RESUMEN
The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are localized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic trafficking and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Meiosis/genética , Saccharomyces cerevisiae/genética , Transporte Activo de Núcleo Celular/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ADN/genética , Carioferinas/genética , Carioferinas/metabolismo , HomeostasisRESUMEN
During meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion of PCH2 or mutation of the ATPase catalytic sites suppress the meiotic block of the zip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. In zip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic Nuclear Export (NES) or Nuclear Localization (NLS) sequences, or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.
Asunto(s)
Citoplasma/genética , Proteínas Nucleares/genética , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Puntos de Control del Ciclo Celular , Membrana Celular/metabolismo , Emparejamiento Cromosómico , Cromosomas Fúngicos , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Meiosis , Microorganismos Modificados Genéticamente , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Complejo de Reconocimiento del Origen/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
An essential feature of meiosis is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks (DSBs) is repaired using an intact homologous non-sister chromatid rather than a sister. Involvement of Mec1 and Tel1, the budding yeast homologs of the mammalian ATR and ATM kinases, in meiotic interhomlog bias has been implicated, but the mechanism remains elusive. Here, we demonstrate that Mec1 and Tel1 promote meiotic interhomolog recombination by targeting the axial element protein Hop1. Without Mec1/Tel1 phosphorylation of Hop1, meiotic DSBs are rapidly repaired via a Dmc1-independent intersister repair pathway, resulting in diminished interhomolog crossing-over leading to spore lethality. We find that Mec1/Tel1-mediated phosphorylation of Hop1 is required for activation of Mek1, a meiotic paralogue of the DNA-damage effector kinase, Rad53p/CHK2. Thus, Hop1 is a meiosis-specific adaptor protein of the Mec1/Tel1 signaling pathway that ensures interhomolog recombination by preventing Dmc1-independent repair of meiotic DSBs.
Asunto(s)
Intercambio Genético , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meiosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Cromosomas Fúngicos/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Mutación , Fosforilación , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. The coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for the accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, the reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is a crucial phosphatase required for the dephosphorylation of multiple CDK1 targets in many eukaryotes. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that the elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion in crossovers when other repair pathways are compromised. We also show that Cdc14 is required for the correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of the evolutionary conserved Cdc14 phosphatase in the regulation of meiotic recombination.
Asunto(s)
Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/genética , Resolvasas de Unión Holliday/genética , Meiosis/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Segregación Cromosómica/genética , Intercambio Genético/genética , Reparación del ADN/genética , ADN Cruciforme/genética , Gametogénesis/genética , Recombinación Homóloga/genética , Mutación/genética , Fosforilación/genética , Saccharomyces cerevisiae/genéticaRESUMEN
The meiotic recombination checkpoint blocks meiotic cell cycle progression in response to synapsis and/or recombination defects to prevent aberrant chromosome segregation. The evolutionarily conserved budding yeast Pch2TRIP13 AAA+ ATPase participates in this pathway by supporting phosphorylation of the Hop1HORMAD adaptor at T318. In the wild type, Pch2 localizes to synapsed chromosomes and to the unsynapsed rDNA region (nucleolus), excluding Hop1. In contrast, in synaptonemal complex (SC)-defective zip1Δ mutants, which undergo checkpoint activation, Pch2 is detected only on the nucleolus. Alterations in some epigenetic marks that lead to Pch2 dispersion from the nucleolus suppress zip1Δ-induced checkpoint arrest. These observations have led to the notion that Pch2 nucleolar localization could be important for the meiotic recombination checkpoint. Here we investigate how Pch2 chromosomal distribution impacts checkpoint function. We have generated and characterized several mutations that alter Pch2 localization pattern resulting in aberrant Hop1 distribution and compromised meiotic checkpoint response. Besides the AAA+ signature, we have identified a basic motif in the extended N-terminal domain critical for Pch2's checkpoint function and localization. We have also examined the functional relevance of the described Orc1-Pch2 interaction. Both proteins colocalize in the rDNA, and Orc1 depletion during meiotic prophase prevents Pch2 targeting to the rDNA allowing unwanted Hop1 accumulation on this region. However, Pch2 association with SC components remains intact in the absence of Orc1. We finally show that checkpoint activation is not affected by the lack of Orc1 demonstrating that, in contrast to previous hypotheses, nucleolar localization of Pch2 is actually dispensable for the meiotic checkpoint.
