Hyperglycemia affects neuronal differentiation and Nestin, FOXO1, and LMO3 mRNA expression of human Wharton's jelly mesenchymal stem cells of children from diabetic mothers.

Pregestational Diabetes Mellitus (PDM) during pregnancy constitutes an unfavorable embryonic and fetal development environment, with a high incidence of congenital malformations (CM). Neural tube defects are the second most common type of CM in children of diabetic mothers (CDM), who also have an elevated risk of developing neurodevelopmental disorders. The mechanisms that lead to these neuronal disorders in CDM are not yet fully understood. The present study aimed to know the effect of hyperglycemia on proliferation, neuronal differentiation percentage, and expression of neuronal differentiation mRNA markers in human umbilical cord Wharton's jelly mesenchymal stem cells (hUCWJMSC) of children from normoglycemic pregnancies (NGP) and PDM. We isolated and characterized hUCWJMSC by flow cytometry, immunofluorescence, RT-PCR and were induced to differentiate into adipocytes, osteocytes, and neurons. Proliferation assays were performed to determine the doubling time, and Nestin, TUBB3, FOXO1, KCNK2, LMO3, and MAP2 mRNA gene expression was assessed by semiquantitative RT-PCR. Hyperglycemia significantly decreased proliferation and neuronal differentiation percentage in NGP and PDM cells treated with 40 mM d-glucose. Nestin mRNA expression decreased under control glycemic conditions, while FOXO1, KCNK2, LMO3, and MAP2 mRNA expression increased during neuronal differentiation in both NGP and PDM cells. On the other hand, under hyperglycemic conditions, Nestin was significantly decreased in cells from NGP but not in cells from PDM, while mRNA expression of FOXO1 and LMO3 was significantly increased in cells from NGP, but not in cells from PDM. We found evidence that maternal PDM, with hyperglycemia in culture, affects the biological properties of fetal cells. All these results could be part of fetal programming.

Implementation of a tunable t-CRISPRi system for gene regulation in Giardia duodenalis.

Giardia duodenalis, is a binuclear and microaerophilic protozoan that causes giardiasis. Up to date, several molecular approaches have been taken to understand the molecular mechanisms of diverse cellular processes in this parasitic protozoan. However, the role of many genes involved in these processes needs further analysis. The CRISPR interference (CRISPRi) system has been widely used, as a constitutive expression system for gene silencing purposes in several parasites, including Giardia. The aim of this work was to implement a tunable t-CRISPRi system in Giardia to silence abundant, moderately and low expressed genes, by constructing an optimized and inducible plasmid for the expression of both gRNA and dCas9. A doxycycline inducible pRan promoter was used to express dCas9 and each gRNA, consistently dCas9 expression and nuclear localization were confirmed by Western-blot and immunofluorescence in transfected trophozoites. The transcriptional repression was performed on α-tubulin (high expression), giardipain-1 (moderate expression) and Sir2 and Sir4 (low expression) genes. The α-tubulin gene knock-down caused by dCas9 doxycycline-induction was confirmed by a decrease in its protein expression which was of 50% and 60% at 24 and 48 h, respectively. This induced morphological alterations in flagella. The giardipain-1 knock down, showed a decrease in protein expression of 40 and 50% at 12 and 24 h, respectively, without affecting trophozoites viability, consistent with this a zymogram analysis on giardipain-1 knock down revealed a decrease in giardipain-1 protease activity. When repressing sirtuins expression, a total repression was obtained but trophozoites viability was compromised. This approach provides a molecular tool for a tailored repression to produce specific gene knockdowns.

Giardia duodenalis carries out canonical homologous recombination and single-strand annealing.

