where there was no previous information on this subject. We detected influenza with reverse transcription coupled with a PCR approach. Of the 534 birds sampled between 2010 and 2012, we detected the influenza A virus in a high proportion of birds (39%). Prevalence was particularly high in land birds (49%) when compared to aquatic birds (26%); there was no difference in overall prevalence between resident (39%) and migratory birds (39%). The high prevalence of the avian influenza virus in land birds was noteworthy in the innermost sampling areas in northern Mexico (Coahuila [82%] and Nuevo Leon [43%]).

Alta prevalencia del virus de la influenza aviar entre aves acuáticas silvestres y aves terrestres de México. Las aves silvestres acuáticas, especialmente las aves anseriformes, han sido consideradas durante mucho tiempo los principales reservorios del virus de la influenza aviar A; sin embargo, muestreos recientes también han encontrado una importante prevalencia de estos virus entre las aves terrestres. Se ha sugerido que la migración es un factor importante en la diseminación del virus de la influenza aviar. El objetivo de este estudio fue estimar la prevalencia de los virus de la influenza A en aves silvestres (aves acuáticas y terrestres; residentes y migratorias) en el este de México, donde convergen las tres rutas migratorias principales de América del Norte y donde no había información previa sobre este tema. Se detectó al virus de influenza mediante transcripción reversa acoplada a PCR. De las 534 aves muestreadas entre los años 2010 y 2012, se detectó al virus de la influenza A en una alta proporción de aves (39%). La prevalencia fue particularmente alta en las aves terrestres (49%) en comparación con las aves acuáticas (26%); no se observó diferencia en la prevalencia general entre las aves residentes (39%) y las migratorias (39%). La alta prevalencia del virus de la influenza aviar en las aves terrestres fue notable en las áreas de muestreo hacia el interior del norte de México (Coahuila [82%] y Nuevo León [43%]).

DNA binding activity studies and computational approach of mutant SRY in patients with 46, XY complete pure gonadal dysgenesis.

Mutations of SRY are the cause of 46,XY complete pure gonadal dysgenesis (PGD) in 10-15% of patients. In this study, DNA was isolated and sequenced from blood leukocytes and from paraffin-embedded gonadal tissue in five patients with 46,XY complete PGD. DNA binding capability was analyzed by three different methods. The structure of the full length SRY and its mutant proteins was carried out using a protein molecular model. DNA analysis revealed two mutations and one synonymous polymorphism: in patient #4 a Y96C mutation, and a E156 polymorphism; in patient #5 a S143G mosaic mutation limited to gonadal tissue. We demonstrated, by all methods used, that both mutant proteins reduced SRY DNA binding activity. The three-dimensional structure of SRY suggested that besides the HMG box, the carboxy-terminal region of SRY interacts with DNA. In conclusion, we identified two SRY mutations and a polymorphism in two patients with 46,XY complete PGD, demonstrating the importance of the carboxy-terminal region of SRY in DNA binding activity.

Regulation of Ca v 3.1 channels by glucocorticoids.

The activity of low voltage-activated Ca(2+) (Ca(V)3) channels is tightly coupled to neurotransmitter and hormone secretion. Previous studies have shown that Ca(V)3 channels are regulated by glucocorticoids (GCs), though the mechanism underlying channel regulation remains unclear. Here, using the pituitary GH(3) cell line as a model, we investigated whether Ca(V)3 channel expression is under the control of GCs, and if their actions are mediated by transcriptional and/or post-transcriptional mechanisms. RT-PCR and western blot analyses showed that Ca(V)3.1 but not Ca(V)3.2 and Ca(V)3.3 channels is expressed in the GH(3) cells, and patch clamp recordings confirmed that Ca(2+) currents through low voltage-activated channels were decreased after chronic treatment with GCs. Consistent with this, total plasma membrane expression of Ca(V)3.1 protein as analyzed by cell-surface biotinylation assays and semi-quantitative western blotting was also down-regulated, while quantitative real-time RT-PCR analysis revealed a significant decrease of Ca(V)3.1 mRNA expression in the treated cells. In contrast, patch-clamp recordings on HEK-293 cells stably expressing recombinant Ca(V)3.1 channels showed that Ca(2+) currents were not affected by GC treatment. These results suggest that decreased transcription is a likely mechanism to explain the inhibitory actions of GCs on the functional expression of native Ca(V)3.1 channels.

Pharmacogenetics of amfepramone in healthy Mexican subjects reveals potential markers for tailoring pharmacotherapy of obesity: results of a randomised trial.

