Estudio del gen de la piruvato: ferredoxin óxidoreductasa en aislados humanos de blastocystis spp

Blastocystis spp. es un parásito intestinal que presenta la mayor prevalencia en los exámenescoproparasitoscópicos; su amplia diversidad morfológica y genética permite identificar diferentesestadios morfológicos y al menos 17 linajes ribosomales conocidos como subtipos (ST), basadosen su identificación por el gen 18S rDNA. Se ha argumentado que el gen 18S rDNA podría no serel marcador de elección para discriminar entre cepas de un mismo ST debido a su ampliopolimorfismo intragenómico. Se ha propuesto que la patogenicidad pudiera ser dependiente del ST,esto y características de la biología del parásito por confirmar han causado incertidumbre paraestablecer sin duda su papel como patógeno. Por otro lado, la piruvato: ferredoxin óxidoreductasa(PFOR) es una enzima conservada que se ha identificado en el MLO de Blastocystis; su papelcentral en la obtención de energía para este y otros organismos anaerobios la hace un blancoprometedor para identificar cepas de este parásito. Objetivo: Evaluar la diversidad genética delgen PFOR en aislados de Blastocystis de niños sintomáticos. Método: Se analizaron 192 muestrasde materia fecal de niños residentes del Estado de México, que presentaban síntomas de diarreay/o dolor abdominal. Los exámenes coproparasitoscópicos revelaron a Blastocystis spp. comoúnico parásito en 21 muestras, las que fueron analizadas por PCR para las regiones de interés delgen 18S rDNA y el gen PFOR. Se obtuvieron las secuencias de los amplicones y se analizaronpara determinar la variabilidad genética de la región de interés para el gen PFOR. Resultados: Elanálisis del gen 18S rDNA mostró que el subtipo ST3 de Blastocystis fue el más frecuente (43%),seguido del ST1 (38%), ST2 (14%) y ST7 (5%). Además, se encontró en dos muestras coinfeccióncon los ST1 y ST2 (10%). La reconstrucción filogenética por inferencia Bayesiana mostró que lassecuencias del gen PFOR se agruparon en tres clústeres, dos de ellos contienen secuencias dediferentes subtipos y solo un clúster agrupó secuencias del subtipo ST3. Los valores de ladiversidad nucleotídica () y el polimorfismo de haplotipo () del análisis del gen 18S rDNA fueronsimilares entre ST1 y ST2 (~0.025 y ~0.03@). Interesantemente, los valores y para elsubtipo ST3 fueron aproximadamente 10 veces más bajos (~0.004 y ~0.005). Para el gen PFORuna tendencia similar fue encontrada; los clústeres I y II tuvieron valores de ( ~0.05 y ~0.05),mientras que para el clúster III se encontró valores seis veces más bajos (~0.008 y ~0.009).Conclusiones: Aunque el marcador PFOR no permitió diferenciar a Blastocystis en subtipos, seencontró una baja variabilidad genética para las muestras del subtipo ST3 usando los marcadoresPFOR y 18S rDNA, lo que sugiere un panorama epidemiológico particular que lo distingue de losotros subtipos

Perspective on the Genetic Response to Antiparasitics: A Review Article

Background: Drugs’ pharmacokinetics and pharmacodynamics can be af- fected by diverse genetic variations, within which simple nucleotide poly- morphisms (SNPs) are the most common. Genetic variability is one of the factors that could explain questions like why a given drug does not have the desired effect or why do adverse drug reactions arise. Methods: In this retrospective observational study, literature search limits were set within PubMed database as well as the epidemiological bulletins published by the Mexican Ministry of Health, from Jan 1st 2001 to Mar 31st 2017 (16 years). Results: Metabolism of antiparasitic drugs and their interindividual re- sponses are mainly modified by variations in cytochrome P450 enzymes. These enzymes show high frequencies of polymorphic variability thus af- fecting the expression of CYP2C, CYP2A, CYP2A6, CYP2D6, CYP2E6 and CYP2A6 isoforms. Research in this field opens the door to new person- alized treatment approaches in medicine. Conclusion: Clinical and pharmacological utility yield by applying phar- macogenetics to antiparasitic treatments is not intended as a mean to im- prove the prescription process, but to select or exclude patients that could present adverse drug reactions as well as to evaluate genetic alterations which result in a diversity of responses, ultimately seeking to provide a more effective and safe treatment; therefore choosing a proper dose for the ap- propriate patient and the optimal treatment duration. Furthermore, phar- macogenetics assists in the development of vaccines. In other words, the aim of this discipline is to find therapeutic targets allowing personalized treatments