Asunto(s)
Puntos de Control del Ciclo Celular , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Meiosis , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnica del Anticuerpo Fluorescente , Complejos Multiproteicos/metabolismo , Mutación , Señales de Localización Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Complejo de Reconocimiento del Origen/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
OBJECTIVE: To evaluate changes in the anterior corneal curvature and aberrometry after scleral contact lens wear in keratoconus (KC) subjects with and without intracorneal ring segments (ICRS). METHODS: Twenty-six subjects diagnosed with keratoconus were selected to participate in the study. Subjects were divided into 2 groups, those with ICRS (KC-ICRS group) and those without ICRS (KC group). Subjects were instructed to wear 16.5-mm scleral lenses for 8 hours. Topographic and aberrometric parameters were evaluated before lens wear and immediately after lens removal. Anterior corneal curvature was evaluated at corneal diameters of 2, 4, 6, and 8 mm, and corneal aberrations were measured at 4-, 6-, and 8-mm pupil diameters. RESULTS: The mean age of subjects was 36.95±8.95 years. In KC group, there was a statistically significant flattening of the central corneal curvature, from 6.98 to 7.09 mm (P<0.05). No changes were found in the central corneal curvature in the KC-ICRS group. The KC group showed anterior corneal curvature flattening, mainly in the nasal quadrant. The KC-ICRS group showed flattening primarily in the inferior hemisphere. In the KC group, spherical aberration for 6-mm pupil increased significantly. In the KC-ICRS group, changes in aberrations were significant for high-order root mean square at 4- and 6-mm pupil diameters (P<0.05), for oblique astigmatism for 4 mm and 6 mm, and for vertical coma for 4-mm pupil (P<0.05). CONCLUSION: Short-term scleral lens wear showed flattening of the anterior corneal surface in all subjects. In the KC group, the flattening was more pronounced in the nasal quadrant while changes were more pronounced inferiorly in KC-ICRS group.
Asunto(s)
Lentes de Contacto de Uso Prolongado , Córnea/patología , Topografía de la Córnea/métodos , Queratocono/terapia , Agudeza Visual , Adulto , Femenino , Estudios de Seguimiento , Humanos , Queratocono/diagnóstico , Queratocono/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Esclerótica , Factores de Tiempo , Tomografía de Coherencia ÓpticaRESUMEN
Aloe vera is a crop of wide economic value of worldwide distribution, and a rich source of quinone components. Recently, antiviral aloe anthraquinones had been reported against human influenza virus. In the present work two anthraquinones, aloesaponarin-I (1) and aloesaponarin-II (2) were isolated from A. vera roots, and six derivatives were obtained by methylation (3), acetylation (4) and O-glycosyl (5-6) reactions starting from (1). Additionally, a new Tetra-O-acetyl-ß-d-glucopyranosyl derivative from 2 was also prepared. All compounds were evaluated against two strains of influenza virus AH1N1 by cytopathic effect reduction assay (CPE). The antiviral activity was determined by the ability of compounds to inhibit virus replication on Madin Darby Canine Kidney cells (MDCK). New derivatives 3-(2´,3´,4´,6´-Tetra-O-acetyl-ß-d-glucopyranosyl-aloesaponarin-I (5) and 3-(2´,3´,4´,6´-Tetra-O-acetyl-ß-d-glucopyranosyl- aloesaponarin-II (7) showed a cytopathic reduction effect against influenza strain A/Yucatán/2370/09 with IC50 of 30.77 and 13.70 µM, and against the virus A/Mexico/InDRE797/10 with IC50 of 62.28 and 19.47 µM, respectively. To assess the effect of derivatives 5 and 7 during one cycle of replication (0-10 h), a time-of-addition experiment was performed. As a result it was found that both compounds were most effective when added 6-10 h post-infection and significantly inhibited viral titre (> 70%) at the concentrations of 50 and 100 µM. Based on the structural analysis of the compounds, it was suggested that the Tetra-O-acetyl-ß-d-glucopyranosyl substituent at the C3 position of the anthraquinone might have an effect against the influenza AH1N1 virus.
