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.

Epigenetic Markers Associated with Schistosomiasis.

It is important to consider the use of the epigenome as source of complementary data for genome knowledge, which is suitable for the diagnosis of schistosomiasis. Usually, a laboratory diagnosis of schistosomiasis is performed by means of 1. Egg detection in the stool or urine by microscopy remains with limited sensitivity; 2. Immunological screening, in which positivity persists after treatment, and 3. Molecular appraisals prevail over the disadvantages of the currently used methods. In this sense, molecular methodologies are being developed based on epigenetic biomarkers, aiming to improve the diagnosis of the disease and clinical treatment as early as possible to prevent the occurrence of serious liver damage.

Análise funcional in silico das variantes no gene da APOB em pacientes com hipercolesterolemia familial / In silico functional analysis of variants in the APOB gene in patients with familial hypercholesterolemia

INTRODUÇÃO: O diagnóstico molecular da hipercolesterolemia familial (HF) é atribuído principalmente as variantes nos genes LDLR, LDLRAP1, APOB e PCSK9. O objetivo deste estudo foi realizar análise in silico para investigar o impacto de variantes sem descrição na literatura no gene APOB observado em pacientes com HF. MÉTODOS: Foram selecionados 141 indivíduos com diagnóstico clínico de HF. As variantes no gene da APOB foram selecionadas após sequenciamento dos éxons de 61 genes utilizando a plataforma MiSeq (Illumina). Os dados foram analisados nos programas Real Time Analysis, MiSeq Reporter, BaseSpace Sequence Hub e VariantStudio. Para a análise in silico, as sequências molde das moléculas da apoB-100 e o LDLr foram selecionadas por modelagem comparativa considerando o maior grau de identidade. As sequências proteicas foram alinhadas e os modelos 3D foram construídos utilizando os programas SEAVIEW e MODELLER v9.21. O gráfico de Ramachandran do modelo de menor energia apresenta 0,5% de outliers e análise de regiões de desordem, como principal validação. Os resultados das conformações de ancoragem foram analisados no software PyMol 2.1. Os estudos de docking molecular foram realizados para identificar o melhor complexo de conformação usando o servidor web clusPRO. RESULTADOS: Após a análise molecular dos 141 pacientes foram identificadas 7 variantes missenses sem descrição na literatura no gene APOB (c.433C>T, c.2630C>T, c.2950G>A, c.5743G>A, c.7367C>A, c.9880T>C e c.10780T>C). Os estudos de docking das variantes demonstraram uma maior afinidade entre o LDLr e a apoB-100 (c.2630C> T; Pro877Leu) em comparação com a proteína não mutada. A troca dos resíduos permaneceu como propriedade físico-química, e comparando as distâncias de ligação das proteínas não-mutadas (5Å) e mutadas (3,5Å), sugere-se uma maior afinidade do complexo (LDLr-apoB-100) para a leucina, tal fato é afirmado pela análise da região de desordens da apoB-100, onde a posição 877 está em uma região desorganizada e flexível. Esta maior afinidade poderia levar a uma menor dissociação intracelular deste complexo, resultando em uma alta taxa de degradação do LDLr pelas enzimas lisossômicas, levando ao aumento da concentração plasmática de LDLc. Para as outras variantes não houve alterações significativas. CONCLUSÃO: Os resultados sugerem que estudos in silico baseados na ferramenta de docking molecular podem melhorar o conhecimento da contribuição genética no desenvolvimento da doença HF. Além disso, a variante APOB c.2630C> T deve ser avaliada in vitropara validação do mecanismo proposto. (AU)

Complex Odontoma: A Case Report with Micro-Computed Tomography Findings.

Odontomas are the most common benign tumors of odontogenic origin. They are normally diagnosed on routine radiographs, due to the absence of symptoms. Histopathologic evaluation confirms the diagnosis especially in cases of complex odontoma, which may be confused during radiographic examination with an osteoma or other highly calcified bone lesions. The micro-CT is a new technology that enables three-dimensional analysis with better spatial resolution compared with cone beam computed tomography. Another great advantage of this technology is that the sample does not need special preparation or destruction in the sectioned area as in histopathologic evaluation. An odontoma with CBCT and microtomography images is presented in a 26-year-old man. It was first observed on panoramic radiographs and then by CBCT. The lesion and the impacted third molar were surgically excised using a modified Neumann approach. After removal, it was evaluated by histopathology and microtomography to confirm the diagnostic hypothesis. According to the results, micro-CT enabled the assessment of the sample similar to histopathology, without destruction of the sample. With further development, micro-CT could be a powerful diagnostic tool in future research.

Effects of elevated CO2 concentration and water deficit on fructan metabolism in Viguiera discolor Baker.

