Functional analysis of ldlr genetic variants identified in hypercholesterolemic patients in brazil, USING CRISPR/CAS9

BACKGROUND AND AIMS: Introduction: The familial hypercholesterolemia (FH) is one of the main causes of cardiovascular diseases, and it is mainly caused by genetic variants at the low-density lipoprotein receptor (LDLR). Although ultrasequencing technology has allowed the identification of several genetic variants, few of them was functional analyzed. The CRISPR/ Cas9 tool promotes precise genetic editing and allows the creation of experimental models, therefore contributing to the functional validation process. Aim: To use the CRISPR/Cas9 tool to perform in vitro functional analysis of LDLR variants identified in FH patients. METHODS: Two missense LDLR variants were selected within a group of variants identified in FH patients, based on in silico data, the affected protein domain and MAF. Three sgRNAs were designed for each of the variants c.551G>A and c.1118G>A, to analyze the accuracy of the sgRNAs. The sgRNAs were inserted on PX458 plasmid, cloned, purified in E. coli DH5a, and then co-transfected with the DNA template at HepG2 cells. The DNAs templates were designed to contain the selected variants. RESULTS: HepG2 cells co-transfected with PX458 constructs and DNA templates showed considerably transfection rate, being possible to visualize it at fluorescence microscopy. However, it was noted that single transfection of sgRNAs showed a higher transfection efficiency than cotransfection. CONCLUSIONS: We designed sgRNA for c.551G>A and c.1118G>A variants, being able to analyze the transfection efficiency. In further steps, we will select new sgRNAs for LDLR variants that have not been described yet, and functional analysis will be performed to determine the clinical relevance of these variants.

SNPs from BCHE and DRD3 genes associated to cocaine abuse amongst violent individuals from Sao Paulo, Brazil.

Violence and drug abuse are highly destructive phenomena found world-wide, especially in Brazil. They seem to rise proportionally to one another and possibly related. Additionally, genetics may also play a role in drug abuse. This study has focused on identifying the use of cocaine within postmortem cases arriving at the Institute of Legal Medicine of Sao Paulo as well as the presence of certain single nucleotide polymorphisms (SNPs) to better understand one's susceptibility to abuse the drug. Both hair and blood samples have been extracted through a simple methanol overnight incubation or a rapid dilute-and-shoot method, respectively. The samples were then analyzed using an UPLC-ESI-MS/MS and genotyped through RT-PCR. Statistical analyses were performed via SPSS software. From 105 postmortem cases, 53% and 51% of the cases shown to be positive for cocaine in hair and blood, respectively. Genetic wise, a significant difference has been observed for SNP rs4263329 from the BCHE gene with higher frequencies of the genotypes A/G and G/G seen in cocaine users (OR=8.91; 95%CI=1.58-50.21; p=0.01). Likewise, also SNP rs6280 from the DRD3 gene presented a significant association, with both genotypes T/C and C/C being more frequent in users (OR=4.96; 95% CI=1.07-23.02; p=0.04). To conclude, a rather high proportion of cocaine has been found, which may suggest a connotation between the use of the drug and risky/violent behaviors. Additionally, significant associations were also found within two SNPs related to cocaine use, however, due to several inherent limitations, these must be confirmed.

Avaliação da atividade antimicrobiana e antioxidante das espécies Plectranthus amboinicus (Lour.) e Mentha x villosa (Huds. ) / Assessment of antimicrobial and antioxidant activity and toxicity of the species Coleus amboinicus (Lour.) and Mentha x Villosa (Huds. )

