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Peptic ulcers (PU) are a breach in the mucosa of the digestive tract and are related to several factors including an altered immune system and an unbalanced diet. Current treatment carries to long-term complications; therefore, the use of medicinal plants is an alternative for several inflammatory diseases including ulcerative lesions. Kalanchoë gastonis-bonnieri, is a succulent plant and, has been used in traditional medicine against gastric ulcers, inflammation, and cancer among others. The main goal of this work was to analyze the anti-ulcerogenic potential of extracts from leaves of K. gastonis-bonnieri in an assay of ethanol-induced gastric ulcers. An ethanolic extract was obtained by maceration from fresh leaves of K. gastonis-bonnieri, and fractions were obtained through bipartition and chemical fractioning. The chemical characterization of the extract was made through HPLC, GC-MS, and NMR. Total extract and fractions showed an anti-ulcerogenic effect in specimens of male ICR mice with a gastric ulcer index (UI) of 3.27-5.47. The recorded effect is attributed to the presence of terpenoid compounds such as β-Amyrin acetate, which showed antioxidant properties and lessened formations of ulcers induced by ethanol administration in mice stomach., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Glioblastoma multiforme (GBM) exhibits genetic alterations that induce the deregulation of oncogenic pathways, thus promoting metabolic adaptation. The modulation of metabolic enzyme activities is necessary to generate nucleotides, amino acids, and fatty acids, which provide energy and metabolic intermediates essential for fulfilling the biosynthetic needs of glioma cells. Moreover, the TCA cycle produces intermediates that play important roles in the metabolism of glucose, fatty acids, or non-essential amino acids, and act as signaling molecules associated with the activation of oncogenic pathways, transcriptional changes, and epigenetic modifications. In this review, we aim to explore how dysregulated metabolic enzymes from the TCA cycle and oxidative phosphorylation, along with their metabolites, modulate both catabolic and anabolic metabolic pathways, as well as pro-oncogenic signaling pathways, transcriptional changes, and epigenetic modifications in GBM cells, contributing to the formation, survival, growth, and invasion of glioma cells. Additionally, we discuss promising therapeutic strategies targeting key players in metabolic regulation. Therefore, understanding metabolic reprogramming is necessary to fully comprehend the biology of malignant gliomas and significantly improve patient survival., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

The metabolic reprogramming that promotes tumorigenesis in glioblastoma is induced by dynamic alterations in the hypoxic tumor microenvironment, as well as in transcriptional and signaling networks, which result in changes in global genetic expression. The signaling pathways PI3K/AKT/mTOR and RAS/RAF/MEK/ERK stimulate cell metabolism, either directly or indirectly, by modulating the transcriptional factors p53, HIF1, and c-Myc. The overexpression of HIF1 and c-Myc, master regulators of cellular metabolism, is a key contributor to the synthesis of bioenergetic molecules that mediate glioma cell transformation, proliferation, survival, migration, and invasion by modifying the transcription levels of key gene groups involved in metabolism. Meanwhile, the tumor-suppressing protein p53, which negatively regulates HIF1 and c-Myc, is often lost in glioblastoma. Alterations in this triad of transcriptional factors induce a metabolic shift in glioma cells that allows them to adapt and survive changes such as mutations, hypoxia, acidosis, the presence of reactive oxygen species, and nutrient deprivation, by modulating the activity and expression of signaling molecules, enzymes, metabolites, transporters, and regulators involved in glycolysis and glutamine metabolism, the pentose phosphate cycle, the tricarboxylic acid cycle, and oxidative phosphorylation, as well as the synthesis and degradation of fatty acids and nucleic acids. This review summarizes our current knowledge on the role of HIF1, c-Myc, and p53 in the genic regulatory network for metabolism in glioma cells, as well as potential therapeutic inhibitors of these factors.

The parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease worldwide. This infection often remains asymptomatic and is related to several health complications. The traditional treatment for trichomoniasis is the use of drugs of the 5-nitroimidazole family, such as metronidazole; however, scientific reports indicate an increasing number of drug-resistant strains. Benzimidazole derivatives could offer an alternative in the search for new anti-trichomonas drugs. In this sense, two attractive candidates are the compounds O 2 N-BZM7 and O 2 N-BZM9 (1 H -benzimidazole derivatives), since, through in vitro tests, they have shown a higher trichomonacide activity. In this study, we determined the effect on the expression level of metabolic genes in T. vaginalis . The results show that genes involved in redox balance ( NADHOX , G6PD::6PGL ) are overexpressed, as well as the gene that participates in the first reaction of glycolysis ( CK ); on the other hand, structural genes such as ACT and TUB are decreased in expression in trophozoites treated with the compound O 2 N-BZM9 , which would probably affect its morphology, motility and virulence. These results align with the trichomonacidal activity of the compounds, with benzimidazole O 2 N-BZM9 being the most potent, with an IC 50 value of 4.8 μM. These results are promising for potential future therapeutic applications.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting an estimated 500 million people worldwide, is a genetic disorder that causes human enzymopathies. Biochemical and genetic studies have identified several variants that produce different ranges of phenotypes; thus, depending on its severity, this enzymopathy is classified from the mildest (Class IV) to the most severe (Class I). Therefore, understanding the correlation between the mutation sites of G6PD and the resulting phenotype greatly enhances the current knowledge of enzymopathies' phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments for patients with G6PD deficiency. In this review, we analyzed and compared the structural and functional data from 21 characterized G6PD variants found in the Mexican population that we previously characterized. In order to contribute to the knowledge regarding the function and structure of the variants associated with G6PD deficiency, this review aimed to determine the molecular basis of G6PD and identify how these mutations could impact the structure, stability, and function of the enzyme and its relation with the clinical manifestations of this disease.

Giardiasis, which is caused by Giardia lamblia infection, is a relevant cause of morbidity and mortality worldwide. Because no vaccines are currently available to treat giardiasis, chemotherapeutic drugs are the main options for controlling infection. Evidence has shown that the nitro drug nitazoxanide (NTZ) is a commonly prescribed treatment for giardiasis; however, the mechanisms underlying NTZ's antigiardial activity are not well-understood. Herein, we identified the glucose-6-phosphate::6-phosphogluconate dehydrogenase (GlG6PD::6PGL) fused enzyme as a nitazoxanide target, as NTZ behaves as a GlG6PD::6PGL catalytic inhibitor. Furthermore, fluorescence assays suggest alterations in the stability of GlG6PD::6PGL protein, whereas the results indicate a loss of catalytic activity due to conformational and folding changes. Molecular docking and dynamic simulation studies suggest a model of NTZ binding on the active site of the G6PD domain and near the structural NADP + binding site. The findings of this study provide a novel mechanistic basis and strategy for the antigiardial activity of the NTZ drug.

Laccases are valuable enzymes as an excellent ecological alternative for bioremediation issues because they can oxidize persistent xenobiotic compounds. The production and characterization of extracellular laccases from saprotrophic fungi from disturbed environments have been scarcely explored, even though this could diversify their functional characteristics and expand the conditions in which they carry out their catalysis. Agrocybe pediades , isolated from a disturbed forest, produces an extracellular laccase in liquid culture. The enzyme was purified, identified and characterized. Copper and hexachlorobenzene do not function as inducers for the laccase produced. Partial amino acid sequences were obtained by LC-MS/MS that share similarity with laccases from other fungi. Purified laccase is a monomer with a molecular mass between 55-60 kDa and had an optimum activity at pH 5.0 and the optimum temperature at 45 °C using 2,6-dimethoxyphenol (2,6-DMP) as substrate. The K m and V max also determined with 2,6-DMP were 100 μM and 285 μmol∙min -1 ∙mg -1 , respectively, showing that the laccase of A . pediades has a higher affinity for this substrate than that of other Agaricales. These features could provide a potential catalyst for different toxic substrates and in the future laccase could be used in environmental recovery processes.