RESUMEN
BACKGROUND/AIMS: The study aimed to describe human papillomavirus (HPV) 58 genetic variability in E6 and E7 oncogenes from women in southeast Mexico and their phylogenetic relationships with the sequences from other geographical regions. METHODS: The E6-E7 region was amplified by nested PCR, and sequenced for identification of polymorphisms, phylogenetic trees construction, and haplotype and fixation tests. RESULTS: HPV58 positive samples were obtained from a repository, 54 were amplified, 47 sequences for the E6 gene, and 51 sequences for the E7 gene were obtained. Fifteen new E6 mutations were found; the most frequent were G279T (G57V; 29.78%), T249G (F47C; 34.04%), and A270G (Y54C; 34.04%), and previously reported c307t (63.82%). For E7, 17 known mutations were found, the most frequent were C632T (T20I), 35.30%, G760A (G63S), 35.30%, and t744g 74.50%. No significant association with the severity of the lesions was found. The polytomy in the E6 tree did not allow proposing phylogenetic relationship, and E7 tree presented defined branches. All sequences were presumably A lineage, most closely related to A1 and/or A3 sublineage. HPV58 variants are not specific for a geographical area. Population and fixation analyses suggest a possible Asian origin of HPV58 from Yucatan. The most frequent E7 haplotype in Yucatan groups with other populations of the world. CONCLUSION: The genetic variability of HPV58 from Mexico was described for the first time. E7 was more conserved than E6. New mutants present exclusively in Yucatan were identified.
RESUMEN
An essential feature of meiosis is Spo11 catalysis of programmed DNA double strand breaks (DSBs). Evidence suggests that the number of DSBs generated per meiosis is genetically determined and that this ability to maintain a pre-determined DSB level, or "DSB homeostasis", might be a property of the meiotic program. Here, we present direct evidence that Rec114, an evolutionarily conserved essential component of the meiotic DSB-machinery, interacts with DSB hotspot DNA, and that Tel1 and Mec1, the budding yeast ATM and ATR, respectively, down-regulate Rec114 upon meiotic DSB formation through phosphorylation. Mimicking constitutive phosphorylation reduces the interaction between Rec114 and DSB hotspot DNA, resulting in a reduction and/or delay in DSB formation. Conversely, a non-phosphorylatable rec114 allele confers a genome-wide increase in both DSB levels and in the interaction between Rec114 and the DSB hotspot DNA. These observations strongly suggest that Tel1 and/or Mec1 phosphorylation of Rec114 following Spo11 catalysis down-regulates DSB formation by limiting the interaction between Rec114 and DSB hotspots. We also present evidence that Ndt80, a meiosis specific transcription factor, contributes to Rec114 degradation, consistent with its requirement for complete cessation of DSB formation. Loss of Rec114 foci from chromatin is associated with homolog synapsis but independent of Ndt80 or Tel1/Mec1 phosphorylation. Taken together, we present evidence for three independent ways of regulating Rec114 activity, which likely contribute to meiotic DSBs-homeostasis in maintaining genetically determined levels of breaks.
Asunto(s)
Roturas del ADN de Doble Cadena , Péptidos y Proteínas de Señalización Intracelular/genética , Meiosis , Proteínas Serina-Treonina Quinasas/genética , Recombinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinasas/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Sinaptonémico/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVES: To compare the clinical performance of the Clearkone hybrid contact lens for the treatment of keratoconus against the habitual contact lens of the patients. METHODS: A total of 33 eyes from 18 patients were fitted with the Clearkone. High- and low-contrast visual acuity (HCVA and LCVA), central corneal thickness (CCT), and contrast sensitivity acuity (CSF) were recorded with habitual lenses (prestudy visit) and after 1 week, 15 days, and 1 month of wear of prescribed Clearkone. Subjective vision and comfort were rated using visual analogue scales (VAS). RESULTS: Three patients discontinued the study, one because of diffuse corneal staining after 1 day of use and the other two because of extreme discomfort. The rest of the patients completed the 1-month study. High contrast visual acuity and LCVA (logMAR) improved significantly from 0.16 ± 0.12 and 0.44 ± 0.22, respectively, with the patient's habitual contact lenses to -0.006 ± 0.058 and 0.23 ± 0.13 after 1 day wearing Clearkone, remaining significant during all follow-up visits (P<0.001; repeated measures analysis of variance [RM-ANOVA]). There were no statistically significant differences in the mean CCT. The improvement of CSF was statistically significant with hybrid contact lenses prescribed compared with the patient's habitual contact lenses (P<0.001; RM-ANOVA test). Improvement in VAS score, with prescribed Clearkone, was statistically significant for comfort (P=0.043; RM-ANOVA test), but not for the subjective vision (P=0.759; RM-ANOVA test). CONCLUSIONS: Clearkone provides an improvement in visual acuity, contrast sensitivity, and subjective comfort in patients with keratoconus when compared with other contact lens options. However, clinicians must get specific training to fit the lens and be aware of potential adverse events.