O uso de plantas medicinais com potencial terapêutico tem motivado a realização de estudos através de ensaios experimentais que visam fornecer informações úteis e de extrema importância. Assim o presente trabalho objetivou avaliar a atividade antimicrobiana e antioxidante das espécies de Coleus amboinicus (Lour.) e Mentha x villosa (Huds). A atividade antimicrobiana foi avaliada pelo método de difusão em disco, cepas bacterianas padronizadas Gram negativas Escherichia coli (ATCC 25922 e ATCC 35218)), cepas Gram positivas Staphylococcus aureus (ATCC 25923) e S. aureus ATCC27853), assim como fungos padrões de Cryptococcus neoformans (pertencentes à coleção de microrganismos do CESMAC). Como controle positivo foi usado o imipenem e fluconazol e como controle negativo um disco com etanol absoluto. A determinação do potencial antioxidante foi realizada utilizando os métodos DPPH, FTC e determinação de compostos fenólicos. Os extratos etanólicos de C. amboinicus e M. x villosa apresentaram excelentes resultados tanto relacionado à atividade antimicrobiana quanto à antioxidante. Nossos resultados mostram a potencialidade das plantas analisadas na prevenção e no combate de doenças...

The popular use of medicinal plants with therapeutic potential has motivated experimental studies aimed at providing useful information of the utmost importance. Thus, in the present study, the antimicrobial and antioxidant activities of the species Coleus amboinicus(Lour.) and Mentha x villosa (Huds.) were assessed. Antimicrobial activity was determined by the disk diffusion method with standard Gram-negative (Escherichia coli: ATCC 25922 and ATCC 35218) and Gram-positive (Staphylococcus aureus: ATCC 25923and ATCC 27853) bacterial strains, as well as a standard strain of the fungus Cryptococcus neoformans (belonging to the CESMAC collection of microorganisms). Discs with imipenen and fluconazole were used as positive controls and a disc with absolute ethanol as a negative control. The antioxidant power was assayed by the DPPH and FTC methods and determination of total phenolic compound contents. The ethanolic extracts of C. and M. amboinicus x villosa showed excellent results with regard to both antimicrobial and antioxidant activities. Our results show the potential of these plants for preventing and fighting disease...

Network thermodynamic model of coupled transport in a multicellular tissue--the islet of Langerhans.

Network thermodynamic modeling via bond graphs was used to describe the water and cryoprotectant additive (CPA) transport in a multicellular tissue. The model is presented as a tool to understand the osmotic behavior of the islets of Langerhans when exposed to ternary aqueous solutions containing an electrolyte and a CPA. It accounts for the effects of the location of cells within the tissue and an interstitial matrix, plus differential permeabilities to water and CPA. The interstitial matrix was assumed to be a porous medium able to store the chemical species being transported. Controlled osmotic stress experiments were conducted on isolated rat pancreas islets to measure the transient volumetric response to step-wise changes in dimethyl sulfoxide, Me2SO, concentration. The model provides a tool for predicting the transient volumetric response of peripheral and interior cells and of interstitial tissue, as well as the build up of solute concentration, during addition and removal of CPAs and freezing and thawing protocols. Inverse solution methods were applied to determine values for standard cell membrane permeability parameters Lp, omega and sigma as well as for the interstitial flow conductivities Kw and Kp'.

Osmotic behavior and transport properties of human islets in a dimethyl sulfoxide solution.

The osmotic responses of isolated human islets were evaluated using a perfusion cryomicroscope device. Individual islet volumes were measured following equilibration with a series of solutions of graded solute concentration. The osmotically inactive volume for human islets was determined to be 25% from a Boyle-van't Hoff plot of these data. A network thermodynamic model was developed via the bond graph method to describe the transport of water and cryoprotective agent in pancreatic islets. The model was curve fit to transient volumetric data for the response of islets to a stepwise exposure to 1 Me2SO at temperatures of 24.0, 3.0, or -3.5 degrees C. Standard membrane transport parameters (Lp, omega, sigma) and interstitial diffusion transport properties (kappa w, kappa p) were calculated from the fitting procedure. The temperature coefficients for membrane transport properties were expressed in terms of activation energies for water (ELp) and Me2SO (E omega). Osmotic challenge experiments conducted with fresh and cryopreserved human islets indicate that frozen/thawed islets exhibit a a slight increase in transport properties.