Protozoan parasites, such as Giardia lamblia and Trichomonas vaginalis , cause the most prevalent infections in humans in developing countries and provoke significant morbidity and mortality in endemic countries. Despite its side-effects, metronidazole is still the drug of choice as a giardiacidal and trichomonacidal tissue-active agent. However, the emergence of metronidazole resistance and its evolved strategies of parasites to evade innate host defenses have hindered the identification and development of new therapeutic strategies against these parasites. Here, we tested five synthesized benzimidazole derivatives as possible drugs for treating giardiasis and trichomoniasis, probing the bifunctional enzyme glucose 6-phosphate dehydrogenase::6-phosphogluconolactone from G. lamblia (GlG6PD::6PGL) and T. vaginalis (TvG6PD::6PGL) as a drug target. The investigated benzimidazole derivatives were H-B2M1 , H-B2M2 , H 2 N-BZM6 , O 2 N-BZM7 , and O 2 N-BZM9 . The recombinant enzymes were used in inhibition assays, and in silico computational predictions and spectroscopic studies were applied to follow the structural alteration of the enzymes and identify the possible mechanism of inhibition. We identified two potent benzimidazole compounds ( O 2 N-BZM7 and O 2 N-BZM9 ), which are capable of inhibiting both protozoan G6PD::6PGL enzymes and in vitro assays with these parasites, showing that these compounds also affect their viability. These results demonstrate that other therapeutic targets of the compounds are the enzymes GlG6PD::6PGL and TvG6PD::6PGL, which contribute to their antiparasitic effect and their possible use in antigiardial and trichomonacidal therapies.

Helicobacter pylori ( H. pylori ) has been proposed as the foremost risk factor for the development of gastric cancer. We found that H. pylori express the enzyme glucose-6-phosphate dehydrogenase (HpG6PD), which participates in glucose metabolism via the pentose phosphate pathway. Thus, we hypothesized that if the biochemical and physicochemical characteristics of HpG6PD contrast with the host G6PD (human G6PD, HsG6PD), HpG6PD becomes a potential target for novel drugs against H. pylori . In this work, we characterized the biochemical properties of the HpG6PD from the H. pylori strain 29CaP and expressed the active recombinant protein, to analyze its steady-state kinetics, thermostability, and biophysical aspects. In addition, we analyzed the HpG6PD in silico structural properties to compare them with those of the HsG6PD. The optimal pH for enzyme activity was 7.5, with a T 1/2 of 46.6 °C, at an optimum stability temperature of 37 °C. The apparent K m values calculated for G6P and NADP + were 75.0 and 12.8 µM, respectively. G6P does not protect HpG6PD from trypsin digestion, but NADP + does protect the enzyme from trypsin and guanidine hydrochloride (Gdn-HCl). The biochemical characterization of HpG6PD contributes to knowledge regarding H. pylori metabolism and opens up the possibility of using this enzyme as a potential target for specific and efficient treatment against this pathogen; structural alignment indicates that the three-dimensional (3D) homodimer model of the G6PD protein from H. pylori is different from the 3D G6PD of Homo sapiens .

A clear association between hypoxia and cancer has heretofore been established; however, it has not been completely developed. In this sense, the understanding of the tumoral microenvironment is critical to dissect the complexity of cancer, including the reduction in oxygen distribution inside the tumoral mass, defined as tumoral hypoxia. Moreover, hypoxia not only influences the tumoral cells but also the surrounding cells, including those related to the inflammatory processes. In this review, we analyze the participation of HIF, NF-κB, and STAT signaling pathways as the main components that interconnect hypoxia and immune response and how they modulate tumoral growth. In addition, we closely examine the participation of the immune cells and how they are affected by hypoxia, the effects of the progression of cancer, and some innovative applications that take advantage of this knowledge, to suggest potential therapies. Therefore, we contribute to the understanding of the complexity of cancer to propose innovative therapeutic strategies in the future.

Gliomas are heterogeneous, solid, and intracranial tumors that originate from glial cells. Malignant cells from the tumor undergo metabolic alterations to obtain the energy required for proliferation and the invasion of the cerebral parenchyma. The alterations in the expression of the genes related to the metabolic pathways can be detected in biopsies of gliomas of different CNS WHO grades. In this study, we evaluated the expression of 16 candidate reference genes in the HMC3 microglia cell line. Then, statistical algorithms such as BestKeeper, the comparative ΔC T method, geNorm, NormFinder, and RefFinder were applied to obtain the genes most suitable to be considered as references for measuring the levels of expression in glioma samples. The results show that PKM and TPI1 are two novel genes suitable for genic expression studies on gliomas. Finally, we analyzed the expression of genes involved in metabolic pathways in clinical samples of brain gliomas of different CNS WHO grades. RT-qPCR analysis showed that in CNS WHO grade 3 and 4 gliomas, the expression levels of HK1 , PFKM , GAPDH , G6PD , PGD1 , IDH1 , FASN , ACACA , and ELOVL2 were higher than those of CNS WHO grade 1 and 2 glioma biopsies. Hence, our results suggest that reference genes from metabolic pathways have different expression profiles depending on the stratification of gliomas and constitute a potential model for studying the development of this type of tumor and the search for molecular targets to treat gliomas.