Asunto(s)
Lentes de Contacto de Uso Prolongado , Queratocono/rehabilitación , Adulto , Análisis de Varianza , Sensibilidad de Contraste/fisiología , Femenino , Humanos , Queratocono/fisiopatología , Masculino , Agudeza Visual/fisiología , Adulto JovenRESUMEN
Meiotic recombination is a key process facilitating the formation of crossovers and the exchange of genetic material between homologous chromosomes in early meiosis. This involves controlled double-strand breaks (DSBs) formation catalyzed by Spo11. DSBs exhibit a preferential location in specific genomic regions referred to as hotspots, and their variability is tied to varying Spo11 activity levels. We have refined a ChIP-Seq technique, called SPO-Seq, to map Spo11-specific DSB formation in Saccharomyces cerevisiae. The chapter describes our streamlined approach and the developed bioinformatic tools for processing data and comparing with existing DSB hotspot maps. Through this combined experimental and computational approach, we aim to enhance our understanding of meiotic recombination and genetic exchange processes in budding yeast, with the potential to expand this methodology to other organisms by applying a few modifications.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Meiosis/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Biología Computacional/métodosRESUMEN
Oogenesis is the central process required to produce viable oocytes in female mammals. It is initiated during embryonic development, and it involves the specification of primordial germ cells (PGCs) and progresses through the activation of the meiotic program, reaching a crucial phase in prophase I before pausing at diplotene around the time of birth. The significance of meiosis, particularly the prophase I stage, cannot be overstated, as it plays a pivotal role in ensuring the formation of healthy gametes, a prerequisite for successful reproduction. While research has explored meiosis across various organisms, understanding how environmental factors, including radiation, drugs, endocrine disruptors, reproductive age, or diet, influence this complex developmental process remains incomplete. In this chapter, we describe an ex vivo culture method to investigate meiotic prophase I and beyond and the disruption of oogenesis by external factors. Using this methodology, it is possible to evaluate the effects of individual xenobiotics by administering chemicals at specific points during oogenesis. This culture technique was optimized to study the effects of two selected endocrine disruptors (vinclozolin and MEHP), demonstrating that vinclozolin exposure delayed meiotic differentiation and MEHP exposure reduced follicle size. This approach also opens avenues for future applications, involving the exploration of established or novel pharmaceutical substances and their influence on essential events during prophase I, such as homologous recombination and chromosome segregation. These processes collectively dictate the ultimate fitness of oocytes, with potential implications for factors relevant to the reproductive age and fertility.
Asunto(s)
Meiosis , Ovario , Animales , Femenino , Ratones , Ovario/citología , Meiosis/efectos de los fármacos , Oogénesis/efectos de los fármacos , Oocitos/citología , Oocitos/efectos de los fármacos , Profase Meiótica I/efectos de los fármacos , Disruptores Endocrinos/farmacología , Oxazoles/farmacología , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacosRESUMEN
The most widely used class of antivirals available for Influenza treatment are the neuraminidase inhibitors (NAI) Oseltamivir and Zanamivir. However, amino acid (AA) substitutions in the neuraminidase may cause reduced inhibition or high antiviral resistance. In Mexico, the current state of knowledge about NAI susceptibility is scarce, in this study we report the results of 14 years of Influenza surveillance by phenotypic and genotypic methods. A total of 255 isolates were assessed with the NAI assay, including Influenza A(H1N1)pdm09, A(H3N2) and Influenza B (IBV). Furthermore, 827 sequences contained in the GISAID platform were analyzed in search of relevant mutations.Overall, five isolates showed highly reduced inhibition or reduced inhibition to Oseltamivir, and two showed reduced inhibition to Zanamivir in the NAI assays. Additionally, five A(H1N1)pdm09 sequences from the GISAID possessed AA substitutions associated to reduced inhibition to Oseltamivir and none to Zanamivir. Oseltamivir resistant A(H1N1)pdm09 harbored the H275Y mutation. No genetic mutations were identified in Influenza A(H3N2) and IBV. Overall, these results show that in Mexico the rate of NAI resistance is low (0.6%), but it is essential to continue the Influenza surveillance in order to understand the drug susceptibility of circulating strains.