In the past decades, the ability of Giardia duodenalis to perform homologous recombination has been suggested, supported by the observations of genomic integration of foreign plasmids and the disruption of genes using CRISPR technology. Unfortunately, the direct study of a HR mechanism has not been addressed, which would be pertinent in a minimalist organism lacking fundamental DNA-repair elements and even complete pathways. In addition, the constant ploidy changes through the life cycle of this parasite highlight the conservation and relevance of homologous recombination in maintaining genomic stability. In this research, we analyzed different recombinable plasmid systems and their outcomes after G. duodenalis transfection, using this approach we determined genomic, intra-plasmid and inter-plasmid recombination, moreover, we examined the presence of the non-conservative single-strand annealing pathway. With the intention of corroborating that the observed processes were done by homologous recombination, we used a chemical inhibitor named Mirin, which specifically inhibits Mre11 3'- 5' exonuclease activity, one of the first steps involved in homologous recombination and fundamental to success in repairing. Overall, these results describe the multiple recombinational substrates used by G. duodenalis to achieve HR and demonstrate the presence and use of single-strand annealing recombination.

Sirtuin GdSir2.4 participates in the regulation of rRNA transcription in the Giardia duodenalis parasite.

Giardia duodenalis is a parasite of great medical interest due to the number of infections it causes worldwide each year. Although research on epigenetic mechanisms in this protist has only begun recently, epigenetic regulation has already been shown to have important roles in encystation, antigenic variation, and resistance to antibiotics in Giardia. In this work, we show that a Giardia ortholog of Sir2, GdSir2.4, is involved in the silencing of rRNA expression. Our results demonstrate that GdSir2.4 localizes to the nucleolus, and its binding to the intergenic spacer region of the rDNA is associated with the deacetylation of the chromatin in this region. Given the importance of the regulation of rRNA expression to maintain adequate levels of ribosomes and genomic stability within the cells, GdSir2.4 can be considered a target to create new therapeutic agents against this parasite.

Ribosomal DNA in the protozoan parasite Giardia duodenalis has a differential chromatin distribution and epigenetic markings across the subunits.

Giardia duodenalis is a parasite that causes a large number of diarrheal diseases around the world. It is noteworthy that in a large number of processes, Giardia requires fewer components than other eukaryotes, even without some organelles such as mitochondria and peroxisomes. Despite this, core histones are known to exist in Giardia and epigenetic marks have been found on them, suggesting that they somehow control the expression of certain genes. The regulation of the expression of ribosomal DNA (rDNA) is essential, since it is required to maintain adequate levels of ribosomes and, given the nature of tandem repeat, it is a feasible area to create genomic instability. In Giardia, it is not known how this process occurs, but as in other eukaryotes, it is suggested through various epigenetic mechanisms. Thus, in the present work we seek to identify how chromatin is distributed through the Giardia rDNA and if there were histone marks that could control its expression.

Nicotinamide induces G2 cell cycle arrest in Giardia duodenalis trophozoites and promotes changes in sirtuins transcriptional expression.

Giardia duodenalis is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. Treatment of giardiasis is limited to nitroheterocyclic compounds as metronidazole and benzimidazoles as albendazole, where remarkably treatment failure is relatively common. Consequently, the need for new options to treat this disease is underscored. We predicted by a bioinformatic approach that nicotinamide inhibits Giardia sirtuins by the nicotinamide exchange pathway, and since sirtuins are involved in cell cycle control, they could be related with arrest and decrease of viability. When trophozoites were treated with nicotinamide (NAM), a strong arrest of Giardia trophozoites in G2 phase was observed and at the same time changes in transcriptional expression of sirtuins were produced. Interestingly, the G2 arrest is not related to double-strand breaks, which strengthens the role of sirtuins in the control of the Giardia cell cycle. Results with NAM-treated trophozoites as predicted demonstrate antigiardial effects and thus open new options for the treatment of giardiasis, either with the combination of nicotinamide with another antigiardial drug, or with the design of specific inhibitors for Giardia sirtuins.

Hyperglycemia differentially affects proliferation, apoptosis, and BNIP3 and p53 mRNA expression of human umbilical cord Wharton's jelly cells from non-diabetic and diabetic pregnancies.