Giardia lamblia is a single-celled eukaryotic parasite with a small genome and is considered an early divergent eukaryote. The pentose phosphate pathway (PPP) plays an essential role in the oxidative stress defense of the parasite and the production of ribose-5-phosphate. In this parasite, the glucose-6-phosphate dehydrogenase (G6PD) is fused with the 6-phosphogluconolactonase (6PGL) enzyme, generating the enzyme named G6PD::6PGL that catalyzes the first two steps of the PPP. Here, we report that the G6PD::6PGL is a bifunctional enzyme with two catalytically active sites. We performed the kinetic characterization of both domains in the fused G6PD::6PGL enzyme, as well as the individual cloned G6PD. The results suggest that the catalytic activity of G6PD and 6PGL domains in the G6PD::6PGL enzyme are more efficient than the individual proteins. Additionally, using enzymatic and mass spectrometry assays, we found that the final metabolites of the catalytic reaction of the G6PD::6PGL are 6-phosphoglucono-δ-lactone and 6-phosphogluconate. Finally, we propose the reaction mechanism in which the G6PD domain performs the catalysis, releasing 6-phosphoglucono-δ-lactone to the reaction medium. Then, this metabolite binds to the 6PGL domain catalyzing the hydrolysis reaction and generating 6-phosphogluconate. The structural difference between the G. lamblia fused enzyme G6PD::6PGL with the human G6PD indicate that the G6PD::6PGL is a potential drug target for the rational synthesis of novels anti- Giardia drugs.

Gliomas are the most aggressive neoplasms that affect the central nervous system, being glioblastoma multiforme (GBM) the most malignant. The resistance of GBM to therapies is attributed to its high rate of cell proliferation, angiogenesis, invasion, and resistance to apoptosis; thus, finding alternative therapeutic approaches is vital. In this work, the anti-proliferative, pro-apoptotic, and anti-invasive effect of the copper coordination compound Casiopeina III-La (Cas III-La) on human U373 MG cells was determined in vitro and in vivo . Our results indicate that Cas III-La exerts an anti-proliferative effect, promoting apoptotic cell death and inactivating the invasive process by generating reactive oxygen species (ROS), inactivating GSK3β, activating JNK and ERK, and promoting the nuclear accumulation of β-catenin. The inhibition of ROS generation by N-acetyl-l-cysteine not only recovered cell migration and viability, but also reduced β-catenin accumulation and JNK and ERK activation. Additionally, Cas III-La significantly reduced tumor volume, cell proliferation and mitotic indices, and increased the apoptotic index in mice xenotransplanted with U373 glioma cells. Thus, Cas III-La is a promising agent to treat GBM., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Low levels of oxygen (hypoxia) have been reported in solid tumours. This hypoxic microenvironment modulates the expression of genes linked to a more aggressive disease. However, it is unclear if the expression of drug-metabolizing enzymes as cytochromes P450 (CYPs) is affected by hypoxia in cancer. We aimed to define which cytochromes are affected by hypoxia using a liver cancer model in vitro. For this purpose, we assessed whole-genome expression microarrays of HepG2 liver cancer cell line from free repository databases, looking for gene expression hypoxia-associated profiles and selected those cytochromes with significant differences. Then, we corroborated their mRNA expression and protein levels by RT-qPCR and western blot, respectively, as well as immunofluorescence. Based on microarray analysis, we found that the expression of CYP2S1 and CYP24A1 were up-regulated with at least twice fold change compared with normoxia. The levels of mRNA and protein of CYP2S1 and CYP24A1 were increased significantly in hypoxic conditions (P < .05), and this tendency was also observed by immunofluorescence assays. Our data show that the expression of cytochromes CYP2S1 and CYP24A1 are induced in hypoxia, being the first time that CYP24A1 expression is associated with tumour hypoxia; which might have consequences in cancer progression and drug resistance. SIGNIFICANCE OF THE STUDY: Hypoxia is among the most important factors for cellular adaptation to stress. Especially in cancer, a major public health issue, hypoxia plays a substantial role in angiogenesis, metastasis and resistance to therapy. Tumoral hypoxia has been described at least in the brain, breast, cervical, liver, renal, lung, pancreatic and renal cancer. However, the understanding of how hypoxia drives cancer progression is still a major challenge. One emerging question is the role of hypoxia over the expression of drug-metabolizing enzymes, with a significant impact on drug treatment. In this context, our paper focus on the effect of hypoxia on CYPs, which is an essential group of drug-metabolizing enzymes. We show that hypoxia induces the expression of two members of the CYPs family: CYP2S1 and CYP24A1. Importantly, CYP2S1 is a major metabolizer of carcinogenic substances being relevant that hypoxia could promote this function. Interestingly, CYP24A1 limits the action of the active form of vitamin D, which is an anti-proliferative factor in cancer. Our evidence shows for the first time that hypoxia can induce CYP24A1 expression, with a potential effect on cancer progression. Our contribution clarifies a particular effect of tumoral hypoxia and the implications will be useful in the understanding of the progression of cancer, the resistance to treatment and the development of alternative therapies., (© 2020 John Wiley & Sons Ltd.)

