Design and Characterization of pMyc/pMax Peptide-Coupled Gold Nanosystems for Targeting Myc in Prostate Cancer Cell Lines.

Myc and Max are essential proteins in the development of prostate cancer. They act by dimerizing and binding to E-box sequences. Disrupting the Myc:Max heterodimer interaction or its binding to E-box sequences to interrupt gene transcription represent promising strategies for treating cancer. We designed novel pMyc and pMax peptides from reference sequences, and we evaluated their ability to bind specifically to E-box sequences using an electrophoretic mobility shift assay (EMSA). Then, we assembled nanosystems (NSs) by coupling pMyc and pMax peptides to AuNPs, and determined peptide conjugation using UV-Vis spectroscopy. After that, we characterized the NS to obtain the nanoparticle's size, hydrodynamic diameter, and zeta potential. Finally, we evaluated hemocompatibility and cytotoxic effects in three different prostate adenocarcinoma cell lines (LNCaP, PC-3, and DU145) and a non-cancerous cell line (Vero CCL-81). EMSA results suggests peptide-nucleic acid interactions between the pMyc:pMax dimer and the E-box. The hemolysis test showed little hemolytic activity for the NS at the concentrations (5, 0.5, and 0.05 ng/µL) we evaluated. Cell viability assays showed NS cytotoxicity. Overall, results suggest that the NS with pMyc and pMax peptides might be suitable for further research regarding Myc-driven prostate adenocarcinomas.

A Full Set of In Vitro Assays in Chitosan/Tween 80 Microspheres Loaded with Magnetite Nanoparticles.

Microspheres have been proposed for different medical applications, such as the delivery of therapeutic proteins. The first step, before evaluating the functionality of a protein delivery system, is to evaluate their biological safety. In this work, we developed chitosan/Tween 80 microspheres loaded with magnetite nanoparticles and evaluated cell damage. The formation and physical-chemical properties of the microspheres were determined by FT-IR, Raman, thermogravimetric analysis (TGA), energy-dispersive X-ray spectroscopy (EDS), dynamic light scattering (DLS), and SEM. Cell damage was evaluated by a full set of in vitro assays using a non-cancerous cell line, human erythrocytes, and human lymphocytes. At the same time, to know if these microspheres can load proteins over their surface, bovine serum albumin (BSA) immobilization was measured. Results showed 7 nm magnetite nanoparticles loaded into chitosan/Tween 80 microspheres with average sizes of 1.431 µm. At concentrations from 1 to 100 µg/mL, there was no evidence of changes in mitochondrial metabolism, cell morphology, membrane rupture, cell cycle, nor sister chromatid exchange formation. For each microgram of microspheres 1.8 µg of BSA was immobilized. The result provides the fundamental understanding of the in vitro biological behavior, and safety, of developed microspheres. Additionally, this set of assays can be helpful for researchers to evaluate different nano and microparticles.

Associations of nicotidamide-N-methyltransferase, FTO, and IRX3 genetic variants with body mass index and resting energy expenditure in Mexican subjects.

