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.

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)

Associação entre o polimorfismo VKORC1: C. 174-136G>A (rs9934438) e a dose semanal de varfarina em pacientes idosos ­ estudo piloto / Association between VKORC1: C.174-136G> A (rs9934438) polymorphism and weekly warfarin dose in elderly patients - pilot study

A varfarina é um anticoagulante utilizado na prevenção e no tratamento de doenças tromboembólicas, com janela terapêutica estreita e, elevado risco de hemorragias. Nos idosos, a principal indicação para tratamento anticoagulante oral é fibrilação atrial, cuja prevalência aumenta com a idade, atingindo 8% após os 80 anos. O exame utilizado para controle da anticoagulação oral é o tempo de protrombina, através do cálculo da razão normalizada internacional (RNI), visando o ajuste de dose e manutenção da faixa terapêutica (RNI= 2 a 3). Os idosos requerem um monitoramento efetivo devido a fatores inerentes da idade. Embora seja conhecido que os fatores genéticos influenciam na resposta terapêutica à varfarina, na rotina da maioria dos hospitais a farmacogenética ainda não é considerada no ajuste de dose. Visando estabelecer uma conduta terapêutica personalizada, o presente estudo tem como objetivo avaliar a associação entre o polimorfismo rs9934438 do gene VKORC1, que codifica a enzima vitamina K epóxido redutase, e a dose semanal de varfarina necessária para atingir o RNI adequado. Até o momento, foram incluídos 52 pacientes com idade superior a 70 anos, de ambos os sexos e em uso de varfarina. A análise do polimorfismofoi realizada através da PCR em tempo real utilizando os reagentes TaqMan™ Sample-to-SNP™ e o sistema de detecção TaqMan® SNP Genotyping Assay. As análises estatísticas foram realizadas utilizando o pacote SPSS v. 16.0 e nível de significância adotado foi de 5%. Dos 52 pacientes incluídos até o momento, 37 (71%) permaneceram na faixa terapêutica (Time in Therapeutic Range, TTR) em pelo menos 50% do tempo de anticoagulação e, para eles a dose semanal de varfarina variou de 15mg a 62,5mg. Apesar de ser um estudo piloto, a distribuição dos genótipos está em equilíbrio gênico, segundo Hardy-Weinberg (AA=19,2%, AG=34,6%, GG= 46,2%, χ2= 3,34 e p=0,067). Para os idosos com TTR ≥50%, a frequência do alelo A foi significantemente maior entre os pacientes que utilizaram doses menores de varfarina (Exato de Fisher, p=0,005). Adicionalmente, os portadores do genótipo AA necessitaram, em média, de aproximadamente metade da dose para atingir a faixa terapêutica quando comparados aos portadores do genótipo GG, 20,5 versus 36,5 mg/semana, respectivamente (ANOVA, p=0,006/ Pós-teste Bonferroni, p=0,039). Os resultados permitem concluir que portadores de alelo A são mais responsivos ao tratamento com varfarina, sugerindo que o perfil genotípico pode ser de grande valor para o direcionamento da dose terapêutica em idosos. (AU)

Open lung approach ventilation abolishes the negative effects of respiratory rate in experimental lung injury.

BACKGROUND: We recently reported that a high respiratory rate was associated with less inflammation than a low respiratory rate, but caused more pulmonary edema in a model of ARDS when an ARDSNet ventilatory strategy was used. We hypothesized that an open lung approach (OLA) strategy would neutralize the independent effects of respiratory rate on lung inflammation and edema. This hypothesis was tested in an ARDS model using two clinically relevant respiratory rates during OLA strategy. METHODS: Twelve piglets were subjected to an experimental model of ARDS and randomized into two groups: LRR (20 breaths/min) and HRR (40 breaths/min). They were mechanically ventilated for 6 h according to an OLA strategy. We assessed respiratory mechanics, hemodynamics, and extravascular lung water (EVLW). At the end of the experiment, wet/dry ratio, regional histology, and cytokines were evaluated. RESULTS: After the ARDS model was established, Cdyn,rs decreased from 21 ± 3.3 to 9.0 ± 1.8 ml/cmH2 O (P < 0.0001). After the lung recruitment maneuver, Cdyn,rs increased to the pre-injury value. During OLA ventilation, no differences in respiratory mechanics, hemodynamics, or EVLW were observed between groups. Wet/dry ratio and histological scores were not different between groups. Cytokine quantification was similar and showed a homogeneous distribution throughout the lung in both groups. CONCLUSION: Contrary to previous findings with the ARDSNet strategy, respiratory rate did not influence lung inflammatory response or pulmonary edema during OLA ventilation in experimental ARDS. This indicates that changing the respiratory rate when OLA ventilation is used will not exacerbate lung injury.