Asunto(s)
Antivirales , Farmacorresistencia Viral , Virus de la Influenza B , Gripe Humana , Neuraminidasa , Oseltamivir , Zanamivir , Farmacorresistencia Viral/genética , Antivirales/farmacología , México/epidemiología , Humanos , Virus de la Influenza B/efectos de los fármacos , Virus de la Influenza B/genética , Gripe Humana/virología , Gripe Humana/tratamiento farmacológico , Gripe Humana/epidemiología , Oseltamivir/farmacología , Zanamivir/farmacología , Neuraminidasa/genética , Neuraminidasa/antagonistas & inhibidores , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Mutación , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/genética , Adulto , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/genética , Adolescente , Niño , Sustitución de Aminoácidos , Adulto Joven , Persona de Mediana Edad , Femenino , Preescolar , Genotipo , Masculino , Anciano , Pruebas de Sensibilidad Microbiana , Proteínas Virales/genéticaRESUMEN
The incidence of anal intraepithelial neoplasias associated with HPV is rising worldwide. In the general population, this pathology is rare, but individuals living with HIV/AIDS are at a significantly higher risk. We aimed to study HPV infection and performed cytological screening to study the epidemiological and behavioral determinants in a group of men and women living with HIV from a region in Mexico with high HIV incidence. This was a cross-sectional study including adults living with HIV/AIDS performed in Merida (Mexico). We invited patients of public HIV/STD clinics and those affiliated with social organizations of people living with HIV to participate in the study. Participants responded to an instrument to assess their risky behaviors and clinical history. Swabs from the anal canal and cervix and anal cytology specimens were obtained by medical staff from women and by self-sampling from men. For the 200 participants, 169 men and 31 women, anal HPV PCR tests resulted in 59.8% positivity (62.6% of men and 45.2% of women), and 17 genotypes were identified. The most frequent high-risk (HR) types for the anal canal were: HPV33 (35.3%), HPV58 (20.6%), HPV66 (18.6%), HPV45 (17.6%), and HPV16 (14.7%). Multiple genotypes were found in over 80% of the participants. Receptive anal intercourse in the previous 12 months, inconsistent condom use, and detectable HIV titers (≥50 cc/mL) were associated with HPV infection (p < 0.05). Cytology (smears and liquid-based) identified that 34.6% of the participants had low-grade squamous intraepithelial lesions (LSILs), and 3.5% had high-grade squamous intraepithelial lesions (HSILs). Neither HPV nor lesions were associated with low CD4+ counts (<200 cells/mm3, p > 0.05). Of the women, 60% were infected in the cervix and 45% in the anal canal, with an agreement of at least one genotype in 90%. The HR-HPV types associated with HSILs were HPV66, 33, 52, 51, 45, 18, and 68.
RESUMEN
High-Risk Human Papillomavirus (HR-HPV) types 16 and 18 are estimated to be responsible for 72.4% of all HPV-related cancers worldwide in both men and women, including cervical, anal, penile, vulval, vaginal and head and neck cancers [1]. Important efforts worldwide have devoted to the study of these genotypes, throughout epidemiology and basic science approaches. Of particular interest are the genes from the early region (E), coding non-structural proteins. Early genes E1 and E2 products are involved in replication and transcription regulation, while E6 and E7 proteins are recognised for their oncogenic potential. In this data report, we described a set of primers based on reference sequences from HPV16 and HPV18 designed to cover the early region of these oncogenic genotypes. The design was based on multiple sequences alignment to identify the less conserved regions along the open reading frames (ORFs) E6, E7, E1 and E2. The design allows a highly stringent real time PCR essay ranged from 123 to 598 bp overlapping products for HPV16 (12 products in total) and from 183 to 526 bp for HPV18 (11 products in total), both spanning the early genomic region. The high annealing temperatures (Ta) and regions selected for primer bind were intended for genotypic specificity, without compromising the qPCR amplification efficiency (≥ 90%). Evaluation of qPCR conditions for primer set was performed using DNA standards as controls, generated from the HPV16 and 18 genomes clones. This provides relevant information for further multiple quantitative real-time PCR analysis (qPCR), using the SYBR green chemistry, which is is more affordable than generating multiple fluorescently labeled probes.