The enzyme nicotidamide-N-methyltransferase (NNMT) regulates adipose tissue energy expenditure through increasing nicotinamide adenosine dinucleotide (NAD+) content. NNMT methylates nicotinamide to N1-methylnicotidamide (MNA-1) using S-adenosyl methionine. The rs694539 NNMT polymorphism is associated with non-alcoholic steatohepatitis, and rs1941404 is associated with hyperlipidemia. The rs1421085 FTO is related to poor eating behaviors, and rs3751723 IRX3 is associated with obesity. To investigate the association of rs694539 and rs1941404 NNMT, rs140285 FTO and rs3751723 IRX3 polymorphisms with MNA-1 concentrations, resting energy expenditure (REE) and BMI, we included clinically healthy Mexican subjects 30 to 50 years old, 100 subjects (35 men/65 women) with BMI > 30 kg/m2 and 100 subjects (32 men/68 women) with BMI < 25 kg/m2. Glucose, lipid profile, insulin, leptin, acylated ghrelin, and MNA-1 (LC-MS) were quantified. Resting energy expenditure (REE) was estimated using indirect calorimetry with a Fitmate instrument. Genotyping was performed using PCR-RFLP, and allelic discrimination was examined using TaqMan probes. MNA-1 concentrations and REE were significantly higher in obese subjects. Subjects with the rs694539AA NNMT genotype (recessive model) had lower weight, BMI, and REE. BMI showed an association with HDL-C, triglycerides, MNA-1, acetylated ghrelin, leptin, insulin concentrations, HOMA-IR, REE, and rs1421085. Subjects with the TC or CC genotypes of rs1421085 FTO showed 6 kg and 2 units of BMI more than did those with the TT wild type. The CG of the rs1421085 and rs3751723 haplotypes was associated with BMI. These findings showed that BMI was strongly associated with REE, rs1421085 FTO and the CG rs1421085 FTO and rs3751723 IRX3 haplotypes. We used the GMDR approach in obesity phenotype to show the interaction of four SNPs and metabolic variables.

Whole exome sequencing identifies multiple novel candidate genes in familial gastroschisis.

BACKGROUND: Genetic association studies for gastroschisis have highlighted several candidate variants. However, genetic basis in gastroschisis from noninvestigated heritable factors could provide new insights into the human biology for this birth defect. We aim to identify novel gastroschisis susceptibility variants by employing whole exome sequencing (WES) in a Mexican family with recurrence of gastroschisis. METHODS: We employed WES in two affected half-sisters with gastroschisis, mother, and father of the proband. Additionally, functional bioinformatics analysis was based on SVS-PhoRank and Ensembl-Variant Effect Predictor. The latter assessed the potentially deleterious effects (high, moderate, low, or modifier impact) from exome variants based on SIFT, PolyPhen, dbNSFP, Condel, LoFtool, MaxEntScan, and BLOSUM62 algorithms. The analysis was based on the Human Genome annotation, GRCh37/hg19. Candidate genes were prioritized and manually curated based on significant phenotypic relevance (SVS-PhoRank) and functional properties (Ensembl-Variant Effect Predictor). Functional enrichment analysis was performed using ToppGene Suite, including a manual curation of significant Gene Ontology (GO) biological processes from functional similarity analysis of candidate genes. RESULTS: No single gene-disrupting variant was identified. Instead, 428 heterozygous variations were identified for which SPATA17, PDE4DIP, CFAP65, ALPP, ZNF717, OR4C3, MAP2K3, TLR8, and UBE2NL were predicted as high impact in both cases, mother, and father of the proband. PLOD1, COL6A3, FGFRL1, HHIP, SGCD, RAPGEF1, PKD1, ZFHX3, BCAS3, EVPL, CEACAM5, and KLK14 were segregated among both cases and mother. Multiple interacting background modifiers may regulate gastroschisis susceptibility. These candidate genes highlight a role for development of blood vessel, circulatory system, muscle structure, epithelium, and epidermis, regulation of cell junction assembly, biological/cell adhesion, detection/response to endogenous stimulus, regulation of cytokine biosynthetic process, response to growth factor, postreplication repair/protein K63-linked ubiquitination, protein-containing complex assembly, and regulation of transcription DNA-templated. CONCLUSION: Considering the likely gene-disrupting prediction results and similar biological pattern of mechanisms, we propose a joint "multifactorial model" in gastroschisis pathogenesis.

Bioinformatic Analysis of Gene Variants from Gastroschisis Recurrence Identifies Multiple Novel Pathogenetic Pathways: Implication for the Closure of the Ventral Body Wall.