Amfepramone (AFP) is an appetite-suppressant drug used in the treatment of obesity. Nonetheless, studies on interindividual pharmacokinetic variability and its association with genetic variants are limited. We employed a pharmacokinetic and pharmacogenetic approach to determine possible metabolic phenotypes of AFP and identify genetic markers that could affect the pharmacokinetic variability in a Mexican population. A controlled, randomized, crossover, single-blind, two-treatment, two-period, and two sequence clinical study of AFP (a single 75 mg dose) was conducted in 36 healthy Mexican volunteers who fulfilled the study requirements. Amfepramone plasma levels were measured using high-performance liquid chromatography mass spectrometry. Genotyping was performed using real-time PCR with TaqMan probes. Four AFP metabolizer phenotypes were found in our population: slow, normal, intermediate, and fast. Additionally, two gene polymorphisms, ABCB1-rs1045642 and CYP3A4-rs2242480, had a significant effect on AFP pharmacokinetics (P < 0.05) and were the predictor factors in a log-linear regression model. The ABCB1 and CYP3A4 gene polymorphisms were associated with a fast metabolizer phenotype. These results suggest that metabolism of AFP in the Mexican population is variable. In addition, the genetic variants ABCB1-rs1045642 and CYP3A4-rs2242480 may partially explain the AFP pharmacokinetic variability.

Myotonic dystrophy CTG repeat expansion alters Ca2+ channel functional expression in PC12 cells.

We previously reported that expression of myotonic dystrophy (DM1) expanded CUG repeats impedes NGF-induced differentiation in a PC12 clone (CTG90 cells). Here, we present evidence for changes in the fractional contribution of distinct voltage-gated Ca(2+) channels, key elements in neurotrophin-promoted differentiation, to the total Ca(2+) current in the CTG90 cells. Patch-clamp recordings showed that the relative proportion of pharmacologically isolated Ca(2+) channel types differed between control and CTG90 cells. Particularly, the functional expression of N-type channels was significantly reduced. Though quantitative real-time RT-PCR revealed that transcripts for the pore-forming subunit encoding the N-type channels remained unchanged, the protein level analyzed by semi-quantitative Western blotting was down-regulated in the CTG90 cells. These data suggest modifications in the processing of N-type Ca(2+) channels in PC12 cells expressing the DM1 mutation.

Selenite restores Pax6 expression in neuronal cells of chronically arsenic-exposed Golden Syrian hamsters.

Arsenic is a worldwide environmental pollutant that generates public health concerns. Various types of cancers and other diseases, including neurological disorders, have been associated with human consumption of arsenic in drinking water. At the molecular level, arsenic and its metabolites have the capacity to provoke genome instability, causing altered expression of genes. One such target of arsenic is the Pax6 gene that encodes a transcription factor in neuronal cells. The aim of this study was to evaluate the effect of two antioxidants, α-tocopheryl succinate (α-TOS) and sodium selenite, on Pax6 gene expression levels in the forebrain and cerebellum of Golden Syrian hamsters chronically exposed to arsenic in drinking water. Animals were divided into six groups. Using quantitative real-time reverse transcriptase (RT)-PCR analysis, we confirmed that arsenic downregulates Pax6 expression in nervous tissues by 53 ± 21% and 32 ± 7% in the forebrain and cerebellum, respectively. In the presence of arsenic, treatment with α-TOS did not modify Pax6 expression in nervous tissues; however, sodium selenite completely restored Pax6 expression in the arsenic-exposed hamster forebrain, but not the cerebellum. Although our results suggest the use of selenite to restore the expression of a neuronal gene in arsenic-exposed animals, its use and efficacy in the human population require further studies.

CYP1A2 phenotype and genotype in a population from the Carboniferous Region of Coahuila, Mexico.

CYP1A2 regulation by polycyclic aromatic hydrocarbons (PAHs) exposure and polymorphism was investigated in 46 male volunteers from the Carboniferous Region in northern Coahuila, Mexico. PAH exposure was estimated by the urinary excretion of 1-hydroxypyrene (1-OHP), whereas the regulatory effects were assessed by the caffeine metabolic ratio (CMR). Genotype was evaluated by determining 5'-flanking region (-2964) and intron I (734) polymorphisms. A statistically significant difference in the urinary 1-OHP geometric means of Barroterán, Cloete and Juárez (2.30, 0.45 and 0.04, respectively) was observed. As for the genotype, the intron I distribution was 0% C/C, 46% C/A and 54% A/A, whereas that of the 5'-flanking region was 26% G/G, 42% G/A and 32% A/A. Both distributions were in agreement with the Hardy-Weinberg equilibrium model. A greater enzyme activity was observed in the A/A compared to C/A individuals according to the CMR (P<0.001), whereas the 5'-flanking region polymorphism showed no effect on CYP1A2 enzymatic activity. These results suggest that intron I polymorphism and PAH exposure are relevant factors that modulate CYP1A2 enzymatic activity.