Diabetes in pregnancy constitutes an unfavorable environment for embryonic and fetal development, where the child has a higher risk of perinatal morbidity and mortality, with high incidence of congenital malformations and predisposition to long-term metabolic diseases that increase with a hypercaloric diet. To analyze whether hyperglycemia differentially affects proliferation, apoptosis, and mRNA expression in cells from children of normoglycemic pregnancies (NGPs) and diabetes mellitus pregnancies (DMPs), we used umbilical cord Wharton jelly cells as a research model. Proliferation assays were performed to analyze growth and determine the doubling time, and the rate of apoptosis was determined by flow cytometry-annexin-V assays. AMPK, BNIP3, HIF1α, and p53 mRNA gene expression was assessed by semi-quantitative RT-PCR. We found that hyperglycemia decreased proliferation in a statistically significant manner in NGP cells treated with 40 mM D-glucose and in DMP cells treated with 30 and 40 mM D-glucose. Apoptosis increased in hyperglycemic conditions in NGP and DMP cells. mRNA expression of BNIP3 and p53 was significantly increased in cells from DMPs but not in cells from NGPs. We found evidence that maternal irregular metabolic conditions, like diabetes with hyperglycemia in culture, affect biological properties of fetal cells. These observations could be a constituent of fetal programming.

DNA damage induced by metronidazole in Giardia duodenalis triggers a DNA homologous recombination response.

The mechanisms underlying metronidazole (MTZ) resistance in Giardia duodenalis have been associated with decreased activity of the enzymes implicated in its activation including nitroductase-1, thioredoxin reductase and pyruvate-ferredoxin oxidoreductase (PFOR). MTZ activation generates radicals that can form adducts with proteins such as thioredoxin reductase and α- and -ß giardins as well as DNA damage resulting in trophozoite's death. The damage induced in DNA requires a straight forward response that may allow parasite survival. Here, we studied changes in histone H2A phosphorylation to evaluate the DNA repair response pathway after induction of double strand break (DSB) by MTZ in Giardia DNA. Our results showed that the DNA repair mechanisms after exposure of Giardia trophozoites to MTZ, involved a homologous recombination pathway. We observed a significant increase in the expression level of proteins GdDMC1B, which carries out Rad51 role in G. duodenalis, and GdMre11, after 12 h of exposure to 3.2 µM MTZ. This increase was concomitant with the generation of DSB in the DNA of trophozoites treated MTZ. Altogether, these results suggest that MTZ-induced DNA damage in Giardia triggers the DNA homologous recombination repair (DHRR) pathway, which may contribute to the parasite survival in the presence of MTZ.

Giardia duodenalis Rad52 protein: biochemical characterization and response upon DNA damage.

Giardia duodenalis is a flagellated binucleated protozoan that colonizes the small intestine in mammals, causing giardiasis, acute or chronic diarrhea. DNA double strand break either endogenously or exogenously generated is a major insult to DNA and its repair by homologous recombination (HR) is crucial for genomic stability. During HR, Rad52 plays key roles in the loading of the Rad51 recombinase, and the annealing of the second double-strand break end to the displaced strand of the D-loop structure. Among the functions found in vitro in yeast and human Rad52 protein are: ssDNA or dsDNA binding activity, ability to anneal bare or RPA coated-ssDNA, as well as multimeric ring formation. In this work, we searched for conserved domains in a putative Rad52 protein from G. duodenalis (GdRad52). Its coding sequence was cloned, expressed and purified to study its biochemical properties. rGdRad52 binds to dsDNA and ssDNA, with greater affinity for the latter. Likewise, rGdRad52 promotes annealing of DNA uncoated and coated with GdRPA1. rGdRad52 interacts with GdDMC1B and with GdRPA1 protein as shown in far western blotting assay. Additionally, rGdRad52 formed multimeric rings as observed by electronic microscopy. Finally, GdRad52 is over expressed in response upon DNA damage inflicted on trophozoites.

In silico data analyses of recombinases GdDMC1A and GdDMC1B from Giardia duodenalis.