Background and Aims: This is the first time that obesity and diabetes mellitus (DM) as protein conformational diseases (PCD) are reported in children and they are typically diagnosed too late, when β-cell damage is evident. Here we wanted to investigate the level of naturally-ocurring or real (not synthetic) oligomeric aggregates of the human islet amyloid polypeptide (hIAPP) that we called RIAO in sera of pediatric patients with obesity and diabetes. We aimed to reduce the gap between basic biomedical research, clinical practice-health decision making and to explore whether RIAO work as a potential biomarker of early β-cell damage., Materials and Methods: We performed a multicentric collaborative, cross-sectional, analytical, ambispective and blinded study; the RIAO from pretreated samples (PTS) of sera of 146 pediatric patients with obesity or DM and 16 healthy children, were isolated, measured by sound indirect ELISA with novel anti-hIAPP cytotoxic oligomers polyclonal antibody (MEX1). We carried out morphological and functional studied and cluster-clinical data driven analysis., Results: We demonstrated by western blot, Transmission Electron Microscopy and cell viability experiments that RIAO circulate in the blood and can be measured by ELISA; are elevated in serum of childhood obesity and diabetes; are neurotoxics and works as biomarkers of early β-cell failure. We explored the range of evidence-based medicine clusters that included the RIAO level, which allowed us to classify and stratify the obesity patients with high cardiometabolic risk., Conclusions: RIAO level increases as the number of complications rises; RIAOs > 3.35 μg/ml is a predictor of changes in the current indicators of β-cell damage. We proposed a novel physio-pathological pathway and shows that PCD affect not only elderly patients but also children. Here we reduced the gap between basic biomedical research, clinical practice and health decision making., Competing Interests: The authors have declared that no competing interests exist.

Glioma is the most frequent and aggressive type of brain neoplasm, being anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM), its most malignant forms. The survival rate in patients with these neoplasms is 15 months after diagnosis, despite a diversity of treatments, including surgery, radiation, chemotherapy, and immunotherapy. The resistance of GBM to various therapies is due to a highly mutated genome; these genetic changes induce a de-regulation of several signaling pathways and result in higher cell proliferation rates, angiogenesis, invasion, and a marked resistance to apoptosis; this latter trait is a hallmark of highly invasive tumor cells, such as glioma cells. Due to a defective apoptosis in gliomas, induced autophagic death can be an alternative to remove tumor cells. Paradoxically, however, autophagy in cancer can promote either a cell death or survival. Modulating the autophagic pathway as a death mechanism for cancer cells has prompted the use of both inhibitors and autophagy inducers. The autophagic process, either as a cancer suppressing or inducing mechanism in high-grade gliomas is discussed in this review, along with therapeutic approaches to inhibit or induce autophagy in pre-clinical and clinical studies, aiming to increase the efficiency of conventional treatments to remove glioma neoplastic cells.