We investigated whether likely pathogenic variants co-segregating with gastroschisis through a family-based approach using bioinformatic analyses were implicated in body wall closure. Gene Ontology (GO)/Panther functional enrichment and protein-protein interaction analysis by String identified several biological networks of highly connected genes in UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, AOX1, NOTCH1, HIST1H2BB, RPS3, THBS1, ADCY9, and FGFR4. SVS-PhoRank identified a dominant model in OR10G4 (also as heterozygous de novo), ITIH3, PLEKHG4B, SLC9A3, ITGA2, AOX1, and ALPP, including a recessive model in UGT1A7, UGT1A6, PER2, PTPRD, and UGT1A3. A heterozygous compound model was observed in CDYL, KDM5A, RASGRP1, MYBPC2, PDE4DIP, F5, OBSCN, and UGT1A. These genes were implicated in pathogenetic pathways involving the following GO related categories: xenobiotic, regulation of metabolic process, regulation of cell adhesion, regulation of gene expression, inflammatory response, regulation of vascular development, keratinization, left-right symmetry, epigenetic, ubiquitination, and regulation of protein synthesis. Multiple background modifiers interacting with disease-relevant pathways may regulate gastroschisis susceptibility. Based in our findings and considering the plausibility of the biological pattern of mechanisms and gene network modeling, we suggest that the gastroschisis developmental process may be the consequence of several well-orchestrated biological and molecular mechanisms which could be interacting with gastroschisis predispositions within the first ten weeks of development.

A clinical-pathogenetic approach on associated anomalies and chromosomal defects supports novel candidate critical regions and genes for gastroschisis.

BACKGROUND: Gastroschisis has been assumed to have a low rate of syndromic and primary malformations. We aimed to systematically review and explore the frequency and type of malformations/chromosomal syndromes and to identify significant biological/genetic roles in gastroschisis. METHODS: Population-based, gastroschisis-associated anomalies/chromosomal defects published 1950-2018 (PubMed/MEDLINE) were independently searched by two reviewers. Associated anomalies/chromosomal defects and selected clinical characteristics were subdivided and pooled by race, system/region, isolated, and associated cases (descriptive analysis and chi-square test were performed). Critical regions/genes from representative chromosomal syndromes including an enrichment analysis using Gene Ontology Consortium/Panther Classification System databases were explored. Fisher's exact test with False Discovery Rate multiple test correction was performed. RESULTS: Sixty-eight articles and 18525 cases as a base were identified (prevalence of 17.9 and 3% for associated anomalies/chromosomal defects, respectively). There were 3596 associated anomalies, prevailing those cardiovascular (23.3%) and digestive (20.3%). Co-occurring anomalies were associated with male, female, American Indian, Caucasian, prenatally diagnosed, chromosomal defects, and mortality (P < 0.00001). Gene clusters on 21q22.11 and 21q22.3 (KRTAP), 18q21.33 (SERPINB), 18q22.1 (CDH7, CDH19), 13q12.3 (FLT1), 13q22.1 (KLF5), 13q22.3 (EDNRB), and 13q34 (COL4A1, COL4A2, F7, F10) were significantly related to biological processes: blood pressure regulation and/or vessel integrity, angiogenesis, coagulation, cell-cell and/or cell-matrix adhesion, dermis integrity, and wound healing (P < 0.05). CONCLUSIONS: Our findings suggest that gastroschisis may result from the interaction of several chromosomal regions in an additive manner as a pool of candidate genes were identified from critical regions supporting a role for vascular disruption, thrombosis, and mesodermal deficiency in the pathogenesis of gastroschisis.

Uridine 5'-diphospho-glucronosyltrasferase: Its role in pharmacogenomics and human disease.

Biotransformation is an enzyme-catalyzed process in which the body converts endogenous compounds, xenobiotics and toxic substances into harmless or easily excreted metabolites. The biotransformation reactions are classified as phase I and II reactions. Uridine 5'-diphospho (UDP)-glucuronosyltransferases (UGTs) are a superfamily of phase II enzymes which have roles in the conjugation of xenobiotics or endogenous compounds, including drugs and bilirubin, with glucuronic acid to make them easier to excrete. The method the human body uses to achieve glucuronidation may be affected by a large interindividual variation due to changes in the sequences of the genes encoding these enzymes. In the last five years, the study of the genetic variants of the UGTs at a molecular level has become important due to its association with several diseases and the ability to predict adverse events due to drug metabolism. In the present review, the structure and the prominent genetic variants of the UGT1A subfamily and their metabolic and clinical implications are described.