Giardia duodenalis is a worldwide protozoa known causing diarrhea in all vertebrates, humans among these. Homologous recombination is a mechanism that provides genomic stability. Two putative recombinases were identified in G. duodenalis genome: GdDMC1A and GdDMC1B. In this article, we describe the identification of conserved domains in GdDMC1A and GdDMC1B, such as: DNA binding domains (Helix-turn-helix motif, loops 1 and 2) and an ATPcap and Walker A and B motifs associated with ATP binding and hydrolysis, phylogenetic analyses among assemblages and three-dimensional structure modeling of these recombinases using bioinformatics tools. Also, experimental data is described about LD50 determination for ionizing radiation in trophozoites of G. duodenalis. Additionally, as recombinases, GdDMC1A and GdDMC1B were used to rescue a defective Saccharomyces cerevisiae Δ rad51 strain under genotoxic conditions and data is described. The data described here are related to the research article entitled "Characterization of recombinase DMC1B and its functional role as Rad51 in DNA damage repair in Giardia duodenalis trophozoites" (Torres-Huerta et al.,) [1].

Characterization of recombinase DMC1B and its functional role as Rad51 in DNA damage repair in Giardia duodenalis trophozoites.

Homologous recombination (HR) is a highly conserved pathway for the repair of chromosomes that harbor DNA double-stranded breaks (DSBs). The recombinase RAD51 plays a key role by catalyzing the pairing of homologous DNA molecules and the exchange of information between them. Two putative DMC1 homologs (DMC1A and DMC1B) have been identified in Giardia duodenalis. In terms of sequences, GdDMC1A and GdDMC1B bear all of the characteristic recombinase domains: DNA binding domains (helix-turn-helix motif, loops 1 and 2), an ATPcap and Walker A and B motifs associated with ATP binding and hydrolysis. Because GdDMC1B is expressed at the trophozoite stage and GdDMC1A is expressed in the cyst stage, we cloned the giardial dmc1B gene and expressed and purified its protein to determine its activities, including DNA binding, ATP hydrolysis, and DNA strand exchange. Our results revealed that it possessed these activities, and they were modulated by divalent metal ions in different manners. GdDMC1B expression at the protein and transcript levels, as well as its subcellular localization in trophozoites upon DNA damage, was assessed. We found a significant increase in GdDMC1B transcript and protein levels after ionizing radiation treatment. Additionally, GdDMC1B protein was mostly located in the nucleus of trophozoites after DNA damage. These results indicate that GdDMC1B is the recombinase responsible for DSBs repair in the trophozoite; therefore, a functional Rad51 role is proposed for GdDMC1B.

Multiplex PCR to detect four different tomato-infecting pathogens.

This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.

Protein kinase C isoforms from Giardia duodenalis: identification and functional characterization of a beta-like molecule during encystment.

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate many different cellular processes such as cell growth and differentiation in eukaryotic cells. Using specific polyclonal antibodies raised against mammalian PKC isoforms, it was demonstrated here for the first time that Giardia duodenalis expresses several PKC isoforms (beta, delta, epsilon, theta and zeta). All PKC isoforms detected showed changes in their expression pattern during encystment induction. In addition, selective PKC inhibitors blocked the encystment in a dose-dependent manner, suggesting that PKC isozymes may play important roles during this differentiation process. We have characterized here the only conventional-type PKC member found so far in Giardia, which showed an increased expression and changes in its intracellular localization pattern during cyst formation. The purified protein obtained by chromatography on DEAE-cellulose followed by size-exclusion chromatography, displayed in vitro kinase activity using histone HI-IIIS as substrate, which was dependent on cofactors required by conventional PKCs, i.e., phospholipids and calcium. An open reading frame in the Giardia Genome Database that encodes a homolog of PKCbeta catalytic domain was identified and cloned. The expressed recombinant protein was also recognized by a mammalian anti-PKCbeta antibody and was referred as giardial PKCbeta on the basis of all these experimental evidence.