Long-term infection passaging of Human Adenovirus 36 in monkey kidney cells

Human Adenovirus 36 (HAdV-36) has been related to diverse effects on metabolism and may attenuate the lipid accumulation in kidneys with increased adiposity. Some of these effects would be related to viral persistence. However, until now, a model of persistent in vitro infection by HAdV-36 is unknown. In this study, we examined the cells of the Vero lineage to explore their permissiveness to long-term HAdV-36 infection. HAdV-36 was productively replicated in Vero cells and maintained long-term infection for up to 35 cell passages. A subculture was obtained from the cells that survived the primary infection at a low MOI (0.5). The production of the extracellular infectious virus with titers ranging from 104 to 106 TCID50/mL and DNA-bearing cells was detected. In long-term infected cells, the intracellular distribution of viral antigen was demonstrated by performing immunolocalization (IFI) and expression of cell-viral antigen in 50% of cells by flow cytometry, using anti-HAdV-36 hyperimmune rabbit serum. Furthermore, E1a and E4orf1 genes in long-term infected passages showed a decreasing trend. Our preliminary results reveal that renal epithelial monkey cells are permissive for the productive infection of HAdV-36. Vero cell culture long-term infection might be a promising model for addressing the fundamental aspects of the HAdV-36 biology that cannot reveal broadly-used cultures, which do not maintain long-term infection in primary or transformed cells

Can the pyruvate: ferredoxin oxidoreductase (PFOR) gene be used as an additional marker to discriminate among Blastocystis strains or subtypes?

Background Blastocystis spp. are the most prevalent intestinal eukaryotes identified in humans, with at least 17 genetic subtypes (ST) based on genes coding for the small-subunit ribosomal RNA (18S). It has been argued that the 18S gene should not be the marker of choice to discriminate between STs of these strains because this marker exhibits high intra-genomic polymorphism. By contrast, pyruvate:ferredoxin oxidoreductase (PFOR) is a relevant enzyme involved in the core energy metabolism of many anaerobic microorganisms such as Blastocystis, which, in other protozoa, shows more polymorphisms than the 18S gene and thus may offer finer discrimination when trying to identify Blastocystis ST. Therefore, the objective of the present study was to assess the suitability of the PFOR gene as an additional marker to discriminate among Blastocystis strains or subtypes from symptomatic carrier children. Methods Faecal samples from 192 children with gastrointestinal symptoms from the State of Mexico were submitted for coprological study. Twenty-one of these samples were positive only for Blastocystis spp.; these samples were analysed by PCR sequencing of regions of the 18S and PFOR genes. The amplicons were purified and sequenced; afterwards, both markers were assessed for genetic diversity. Results The 18S analysis showed the following frequencies of Blastocystis subtypes: ST3 = 43%; ST1 = 38%; ST2 = 14%; and ST7 = 5%. Additionally, using subtype-specific primer sets, two samples showed mixed Blastocystis ST1 and ST2 infection. For PFOR, Bayesian inference revealed the presence of three clades (I-III); two of them grouped different ST samples, and one grouped six samples of ST3 (III). Nucleotide diversity (π) and haplotype polymorphism (θ) for the 18S analysis were similar for ST1 and ST2 (π = ~0.025 and θ = ~0.036); remarkably, ST3 showed almost 10-fold lower values. For PFOR, a similar trend was found: clade I and II had π = ~0.05 and θ = ~0.05, whereas for clade III, the values were almost 6-fold lower. Conclusions Although the fragment of the PFOR gene analysed in the present study did not allow discrimination between Blastocystis STs, this marker grouped the samples in three clades with strengthened support, suggesting that PFOR may be under different selective pressures and evolutionary histories than the 18S gene. Interestingly, the ST3 sequences showed lower variability with probable purifying selection in both markers, meaning that evolutionary forces drive differential processes among Blastocystis STs