This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis ( T. vaginalis ). The glucose-6-phosphate dehydrogenase ( g6pd ) gene from T . vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tv g6pd :: 6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP + ) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP + were performed. These results revealed that the protein becomes more stable in the presence of the NADP + . In addition, we determined the dissociation constant for the binding ( K d ) of NADP + in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to the knowledge of the function and structure of three single natural variants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and a double mutant (G6PD Mount Sinai), each localized in a different region of the three-dimensional (3D) structure. In the functional characterization of the mutants, we observed a decrease in specific activity, protein expression and purification, catalytic efficiency, and substrate affinity in comparison with wild-type (WT) G6PD. Moreover, the analysis of the effect of all mutations on the structural stability showed that its presence increases denaturation and lability with temperature and it is more sensible to trypsin digestion protease and guanidine hydrochloride compared with WT G6PD. This could be explained by accelerated degradation of the variant enzymes due to reduced stability of the protein, as is shown in patients with G6PD deficiency.

Giardia lambia is a flagellated protozoan parasite that lives in the small intestine and is the causal agent of giardiasis. It has been reported that G. lamblia exhibits glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway (PPP). Our group work demonstrated that the g6pd and 6pgl genes are present in the open frame that gives rise to the fused G6PD::6PGL protein; where the G6PD region is similar to the 3D structure of G6PD in Homo sapiens . The objective of the present work was to show the presence of the structural NADP + binding site on the fused G6PD::6PGL protein and evaluate the effect of the NADP + molecule on protein stability using biochemical and computational analysis. A protective effect was observed on the thermal inactivation, thermal stability, and trypsin digestions assays when the protein was incubated with NADP + . By molecular docking, we determined the possible structural-NADP + binding site, which is located between the Rossmann fold of G6PD and 6PGL. Finally, molecular dynamic (MD) simulation was used to test the stability of this complex; it was determined that the presence of both NADP + structural and cofactor increased the stability of the enzyme, which is in agreement with our experimental results.

Gluconacetobacter diazotrophicus PAL5 (GDI) is an endophytic bacterium with potential biotechnological applications in industry and agronomy. The recent description of its complete genome and its principal metabolic enzymes suggests that glucose metabolism is accomplished through the pentose phosphate pathway (PPP); however, the enzymes participating in this pathway have not yet been characterized in detail. The objective of the present work was to clone, purify, and biochemically and physicochemically characterize glucose-6-phosphate dehydrogenase (G6PD) from GDI. The gene was cloned and expressed as a tagged protein in E . coli to be purified by affinity chromatography. The native state of the G6PD protein in the solution was found to be a tetramer with optimal activity at pH 8.8 and a temperature between 37 and 50 °C. The apparent Km values for G6P and nicotinamide adenine dinucleotide phosphate (NADP + ) were 63 and 7.2 μM, respectively. Finally, from the amino acid sequence a three-dimensional (3D) model was obtained, which allowed the arrangement of the amino acids involved in the catalytic activity, which are conserved (RIDHYLGKE, GxGGDLT, and EKPxG) with those of other species, to be identified. This characterization of the enzyme could help to identify new environmental conditions for the knowledge of the plant-microorganism interactions and a better use of GDI in new technological applications.

Glioblastoma multiforme is the most malignant and aggressive type of brain tumor, with a mean life expectancy of less than 15 months. This is due in part to the high resistance to apoptosis and moderate resistant to autophagic cell death in glioblastoma cells, and to the poor therapeutic response to conventional therapies. Autophagic cell death represents an alternative mechanism to overcome the resistance of glioblastoma to pro-apoptosis-related therapies. Nevertheless, apoptosis induction plays a major conceptual role in several experimental studies to develop novel therapies against brain tumors. In this review, we outline the different components of the apoptotic and autophagic pathways and explore the mechanisms of resistance to these cell death pathways in glioblastoma cells. Finally, we discuss drugs with clinical and preclinical use that interfere with the mechanisms of survival, proliferation, angiogenesis, migration, invasion, and cell death of malignant cells, favoring the induction of apoptosis and autophagy, or the inhibition of the latter leading to cell death, as well as their therapeutic potential in glioma, and examine new perspectives in this promising research field.