Nanoparticles for death­induced gene therapy in cancer (Review).

Due to the high toxicity and side effects of the use of traditional chemotherapy in cancer, scientists are working on the development of alternative therapeutic technologies. An example of this is the use of death­induced gene therapy. This therapy consists of the killing of tumor cells via transfection with plasmid DNA (pDNA) that contains a gene which produces a protein that results in the apoptosis of cancerous cells. The cell death is caused by the direct activation of apoptosis (apoptosis­induced gene therapy) or by the protein toxic effects (toxin­induced gene therapy). The introduction of pDNA into the tumor cells has been a challenge for the development of this therapy. The most recent implementation of gene vectors is the use of polymeric or inorganic nanoparticles, which have biological and physicochemical properties (shape, size, surface charge, water interaction and biodegradation rate) that allow them to carry the pDNA into the tumor cell. Furthermore, nanoparticles may be functionalized with specific molecules for the recognition of molecular markers on the surface of tumor cells. The binding between the nanoparticle and the tumor cell induces specific endocytosis, avoiding toxicity in healthy cells. Currently, there are no clinical protocols approved for the use of nanoparticles in death­induced gene therapy. There are still various challenges in the design of the perfect transfection vector, however nanoparticles have been demonstrated to be a suitable candidate. This review describes the role of nanoparticles used for pDNA transfection and key aspects for their use in death­induced gene therapy.

A pharmacogenetic pilot study reveals MTHFR, DRD3, and MDR1 polymorphisms as biomarker candidates for slow atorvastatin metabolizers.

BACKGROUND: The genetic variation underlying atorvastatin (ATV) pharmacokinetics was evaluated in a Mexican population. Aims of this study were: 1) to reveal the frequency of 87 polymorphisms in 36 genes related to drug metabolism in healthy Mexican volunteers, 2) to evaluate the impact of these polymorphisms on ATV pharmacokinetics, 3) to classify the ATV metabolic phenotypes of healthy volunteers, and 4) to investigate a possible association between genotypes and metabolizer phenotypes. METHODS: A pharmacokinetic study of ATV (single 80-mg dose) was conducted in 60 healthy male volunteers. ATV plasma concentrations were measured by high-performance liquid chromatography mass spectrometry. Pharmacokinetic parameters were calculated by the non-compartmental method. The polymorphisms were determined with the PHARMAchip® microarray and the TaqMan® probes genotyping assay. RESULTS: Three metabolic phenotypes were found in our population: slow, normal, and rapid. Six gene polymorphisms were found to have a significant effect on ATV pharmacokinetics: MTHFR (rs1801133), DRD3 (rs6280), GSTM3 (rs1799735), TNFα (rs1800629), MDR1 (rs1045642), and SLCO1B1 (rs4149056). The combination of MTHFR, DRD3 and MDR1 polymorphisms associated with a slow ATV metabolizer phenotype. CONCLUSION: Further studies using a genetic preselection method and a larger population are needed to confirm these polymorphisms as predictive biomarkers for ATV slow metabolizers. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry: ACTRN12614000851662, date registered: August 8, 2014.

An investigation into the MMP1 gene promoter region polymorphism--1607 2G with recessive dystrophic epidermolysis bullosa disease severity in northeastern Mexican patients.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe genetic skin blistering disorder caused by mutations in the gene COL7A1 encoding type VII collagen. Most of the patients' clinical severity depends in part on the nature and location of the mutations, ranging from the mild form described as RDEBother-generalized (RDEB-O) to the more aggressive phenotype described as RDEBsevere-generalized (RDEB-sev gen). However, interfamilial and interindividual differences in subjects with identical COL7A1 mutations suggest the presence of modifier elements, which may influence severity. There is a single nucleotide polymorphism (SNP) at the promoter of the MMP1 gene-encoding matrix metalloproteinase type 1, which has been studied as a genetic disease modifier in different patient cohorts with different findings. METHODS: We tested the SNP in 30 patients with RDEB and 130 controls whose four grandparents were born in northeastern Mexico. Patients were clinically classified as RDEB-sev gen and RDEB-O by three dermatologists. The SNPStats, RXC, and SPSS software were used to perform statistical testing. RESULTS: The allele frequencies for 2G were 0.607, 0.562, and 0.642 for RDEB-O, RDEB-sev gen, and the control group, respectively. When the genotype frequencies were compared, there was no significant difference between RDEB-sev gen (OR = 0.38, CI 95% 0.12-1.21), RDEB-O (OR = 1.03, CI 95% 0.21-4.96), and the control group. CONCLUSION: We found no significant association in relation to the severity of the study subjects and the SNP at the promoter of the MMP1 gene.