Myotonic dystrophy 1 in the nervous system: from the clinic to molecular mechanisms.

Myotonic dystrophy type 1 (DM1) is a dominant neuromuscular disorder caused by the expansion of trinucleotide CTG repeats in the 3'-untranslated region (3'-UTR) of the DMPK gene. Prominent features of classical DM1 are muscle wasting and myotonia, whereas mental retardation is distinctive for congenital DM1. The main nervous system symptoms of DM1 are cognitive impairment, neuroendocrine dysfunction, and personality and behavior abnormalities. It is thought that expansion of CTG repeats causes DM1 pathology through different molecular mechanisms; however, a growing body of evidence indicates that an RNA gain-of-function mechanism plays a major role in the disease development. At the skeletal muscle level, three main molecular events can be distinguished in this model: 1) formation of nuclear foci that are composed at least of mutant DMPK mRNA and recruited RNA-binding proteins, such as splicing regulators and transcription factors; 2) disturbance of alternative splicing of specific genes; and 3) impairment of cell differentiation. Contrasting with the substantial advances in understanding DM1 muscle pathology, the molecular basis of DM1 in the nervous system has just started to be revealed. This review focuses in the DM1 nervous system pathology and provides an overview of the genetic and molecular studies analyzing the effects of the DMPK gene CUG expanded repeats on cell function in neuronal systems. A comparison between the molecular mechanisms of DM1 in the skeletal muscle and those identified in DM1 nervous system models is provided. Finally, future directions in the study of DM1 in the nervous system are discussed.

Pregnane X receptor-dependent induction of the CYP3A4 gene by o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane.

CYP3A4, the predominant cytochrome P450 (P450) expressed in human liver and intestine, contributes to the metabolism of approximately half the drugs in clinical use today. CYP3A4 catalyzes the 6beta-hydroxylation of a number of steroid hormones and is involved in the bioactivation of environmental procarcinogens. The expression of CYP3A4 is affected by several stimuli, including environmental factors such as insecticides and pesticides. The o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane (DDT) isomer of DDT comprises approximately 20% of technical grade DDT, which is an organochloride pesticide. We have recently shown that o,p'-DDT exposure increases CYP3A4 mRNA levels in HepG2 cells. To determine the mechanism by which o,p'-DDT induces CYP3A4 expression, transactivation and electrophoretic mobility shift assays were carried out, revealing that o,p'-DDT activates the CYP3A4 gene promoter through the pregnane X receptor (PXR). CYP3A4 gene promoter activation resulted in both an increase in CYP3A4 mRNA levels and an increase in the total CYP3A4 activity in HepG2 cells. We also observed induction of CYP3A4 and mouse Cyp3a11 mRNA in the intestine of CYP3A4-transgenic mice after exposure to 1 mg/kg o,p'-DDT. At higher doses, a decrease of CYP3A4 inducibility was observed together with an increase in levels of interleukin 6 mRNA, a proinflammatory cytokine that strongly represses CYP3A4 transcription. The present study indicates that regulation of other genes under PXR control may be altered by o,p'-DDT exposure.

Dystrophin Dp71 expression is down-regulated during myogenesis: role of Sp1 and Sp3 on the Dp71 promoter activity.

Dp71 expression is present in myoblasts but declines during myogenesis to avoid interfering with the function of dystrophin, the predominant Duchenne muscular dystrophy gene product in differentiated muscle fibers. To elucidate the transcriptional regulatory mechanisms operating on the developmentally regulated expression of Dp71, we analyzed the Dp71 expression and promoter activity during myogenesis of the C2C12 cells. We demonstrated that the cellular content of Dp71 transcript and protein decrease in myotubes as a consequence of the negative regulation that the differentiation stimulus exerts on the Dp71 promoter. Promoter deletion analysis showed that the 224-bp 5'-flanking region, which contains several Sp-binding sites (Sp-A to Sp-D), is responsible for the Dp71 promoter basal activity in myoblasts as well as for down-regulation of the promoter in differentiated cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that Sp1 and Sp3 transcription factors specifically bind to the Sp-binding sites in the minimal Dp71 promoter region. Site-directed mutagenesis assay revealed that Sp-A is the most important binding site for the proximal Dp71 promoter activity. Additionally, cotransfection of the promoter construct with Sp1- and Sp3-expressing vectors into Drosophila SL2 cells, which lack endogenous Sp family, confirmed that these proteins activate specifically the minimal Dp71 promoter. Endogenous Sp1 and Sp3 proteins were detected only in myoblasts and not in myotubes, which indicates that the lack of these factors causes down-regulation of the Dp71 promoter activity in differentiated cells. In corroboration, efficient promoter activity was restored in differentiated muscle cells by exogenous expression of Sp1 and Sp3.