RESUMEN
Taurine, a cysteine-derived zwitterionic sulfonic acid, is a common ingredient in energy drinks and is naturally found in fish and other seafood. In humans, taurine is produced mainly in the liver, and it can also be obtained from food. In target tissues, such as the retina, heart, and skeletal muscle, it functions as an essential antioxidant, osmolyte, and antiapoptotic agent. Taurine is also involved in energy metabolism and calcium homeostasis. Taurine plays a considerable role in bone growth and development, and high-profile reports have demonstrated the importance of its metabolism for bone health. However, these reports have not been collated for more than 10 years. Therefore, this review focuses on taurine-bone interactions and covers recently discovered aspects of taurine's effects on osteoblastogenesis, osteoclastogenesis, bone structure, and bone pathologies (e.g., osteoporosis and fracture healing), with due attention to the taurine-cartilage relationship.
Asunto(s)
Osteoporosis , Taurina , Animales , Cartílago/metabolismo , Humanos , Músculo Esquelético/metabolismo , Osteogénesis , Taurina/metabolismoRESUMEN
OBJECTIVE: To analyze the presenceof human papillomavirus in prostate and its associationwith prostate cancer. METHODS: A case-control study was conducted.Tissue samples with benign hyperplasia and prostatecancer were collected. Risk factors related to prostatecancer and human papillomavirus were assessedby a medical interview. Prostate tissue was obtainedby transrectal biopsy or transurethral resection. Theidentification of viral genome was assessed by the amplificationof 450 pb., from L1 gene. Real time PCR wasused to identified HPV genotypes 16 and 18. For dataanalysis, the χ2 test, Student's T test or Mann-WhitneyU test and OR were computed. RESULTS: Thirty and 99 with benign prostatehyperplasia were included in a 1:3 ratio, with a meanage of 69.44±9.22 years. The global prevalence of humanpapillomavirus was 15.2% being similar in bothcases (15.6%) and controls (15.1%) with no significantdifference (p = 0.572). Forty percent of the infectionswere persistent. From all positive samples, only in the40% were identified some of the genotypes analyzed(16 and 18). The group of patients with Gleason scorede > 7 had a virus prevalence of 16%. CONCLUSIONS: The results show the presence ofthe human papillomavirus genome in prostate tissuewith and without neoplasia; no association was foundbetween infection and prostate cancer.
OBJETIVOS: Analizar la presencia delvirus de papiloma humano en próstata y su asociacióncon cáncer.MATERIAL Y MÉTODOS: Se realizó un estudiode casos y controles, para lo cual se colectaronmuestras de tejido con hiperplasia benigna y concáncer de próstata. Se realizó una historia clínicapara conocer la presencia de los factores de riesgoasociados al cáncer de próstata, así como los relacionadoscon el virus. El tejido prostático fue obtenidopor biopsia transrectal o resección transuretral.La identificación del genoma viral se realizó amplificandoun fragmento de 450 del gen L1 por mediode una PCR clásica. Para la identificación de los genotipos16 y 18, se utilizó PCR tiempo real. Para elanálisis de los datos se utilizó la prueba de χ2, pruebade T de Student o U de Mann-Whitney y cálculode OR. RESULTADOS: Se incluyeron 32 pacientes concáncer de próstata y 99 con hiperplasia benigna depróstata en una relación 1:3. La media de edad fuede 69.44±9.22 años. La prevalencia global del virusde papiloma humano fue de 15.2% siendo similar en los casos y entre casos (15.6%) y controles (15.1%),no existiendo diferencia significativa (p=0.572). El40% de las infecciones eran persistentes. Del totalde las muestras positivas, solamente en el 40% seencontró alguno de los dos genotipos analizados (16y 18). Los pacientes con un puntaje de Gleason > 7tuvieron una prevalencia del virus de 16%. CONCLUSIONES: Los resultados ponen de manifiestola presencia del genoma del virus del papilomahumano en próstata con y sin neoplasia, no se encontróasociación entre la infección y el cáncer de próstata.