The tumor necrosis factor α (-308 A/G) polymorphism is associated with cystic fibrosis in Mexican patients.

UNLABELLED: Environmental and genetic factors may modify or contribute to the phenotypic differences observed in multigenic and monogenic diseases, such as cystic fibrosis (CF). An analysis of modifier genes can be helpful for estimating patient prognosis and directing preventive care. The aim of this study is to determine the association between seven genetic variants of four modifier genes and CF by comparing their corresponding allelic and genotypic frequencies in CF patients (n = 81) and control subjects (n = 104). Genetic variants of MBL2 exon 1 (A, B, C and D), the IL-8 promoter (-251 A/T), the TNFα promoter (TNF1/TNF2), and SERPINA1 (PI*Z and PI*S) were tested in CF patients and control subjects from northeastern Mexico by PCR-RFLP. RESULTS: The TNF2 allele (P = 0.012, OR 3.43, 95% CI 1.25-9.38) was significantly associated with CF under the dominant and additive models but was not associated with CF under the recessive model. This association remained statistically significant after adjusting for multiple tests using the Bonferroni correction (P = 0.0482). The other tested variants and genotypes did not show any association with the disease. CONCLUSION: An analysis of seven genetic variants of four modifier genes showed that one variant, the TNF2 allele, appears to be significantly associated with CF in Mexican patients.

Nanoparticles vs cancer: a multifuncional tool.

Nanotechnology is revolutionizing the development of different branches of science. The investigation of nanomaterials in the battle against cancer is not an exception. The main goal of this contribution is to bring an overview about the types of organic and inorganic nanomaterials that are under investigation for its applications in different aspects of cancer therapy: detection, diagnosis, contrast agents, controlled drug delivery, and hyperthermia. This review also includes fundamental aspects such as basic properties and synthesis of nanoparticles, with an emphasis on the use of selfassembed systems such as micelles, microemulsions, nanoemulsions, and liposomes.

Genetic structure of Mexican Mestizos with type 2 diabetes mellitus based on three STR loci.

BACKGROUND: The aims of this population genetics study were: 1) to ascertain whether Mexicans with type 2 diabetes mellitus (DM) were genetically homogeneous and 2) to compare the genetic structure of this selected population with the previously reported data of four random populations (Nuevo León, Hispanics, Chihuahua, and Central Region of Mexico). METHODS: A sample of 103 unrelated individuals with DM and whose 4 grandparents were born in five zones of Mexico was interviewed in 32 Medical Units in the Mexican Institute of Social Security (IMSS). The non-coding STRs D16S539, D7S820, and D13S317 were analyzed. RESULTS: Genotype distribution was in agreement with Hardy-Weinberg expectations for all three markers. Allele frequencies were found to be similar between the selected population and the four random populations. Gene diversity analysis suggested that more than 99.57% of the total gene diversity could be attributed to variation between individuals within the population and 0.43% between the populations. CONCLUSIONS: According to the present and previous studies using molecular and non-molecular nuclear DNA markers not associated with any disease, the Mexican Mestizo population is found to be genetically homogeneous and therefore the genetic causes of DM are less heterogeneous, thereby simplifying genetic epidemiological studies as has been found in a previous study with the same design in Mexican women with breast cancer.