Zero expiratory pressure and low oxygen concentration promote heterogeneity of regional ventilation and lung densities.

BACKGROUND: It is not well known what is the main mechanism causing lung heterogeneity in healthy lungs under mechanical ventilation. We aimed to investigate the mechanisms causing heterogeneity of regional ventilation and parenchymal densities in healthy lungs under anesthesia and mechanical ventilation. METHODS: In a small animal model, synchrotron imaging was used to measure lung aeration and regional-specific ventilation (sV̇). Heterogeneity of ventilation was calculated as the coefficient of variation in sV̇ (CVsV̇ ). The coefficient of variation in lung densities (CVD ) was calculated for all lung tissue, and within hyperinflated, normally and poorly aerated areas. Three conditions were studied: zero end-expiratory pressure (ZEEP) and FI O2 0.21; ZEEP and FI O2 1.0; PEEP 12 cmH2 O and FI O2 1.0 (Open Lung-PEEP = OLP). RESULTS: The mean tissue density at OLP was lower than ZEEP-1.0 and ZEEP-0.21. There were larger subregions with low sV̇ and poor aeration at ZEEP-0.21 than at OLP: 12.9 ± 9.0 vs. 0.6 ± 0.4% in the non-dependent level, and 17.5 ± 8.2 vs. 0.4 ± 0.1% in the dependent one (P = 0.041). The CVsV̇ of the total imaged lung at PEEP 12 cmH2 O was significantly lower than on ZEEP, regardless of FI O2 , indicating more heterogeneity of ventilation during ZEEP (0.23 ± 0.03 vs. 0.54 ± 0.37, P = 0.049). CVD changed over the different mechanical ventilation settings (P = 0.011); predominantly, CVD increased during ZEEP. The spatial distribution of the CVD calculated for the poorly aerated density category changed with the mechanical ventilation settings, increasing in the dependent level during ZEEP. CONCLUSION: ZEEP together with low FI O2 promoted heterogeneity of ventilation and lung tissue densities, fostering a greater amount of airway closure and ventilation inhomogeneities in poorly aerated regions.

There is no cephalocaudal gradient of computed tomography densities or lung behavior in supine patients with acute respiratory distress syndrome.

BACKGROUND: There is debate whether pressure transmission within the lungs and alveolar collapse follow a hydrostatic pattern or the compression exerted by the weight of the heart and the diaphragm causes collapse localized in the areas adjacent to these structures. The second hypothesis proposes the existence of a cephalocaudal gradient in alveolar collapse. We aimed to define whether or not lung density and collapse follow a 'liquid-like' pattern with homogeneous isogravitational layers along the cephalocaudal axis in acute respiratory distress syndrome lungs. METHODS: Acute respiratory distress syndrome patients were submitted to full lung computed tomography scans at positive end-expiratory pressure (PEEP) zero (before) and 25 cmH2 O after a maximum-recruitment maneuver. PEEP was then decreased by 2 cmH2 O every 4 min, and a semi-complete scan performed at the end of each PEEP step. RESULTS: Lung densities were homogeneous within each lung layer. Lung density increased along the ventrodorsal axis toward the dorsal region (ß = 0.49, P < 0.001), while there was no increase, but rather a slight decrease, toward the diaphragm along the cephalocaudal axis and toward the heart. Higher PEEP attenuated density gradients. At PEEP 18 cmH2 O, dependent lung regions started to collapse massively, while best compliance was only reached at a lower PEEP. CONCLUSIONS: We could not detect cephalocaudal gradients in lung densities or in alveolar collapse. Likely, external pressures applied on the lung by the chest wall, organs, and effusions are transmitted throughout the lung in a hydrostatic pattern with homogeneous consequences at each isogravitational layer. A single cross-sectional image of the lung could fully represent the heterogeneous mechanical properties of dependent and non-dependent lung regions.