The deficiency of glucose‑6‑phosphate dehydrogenase (G6PD) is one of the most common inborn errors of metabolism worldwide. This congenital disorder generally results from mutations that are spread throughout the entire gene of G6PD. Three single-point mutations for G6PD have been reported in the Mexican population and named Veracruz (Arg365His), G6PD Seattle (Asp282His), and G6PD Mexico DF (Thr65Ala), whose biochemical characterization have not yet been studied. For this reason, in this work we analyzed the putative role of the three mutations to uncover the functional consequences on G6PD activity. To this end, was developed a method to clone, overexpress, and purify recombinant human G6PD. The results obtained from all variants showed a loss of catalysis by 80 to 97% and had a decrease in affinity for both physiological substrates with respect to the wild type (WT) G6PD. Our results also showed that the three mutations affected three-dimensional structure and protein stability, suggesting an unstable structure with low conformational stability that affected its G6PD functionality. Finally, based on the biochemical characterization of the unclassified G6PD Mexico DF, we suggest that this variant could be grouped as a Class I variant, because biochemical data are similar with other Class I G6PDs., (Copyright © 2018. Published by Elsevier B.V.)

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway and is highly relevant in the metabolism of Giardia lamblia. Previous reports suggested that the G6PD gene is fused with the 6-phosphogluconolactonase (6PGL) gene ( 6pgl ). Therefore, in this work, we decided to characterize the fused G6PD-6PGL protein in Giardia lamblia. First, the gene of g6pd fused with the 6pgl gene ( 6gpd :: 6pgl ) was isolated from trophozoites of Giardia lamblia and the corresponding G6PD::6PGL protein was overexpressed and purified in Escherichia coli . Then, we characterized the native oligomeric state of the G6PD::6PGL protein in solution and we found a catalytic dimer with an optimum pH of 8.75. Furthermore, we determined the steady-state kinetic parameters for the G6PD domain and measured the thermal stability of the protein in both the presence and absence of guanidine hydrochloride (Gdn-HCl) and observed that the G6PD::6PGL protein showed alterations in the stability, secondary structure, and tertiary structure in the presence of Gdn-HCl. Finally, computer modeling studies revealed unique structural and functional features, which clearly established the differences between G6PD::6PGL protein from G. lamblia and the human G6PD enzyme, proving that the model can be used for the design of new drugs with antigiardiasic activity. These results broaden the perspective for future studies of the function of the protein and its effect on the metabolism of this parasite as a potential pharmacological target.

Medulloblastoma is the most common type of solid brain tumor in children. This type of embryonic tumor is highly heterogeneous and has been classified into 4 molecular subgroups based on their gene expression profiles: WNT, SHH, Group 3 (G3) and Group 4 (G4). WNT and SHH tumors exhibit the specific dysregulation of genes and pathways, whereas G3 and G4 tumors, two of the more frequent subtypes, are the least characterized. Thus, novel markers to aid in the diagnosis, prognosis and management of medulloblastoma are required. In the present study, microarray gene expression data was downloaded from the Gene Expression Omnibus database, including data from the 4 subgroups of medulloblastoma and healthy cerebellum tissue (CT). The data was utilized in an in silico analysis to characterize each subgroup at a transcriptomic level. Using Partek Genomics Suite software, the data were visualized via hierarchical clustering and principal component analysis. The differentially expressed genes were uploaded to the MetaCore portal to perform enrichment analysis using CT gene expression as baseline, with fold change thresholds of <-5 and >5 for differential expression. The data mining analysis of microarray gene expression data enabled the identification of a range of dysregulated molecules associated with each subgroup of medulloblastoma. G4 is the most heterogeneous subgroup, as no definitive pathway defines its pathogenesis; analysis of the gene expression profiles were associated with the G4α and G4β subcategories. TOX high mobility group box family member 3, synuclein α interacting protein and, potassium voltage-gated channel interacting protein 4 were identified as three novel potential markers for distinguishing the α and β subcategories of G4. These genes may be associated with medulloblastoma pathogenesis, and thus may provide a basis for researching novel targeted treatment strategies for G4 medulloblastoma.