Elevated [CO2 ] is suggested to mitigate the negative effects of water stress in plants; however responses vary among species. Fructans are recognised as protective compounds against drought and other stresses, as well as having a role as reserve carbohydrates. We analysed the combined effects of elevated [CO2 ] and water deficit on fructan metabolism in the Cerrado species Viguiera discolor Baker. Plants were cultivated for 18 days in open-top chambers (OTC) under ambient (∼380 ppm), and high (∼760 ppm) [CO2 ]. In each OTC, plants were submitted to three treatments: (i) daily watering (control), (ii) withholding water (WS) for 18 days and (iii) re-watering (RW) on day 11. Analyses were performed at time 0 and days 5, 8, 11, 15 and 18. High [CO2 ] increased photosynthesis in control plants and increased water use efficiency in WS plants. The decline in soil water content was more distinct in WS 760 (WS under 760 ppm), although the leaf and tuberous root water status was similar to WS 380 plants (WS under 380 ppm). Regarding fructan active enzymes, 1-SST activity decreased in WS plants in both CO2 concentrations, a result consistent with the decline in photosynthesis and, consequently, in substrate availability. Under WS and both [CO2 ] treatments, 1-FFT and 1-FEH seemed to act in combination to generate osmotically active compounds and thus overcome water deficit. The proportion of hexoses to sucrose, 1-kestose and nystose (SKN) was higher in WS plants. In WS 760, this increase was higher than in WS 380, and was not accompanied by decreases in SKN at the beginning of the treatment, as observed in WS 380 plants. These results suggest that the higher [CO2 ] in the atmosphere contributed to maintain, for a longer period, the pool of hexoses and of low DP fructans, favouring the maintenance of the water status and plant survival under drought.

Efeitos de diferentes fontes de energia sobre taxa ovulatória, fertilidade e sobrevivência embrionária em marrãs cíclicas / Effects of different energy sources on ovulation rate, pregnancy rate and embryo survival in cyclic gilts

Cinqüenta e quatro marrãs cíclicas, uniformizadas quanto à linhagem, família, ganho de peso, espessura de toucinho, peso, precocidade sexual, número de cios e escore clínico, foram alocadas em dois grupos experimentais com dietas isocalóricas, isoprotéicas e isolisínicas. Duas fontes de energia foram testadas: amido de milho (T1) e óleo de soja (T2). Sincronizou-se o segundo estro com allyl-trenbolone, para inseminação no terceiro estro. Foi realizada cateterização não cirúrgica em 21 marrãs, submetidas a dois ciclos de coleta para dosagem de glicose e insulina, aos 14 e 21 dias do ciclo. Todas as marrãs foram abatidas aos 28,6 dias de gestação média, para análises biométricas do trato reprodutivo. Marrãs do T1 apresentaram maior taxa ovulatória em relação às do T2 (16,52 vs 14,70, P<0,01). Não houve diferença entre os tratamentos nas taxas de prenhez e sobrevivência embrionária. É possível alterar a eficiência reprodutiva por intermédio de manipulação dietética, mesmo em marrãs em estado anabólico. O uso do amido de milho na fase pré-cobertua melhorou a eficiência reprodutiva dos animais avaliados.

Fifty four cyclic gilts were randomly selected and uniformized according to genetic background, litter of origin, weight gain, backfat, number of cycles and clinical score. Gilts were alloted to one of two groups fed isocaloric, isoproteic and isolysinic diets. Two energy sources were tested: corn starch (T1) and soybean oil (T2). Second estrus was synchronized with oral allyl-trenbolone, so that insemination was carried out at third estrus. Indweeling catheters were implanted by non-surgical technic in 21 gilts, which were submitted to consecutive blood samplings for glicose and insulin determination. Timing of ovulation was estimated by means of ultrasonography. All gilts were slaughtered at an average gestation lenght of 28.6 days. Starch-fed gilts (T1) showed higher ovulation rates than T2 gilts (16.52 vs 14.70; P<0.01). There was no effect of treatments on pregnancy rate and embryo survival. Results indicate it is possible to manipulate reproductive efficiency through diet even in anabolic experimental models like cyclic gilts. Feeding starch as main energy source during pre-mating flushing phase improved reproductive efficiency of cyclic gilts.

No relationship between HTLV-1 infection and filariasis--Serological study on patients with filariasis in Recife, Brazil.

The seroprevalence of antibodies against human T-cell leukemia virus was determined by ELISA in 68 patients with filarial infestation living in an endemic area. The total seropositivity was 2.9% and the HTLV-1-positive cases were detected in 2 microfilaremic patients 12 and 40 years old. This value is very close to that obtained for healthy individuals in the same region and age groups. This result suggests that there is no relationship between filariasis and HTLV-1 infection as previously proposed.