High respiratory rate is associated with early reduction of lung edema clearance in an experimental model of ARDS.

BACKGROUND: The independent impact of respiratory rate on ventilator-induced lung injury has not been fully elucidated. The aim of this study was to investigate the effects of two clinically relevant respiratory rates on early ventilator-induced lung injury evolution and lung edema during the protective ARDSNet strategy. We hypothesized that the use of a higher respiratory rate during a protective ARDSNet ventilation strategy increases lung inflammation and, in addition, lung edema associated to strain-induced activation of transforming growth factor beta (TGF-ß) in the lung epithelium. METHODS: Twelve healthy piglets were submitted to a two-hit lung injury model and randomized into two groups: LRR (20 breaths/min) and HRR (40 breaths/min). They were mechanically ventilated during 6 h according to the ARDSNet strategy. We assessed respiratory mechanics, hemodynamics, and extravascular lung water (EVLW). At the end of the experiment, the lungs were excised and wet/dry ratio, TGF-ß pathway markers, regional histology, and cytokines were evaluated. RESULTS: No differences in oxygenation, PaCO2 levels, systemic and pulmonary arterial pressures were observed during the study. Respiratory system compliance and mean airway pressure were lower in LRR group. A decrease in EVLW over time occurred only in the LRR group (P < 0.05). Wet/dry ratio was higher in the HRR group (P < 0.05), as well as TGF-ß pathway activation. Histological findings suggestive of inflammation and inflammatory tissue cytokines were higher in LRR. CONCLUSION: HRR was associated with more pulmonary edema and higher activation of the TGF-ß pathway. In contrast with our hypothesis, HRR was associated with less lung inflammation.

Real-time ventilation and perfusion distributions by electrical impedance tomography during one-lung ventilation with capnothorax.

BACKGROUND: Carbon dioxide insufflation into the pleural cavity, capnothorax, with one-lung ventilation (OLV) may entail respiratory and hemodynamic impairments. We investigated the online physiological effects of OLV/capnothorax by electrical impedance tomography (EIT) in a porcine model mimicking the clinical setting. METHODS: Five anesthetized, muscle-relaxed piglets were subjected to first right and then left capnothorax with an intra-pleural pressure of 19 cm H2 O. The contra-lateral lung was mechanically ventilated with a double-lumen tube at positive end-expiratory pressure 5 and subsequently 10 cm H2 O. Regional lung perfusion and ventilation were assessed by EIT. Hemodynamics, cerebral tissue oxygenation and lung gas exchange were also measured. RESULTS: During right-sided capnothorax, mixed venous oxygen saturation (P = 0.018), as well as a tissue oxygenation index (P = 0.038) decreased. There was also an increase in central venous pressure (P = 0.006), and a decrease in mean arterial pressure (P = 0.045) and cardiac output (P = 0.017). During the left-sided capnothorax, the hemodynamic impairment was less than during the right side. EIT revealed that during the first period of OLV/capnothorax, no or very minor ventilation on the right side could be seen (3 ± 3% vs. 97 ± 3%, right vs. left, P = 0.007), perfusion decreased in the non-ventilated and increased in the ventilated lung (18 ± 2% vs. 82 ± 2%, right vs. left, P = 0.03). During the second OLV/capnothorax period, a similar distribution of perfusion was seen in the animals with successful separation (84 ± 4% vs. 16 ± 4%, right vs. left). CONCLUSION: EIT detected in real-time dynamic changes in pulmonary ventilation and perfusion distributions. OLV to the left lung with right-sided capnothorax caused a decrease in cardiac output, arterial oxygenation and mixed venous saturation.