High-Density SNP Screening of the Major Histocompatibility Complex in Systemic Lupus Erythematosus Demonstrates Strong Evidence for Independent Susceptibility Regions

A substantial genetic contribution to systemic lupus erythematosus (SLE) risk is conferred by major histocompatibility complex (MHC) gene(s) on chromosome 6p21. Previous studies in SLE have lacked statistical power and genetic resolution to fully define MHC influences. We characterized 1,610 Caucasian SLE cases and 1,470 parents for 1,974 MHC SNPs, the highly polymorphic HLA-DRB1 locus, and a panel of ancestry informative markers. Single-marker analyses revealed strong signals for SNPs within several MHC regions, as well as with HLA-DRB1 (global p = 9.99×10−16). The most strongly associated DRB1 alleles were: *0301 (odds ratio, OR = 2.21, p = 2.53×10−12), *1401 (OR = 0.50, p = 0.0002), and *1501 (OR = 1.39, p = 0.0032). The MHC region SNP demonstrating the strongest evidence of association with SLE was rs3117103, with OR = 2.44 and p = 2.80×10−13. Conditional haplotype and stepwise logistic regression analyses identified strong evidence for association between SLE and the extended class I, class I, class III, class II, and the extended class II MHC regions. Sequential removal of SLE–associated DRB1 haplotypes revealed independent effects due to variation within OR2H2 (extended class I, rs362521, p = 0.006), CREBL1 (class III, rs8283, p = 0.01), and DQB2 (class II, rs7769979, p = 0.003, and rs10947345, p = 0.0004). Further, conditional haplotype analyses demonstrated that variation within MICB (class I, rs3828903, p = 0.006) also contributes to SLE risk independent of HLA-DRB1*0301. Our results for the first time delineate with high resolution several MHC regions with independent contributions to SLE risk. We provide a list of candidate variants based on biologic and functional considerations that may be causally related to SLE risk and warrant further investigation

Perspective on the Genetic Response to Antiparasitics: A Review Article

Background: Drugs’ pharmacokinetics and pharmacodynamics can be af- fected by diverse genetic variations, within which simple nucleotide poly- morphisms (SNPs) are the most common. Genetic variability is one of the factors that could explain questions like why a given drug does not have the desired effect or why do adverse drug reactions arise. Methods: In this retrospective observational study, literature search limits were set within PubMed database as well as the epidemiological bulletins published by the Mexican Ministry of Health, from Jan 1st 2001 to Mar 31st 2017 (16 years). Results: Metabolism of antiparasitic drugs and their interindividual re- sponses are mainly modified by variations in cytochrome P450 enzymes. These enzymes show high frequencies of polymorphic variability thus af- fecting the expression of CYP2C, CYP2A, CYP2A6, CYP2D6, CYP2E6 and CYP2A6 isoforms. Research in this field opens the door to new person- alized treatment approaches in medicine. Conclusion: Clinical and pharmacological utility yield by applying phar- macogenetics to antiparasitic treatments is not intended as a mean to im- prove the prescription process, but to select or exclude patients that could present adverse drug reactions as well as to evaluate genetic alterations which result in a diversity of responses, ultimately seeking to provide a more effective and safe treatment; therefore choosing a proper dose for the ap- propriate patient and the optimal treatment duration. Furthermore, phar- macogenetics assists in the development of vaccines. In other words, the aim of this discipline is to find therapeutic targets allowing personalized treatments

Overall survival in the OlympiA phase III trial of adjuvant olaparib in patients with germline pathogenic variants in BRCA1/2 and high-risk, early breast cancer

International audienc