Asunto(s)
Alphapapillomavirus , Hiperplasia Prostática , Neoplasias de la Próstata , Alphapapillomavirus/genética , Estudios de Casos y Controles , Humanos , Masculino , Papillomaviridae/genéticaRESUMEN
Several vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for controlling the coronavirus disease 2019 (COVID-19) pandemic worldwide. Antibody response is essential to understand the immune response to different viral targets after vaccination with different vaccine platforms. Thus, the main aim of this study was to describe how vaccination with two distinct SARS-CoV-2 vaccine preparations elicit IgG antibody specific responses against two antigenically relevant SARS-CoV-2 viral proteins: the receptor-binding domain (RBD) and the full-length spike (S). To do so, SARS-CoV-2 protein specific in-house enzyme-linked immunosorbent assays (ELISAs) were standardized and tested against serum samples collected from 89 adults, recipients of either a single-dose of the Spike-encoding mRNA-based Pfizer/BioNTech (Pf-BNT) (70%, 62/89) or the Spike-encoding-Adenovirus-5-based CanSino Biologics Inc. (CSBIO) (30%, 27/89) in Merida, Mexico. Overall, we identified an IgG seroconversion rate of 88% (68/78) in all vaccinees after more than 25 days post-vaccination (dpv). Anti-RBD IgG-specific responses ranged from 90% (46/51) in the Pf-BNT vaccine at 25 dpv to 74% (20/27) in the CSBIO vaccine at 42 dpv. Compared to the S, the RBD IgG reactivity was significantly higher in both Pf-BNT (p < 0.004) and CSBIO (p < 0.003) vaccinees. Interestingly, in more than 50% of vaccine recipients, with no history of COVID-19 infection, antibodies against the nucleocapsid (N) protein were detected. Thus, participants were grouped either as naïve or pre-exposed vaccinees. Seroconversion rates after 25 and more dpv varies between 100% in Pf-BNT (22/22) and 75% (9/12) in CSBIO pre-exposed vaccinees, and 89% (26/29) and 73% (11/15) in Pf-BNT and CSBIO naïve vaccine recipients, respectively. In summary, observed seroconversion rates varied depending on the type of vaccine, previous infection with SARS-CoV-2, and the target viral antigen. Our results indicate that both vaccine preparations can induce detectable levels of IgG against the RBD or Spike in both naïve and SARS-CoV-2 pre-exposed vaccinees. Our study provides valuable and novel information about the serodiagnosis and the antibody response to vaccines in Mexico.
RESUMEN
Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction.
Asunto(s)
ADN Ribosómico/genética , Genes de ARNr , No Disyunción Genética , ARN Ribosómico/biosíntesis , Transcripción Genética/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Segregación Cromosómica , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Conversión Génica/fisiología , Eliminación de Gen , Dosificación de Gen , Genes cdc , Modelos Genéticos , Mutación , ARN Polimerasa II/metabolismo , Levaduras/citologíaRESUMEN
During meiosis, the budding yeast polo-like kinase Cdc5 is a crucial driver of the prophase I to meiosis I (G2/M) transition. The meiotic recombination checkpoint restrains cell cycle progression in response to defective recombination to ensure proper distribution of intact chromosomes to the gametes. This checkpoint detects unrepaired DSBs and initiates a signaling cascade that ultimately inhibits Ndt80, a transcription factor required for CDC5 gene expression. Previous work revealed that overexpression of CDC5 partially alleviates the checkpoint-imposed meiotic delay in the synaptonemal complex-defective zip1Δ mutant. Here, we show that overproduction of a Cdc5 version (Cdc5-ΔN70), lacking the N-terminal region required for targeted degradation of the protein by the APC/C complex, fails to relieve the zip1Δ-induced meiotic delay, despite being more stable and reaching increased protein levels. However, precise mutation of the consensus motifs for APC/C recognition (D-boxes and KEN) has no effect on Cdc5 stability or function during meiosis. Compared to the zip1Δ single mutant, the zip1Δ cdc5-ΔN70 double mutant exhibits an exacerbated meiotic block and reduced levels of Ndt80 consistent with persistent checkpoint activity. Finally, using a CDC5-inducible system, we demonstrate that the N-terminal region of Cdc5 is essential for its checkpoint erasing function. Thus, our results unveil an additional layer of regulation of polo-like kinase function in meiotic cell cycle control.