Hyperoxia affects the regional pulmonary ventilation/perfusion ratio: an electrical impedance tomography study.

BACKGROUND: The way in which hyperoxia affects pulmonary ventilation and perfusion is not fully understood. We investigated how an increase in oxygen partial pressure in healthy young volunteers affects pulmonary ventilation and perfusion measured by thoracic electrical impedance tomography (EIT). METHODS: Twelve semi-supine healthy male volunteers aged 21-36 years were studied while breathing room air and air-oxygen mixtures (FiO2) that resulted in predetermined transcutaneous oxygen partial pressures (tcPO2) of 20, 40 and 60 kPa. The magnitude of ventilation (ΔZv) and perfusion (ΔZQ)-related changes in cyclic impedance variations, were determined using an EIT prototype equipped with 32 electrodes around the thorax. Regional changes in ventral and dorsal right lung ventilation (V) and perfusion (Q) were estimated, and V/Q ratios calculated. RESULTS: There were no significant changes in ΔZv with increasing tcPO2 levels. ΔZQ in the dorsal lung increased with increasing tcPO2 (P = 0.01), whereas no such change was seen in the ventral lung. There was a simultaneous decrease in V/Q ratio in the dorsal region during hyperoxia (P = 0.04). Two subjects did not reach a tcPO2 of 60 kPa despite breathing 100% oxygen. CONCLUSION: These results indicate that breathing increased concentrations of oxygen induces pulmonary vasodilatation in the dorsal lung even at small increases in FiO2. Ventilation remains unchanged. Local mismatch of ventilation and perfusion occurs in young healthy men, and the change in ventilation/perfusion ratio can be determined non-invasively by EIT.

Capnography reflects ventilation/perfusion distribution in a model of acute lung injury.

BACKGROUND: Changes in the shape of the capnogram may reflect changes in lung physiology. We studied the effect of different ventilation/perfusion ratios (V/Q) induced by positive end-expiratory pressures (PEEP) and lung recruitment on phase III slope (S(III)) of volumetric capnograms. METHODS: Seven lung-lavaged pigs received volume control ventilation at tidal volumes of 6 ml/kg. After a lung recruitment maneuver, open-lung PEEP (OL-PEEP) was defined at 2 cmH(2)O above the PEEP at the onset of lung collapse as identified by the maximum respiratory compliance during a decremental PEEP trial. Thereafter, six distinct PEEP levels either at OL-PEEP, 4 cmH(2)O above or below this level were applied in a random order, either with or without a prior lung recruitment maneuver. Ventilation-perfusion distribution (using multiple inert gas elimination technique), hemodynamics, blood gases and volumetric capnography data were recorded at the end of each condition (minute 40). RESULTS: S (III) showed the lowest value whenever lung recruitment and OL-PEEP were jointly applied and was associated with the lowest dispersion of ventilation and perfusion (Disp(R-E)), the lowest ratio of alveolar dead space to alveolar tidal volume (VD(alv)/VT(alv)) and the lowest difference between arterial and end-tidal pCO(2) (Pa-ETCO(2)). Spearman's rank correlations between S(III) and Disp(R-E) showed a ρ=0.85 with 95% CI for ρ (Fisher's Z-transformation) of 0.74-0.91, P<0.0001. CONCLUSION: In this experimental model of lung injury, changes in the phase III slope of the capnograms were directly correlated with the degree of ventilation/perfusion dispersion.