Human islet amyloid peptide (hIAPP 1-37 ) aggregation is an early step in Diabetes Mellitus. We aimed to evaluate a family of pharmaco-chaperones to act as modulators that provide dynamic interventions and the multi-target capacity (native state, cytotoxic oligomers, protofilaments and fibrils of hIAPP 1-37 ) required to meet the treatment challenges of diabetes. We used a cross-functional approach that combines in silico and in vitro biochemical and biophysical methods to study the hIAPP 1-37 aggregation-oligomerization process as to reveal novel potential anti-diabetic drugs. The family of pharmaco-chaperones are modulators of the oligomerization and fibre formation of hIAPP 1-37 . When they interact with the amino acid in the amyloid-like steric zipper zone, they inhibit and/or delay the aggregation-oligomerization pathway by binding and stabilizing several amyloid structures of hIAPP 1-37 . Moreover, they can protect cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP 1-37 oligomers. The modulation of proteostasis by the family of pharmaco-chaperones A - F is a promising potential approach to limit the onset and progression of diabetes and its comorbidities., Competing Interests: The author declares having filled two patent applications WO2010118706 A2 and WO2014131374 A1.

Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect-in a rapid, sensitive, specific, and reproducible way-the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G . lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G . lamblia . This methodology will be a powerful tool for studying gene regulation in G. lamblia , and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia ., Competing Interests: The authors declare no conflict of interest.

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme in the pentose phosphate pathway which produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain an adequate reducing environment in the cells and is especially important in red blood cells (RBC). Given its central role in the regulation of redox state, it is understandable that mutations in the gene encoding G6PD can cause deficiency of the protein activity leading to clinical manifestations such as neonatal jaundice and acute hemolytic anemia. Recently, an extensive review has been published about variants in the g6pd gene; recognizing 186 mutations. In this work, we review the state of the art in G6PD deficiency, describing 217 mutations in the g6pd gene; we also compile information about 31 new mutations, 16 that were not recognized and 15 more that have recently been reported. In order to get a better picture of the effects of new described mutations in g6pd gene, we locate the point mutations in the solved three-dimensional structure of the human G6PD protein. We found that class I mutations have the most deleterious effects on the structure and stability of the protein., Competing Interests: The authors declare no conflict of interest.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.

The human breast adenocarcinoma cell line MDA-MB-231 has the triple-negative breast cancer (TNBC) phenotype, which is an aggressive subtype with no specific treatment. MDA-MB-231 cells express neurotensin receptor type 1 (NTSR1), which makes these cells an attractive target of therapeutic genes that are delivered by the neurotensin (NTS)-polyplex nanocarrier via the bloodstream. We addressed the relevance of this strategy for TNBC treatment using NTS-polyplex nanoparticles harboring the herpes simplex virus thymidine kinase (HSVtk) suicide gene and its complementary prodrug ganciclovir (GCV). The reporter gene encoding green fluorescent protein (GFP) was used as a control. NTS-polyplex successfully transfected both genes in cultured MDA-MB-231 cells. The transfection was demonstrated pharmacologically to be dependent on activation of NTSR1. The expression of HSVtk gene decreased cell viability by 49% (P<0.0001) and induced apoptosis in cultured MDA-MB-231 cells after complementary GCV treatment. In the MDA-MB-231 xenograft model, NTS-polyplex nanoparticles carrying either the HSVtk gene or GFP gene were injected into the tumors or via the bloodstream. Both routes of administration allowed the NTS-polyplex nanoparticles to reach and transfect tumorous cells. HSVtk expression and GCV led to apoptosis, as shown by the presence of cleaved caspase-3 and Apostain immunoreactivity, and significantly inhibited the tumor growth (55-60%) (P<0.001). At the end of the experiment, the weight of tumors transfected with the HSVtk gene was 55% less than that of control tumors (P<0.05). The intravenous transfection did not induce apoptosis in peripheral organs. Our results offer a promising gene therapy for TNBC using the NTS-polyplex nanocarrier.