Determinação da circunferência escrotal em touros de corte no Rio Grande do Sul / Measurement of scrotal circumference in beef bulls in Rio Grande do Sul

The variability of scrotal circumference and the effects of age and breed as well as the relationship between testicular weight (TW), scrotal circumference (SC), and live weight (BW) were investigated in bulls. Devon, Polled Hereford, and Red Angus breeds showed higher SC values than the other British, Continental, Synthetic, and Zebu studied breeds. The percentage of culled bulls because of low SC ranged from 7.01 to 24.25 percent. The increase of SC was proportional to the increase in TW and they were highly correlated (r²= 0.90). The results suggest that SC in young bulls is the best way to predict TW and, therefore, a useful tool for their selection.

A variabilidade da circunferência escrotal e os efeitos de idade e raça,assim como, as relações existentes entre o peso testicular (PT), a circunferência escrotal (CE) e o peso vivo (PV) foram investigados em touros. As raças Devon, Polled Hereford e Red Angus apresentaram valores de CE mais altos que as demais raças avaliadas, tanto inglesas, como continentais, sintéticas e zebuínas. O percentual descartado de touros devido à baixa CE variou de 7,0 a 24,2 por cento. O aumento da CE acompanhou o aumento do PT dos animais, e estes foram altamente relacionados, r² = 0,90. Os resultados sugerem que, em touros jovens, a CE é a melhor forma de predizer o PT e, portanto, uma ferramenta útil para selecionar touros.

Atelectasis observed by computerized tomography after Caesarean section.

BACKGROUND: Atelectasis after either vaginal or Caesarean delivery has not been adequately quantified. This study addresses the hypothesis that atelectasis may be worse in women who undergo Caesarean section when compared with vaginal delivery under regional anaesthesia. METHODS: Twenty healthy non-smoking women submitted to a chest computed tomography (CT) 2 h after delivery in a University Hospital, who had experienced vaginal delivery (n=10) under combined spinal-epidural analgesia or a Caesarean section (n=10) under spinal anaesthesia, were evaluated. The percentage cross-sectional area of atelectasis in dependent lung regions were measured from the CT images obtained at cross-section of the xiphoid process and the top of the diaphragm. RESULTS: The percentage cross-sectional area of atelectasis was 3.95% in the vaginal delivery group and 14.1% in the Caesarean group (P<0.001, Mann-Whitney rank sum test). CONCLUSIONS: These results suggested that pulmonary atelectasis is greater after Caesarean section delivery under spinal anaesthesia than after vaginal delivery with combined spinal-epidural analgesia.

[Nursing, a quality service? The patient answers]. / Enfermagem, um serviço de qualidade? O paciente responde.

Being quality an updated concept in several productive sectors, we have tried to study this concept in nursing service environment at the patient's point of view. For reaching this goal, a questionnaire has been made upon which 45 Hospital de Clínicas de Porto Alegre interned patients have produced their opinions. It has been shown that generally speaking, the nursing service has been satisfactory although there is some critic from some clients and improvement could be worked out.

Estudo clinico-anatomopatologico de 260 placentas examinadas pelo servico de anatomia patologica da Maternidade Nossa Senhora de Lourdes no periodo de janeiro/78 a janeiro/79. / Clinical-histopathologic study of 260 placentae examined by the Pathologic Anatomy Service of Maternidade Nossa Senhora de Lourdes from January 1978 to January 1979.

Fez-se um levantamento de 260 placentas examinadas pelo Servico de Anatomia Patologica da Maternidade Nossa Senhora de Lourdes, Goiania, no periodo de janeiro de 1978 a janeiro de 1979. Procurou-se relacionar os achados histopatologicos aos aspectos clinicos e obstetricos das pacientes, bem como com os motivos que levaram o tocologo a solicitar o exame anatomopatologico de placenta. Observou-se que 55,76% das placentas apresentaram alteracoes divididas em oito categorias: infeccoes inespecificas, sofrimento vascular, incompatibilidade sanguinea materno-fetal, placenta imatura, tumores benignos, patologias do cordao umbilical e sofrimento fetal cronico, constatando-se a existencia de relacao entre os varios tipos de alteracoes e os quadros clinico e obstetrico das pacientes, confirmando, uma vez mais, a importancia do exame anatomopatologico de placenta na investigacao e conclusao diagnostica em obstetricia.}