Cation-exchange fast protein liquid chromatography separated whole casein into β-casein A2, A1, and B, K-casein, αs1-casein, and αs2-casein fractions as well as γ-caseins and several unidentified peaks using a urea-acetate buffer at pH 5 and a NaCl gradient. The whole casein fractions eluted in the following order: breakdown products of β-casein and unidentified peaks; β-casein A2, Al, and B; additional breakdown products of β-casein and unidentified peaks; K-casein; αs1-casein; and αs2-casein. The calculated composition of the four major caseins correlated well with values obtained using anion-exchange fast protein liquid chromatography at pH 7. An acid-PAGE gel confirmed that the three β-casein peaks were variants of β-casein. Incubating herd bulk whole casein with neuraminidase (EC 3.2.1.18) removed carbohydrate from K-casein. Anion-exchange fast protein liquid chromatography separated whole casein into β-casein breakdown products, K-casein A and B, β-casein, αs2-casein, and αs1-casein peaks as well as three unidentified fractions using bis-Tris-propane-urea buffer at pH 7 and a NaCl gradient. Fractions of whole casein eluted in the following order: breakdown products of β-casein and unidentified fractions A and B; K-casein fraction; unidentified C fraction; β-casein; αs2-casein; and αs1-casein. Following treatment with neuraminidase, K-casein eluted as K-casein B and A rather than a series of peaks. Casein samples from individual cows containing known combinations of K-casein A and B confirmed that the peaks were K-casein variants. Isoelectric focusing on a PhastSystem™ separated K-casein A and B; β-casein A1, A2, A3, and B; αs1-casein Band C; β-lactoglobulin A and B; αs2-casein A; and α-lactalbumin B. Minimal preparation and a short separation time enabled many whole milk and whole casein samples to be phenotyped daily. Stepwise regression equations derived to predict samples as homozygous or heterozygous for K-casein A and B and β-casein A1, A2, and B had coefficient of determination values of .18, .58, .82, and .72 for K-casein A and B, β-casein A1, β-casein A2, and β-casein B. Although amino acid analysis can identify whether β-casein A1, A2, or B variants are present, it cannot identify whether K-casein A and B variants are present. Percentages of K-casein, β-casein, αs1-casein, and αs2-casein obtained with isoelectric focusing, cation-exchange fast protein liquid chromatography, and anion-exchange fast protein liquid chromatography compare well with published results. Isoelectric focusing and anion-exchange fast protein liquid chromatography methods separated K-casein into its A and B variants. Isoelectric focusing and cation-exchange fast protein liquid chromatography separated β-casein into its A1, A2, and B variants. Individual cows homozygous for K-casein A or B expressed the same amount of K-casein. When results from individual cows heterozygous for K-casein are combined, the two alleles are expressed equally; on an individual cow basis, however, some cows expressed more K-casein B than K-casein A. Individual cows homozygous for β-casein A1, A2 or B expressed the same amount of β-casein. When the results for individual cows heterozygous for β-casein are combined, the two β-casein alleles are expressed equally. In milk from individual cows typed β-casein A2B, slightly more B than A2 was expressed with cation-exchange fast protein liquid chromatography.

Žmonių populiacijų tyrimai atskleidžia jų fizinio pajėgumo genetinę įvairovę. Tokių ypatumų turi ir Lietuvos populiacija. Sportininkų individualaus genomo žinojimas ypač svarbus sporto teorijai, praktikai ir medicinai. Darbas skirtas svarbiausiems klausimams, susijusiems su genetinių veiksnių įtaka sportinio fizinio pajėgumo komponentams. Darbo metu buvo sukaupta Lietuvos didelio meistriškumo sportininkų imtis, kuri ištirta genetiškai ir pagal fenotipą. Sukurtoje Lietuvos didelio meistriškumo įvairių sporto šakų sportininkų DNR mėginių biobazėje sukaupta informacija apie sportininkų genotipus ir sportininkų fizinio išsivystymo bei funkcinio pajėgumo fenotipiniai duomenys. Parinkti stipriausi genai kandidatai, siejami su žmogaus fiziniu pajėgumu. Didelio meistriškumo sportininkai pirmą kartą Lietuvoje buvo tirti pagal 6 genų kandidatų DNR žymenų alelių, dažniausiai asocijuojamų su fiziniu pajėgumu, paplitimą. Tirtų genų kandidatų žymenų genotipų/alelių dažnių įvairovė išskirtose sportininkų grupėse ir bendroje Lietuvos populiacijoje turi savitumų. Visų tirtų Lietuvos sportininkų fizinio išsivystymo ir funkcinio pajėgumo rodikliai atitinka didelio meistriškumo sportininkų lygį. Fenotipinių rodiklių statistinė analizė parodė sportininkų organizmo įgimtus gebėjimus ir prisitaikymą prie fizinių krūvių. Kiekvienos išskirtos sporto šakų grupės sportininkams būdinga genotipų/alelių kombinacija. Tirtų genetinių variantų genotipai turi skirtingos įtakos vyrų bei moterų fiziniam... [toliau žr. visą tekstą] / The genetic diversity of physical capacity in the Lithuanian population has a pattern similar to that of other populations. The knowledge of the individual genomes of the athletes is especially important for sports theory, practice and medicine. This research is devoted to the issue of the effect of genetic factors on the components of sporting physical capacity. We have accumulated a sample of the Lithuanian elite athletes which was studied genetically according to a phenotype. We have created a DNA biobase of the Lithuanian elite athletes of various sporting disciplines and collected information about the genotypes and phenotypes of physical development and functional capacity of the athletes. This is the first time in Lithuania that the elite athletes were investigated according to allelic distribution of 6 candidate gene variants most associated with physical capacity. The genetic diversity of the physical capacity in the Lithuanian population has a pattern manifested by variation in the allele/genotype frequencies of the selected candidate gene markers in the Lithuanian athlete groups and general population. The indexes of physical development and functional capacity of the Lithuanian athletes correspond to the elite levels. Inherited qualities and adaptation to physical loads of the athletes can be assessed by statistical analysis of phenotypic indexes. Each group of athletes investigated had a typical genotype/allele combination. The genotypes of the gene variants... [to full text]

The genetic diversity of physical capacity in the Lithuanian population has a pattern similar to that of other populations. The knowledge of the individual genomes of the athletes is especially important for sports theory, practice and medicine. This research is devoted to the issue of the effect of genetic factors on the components of sporting physical capacity. We have accumulated a sample of the Lithuanian elite athletes which was studied genetically according to a phenotype. We have created a DNA biobase of the Lithuanian elite athletes of various sporting disciplines and collected information about the genotypes and phenotypes of physical development and functional capacity of the athletes. This is the first time in Lithuania that the elite athletes were investigated according to allelic distribution of 6 candidate gene variants most associated with physical capacity. The genetic diversity of the physical capacity in the Lithuanian population has a pattern manifested by variation in the allele/genotype frequencies of the selected candidate gene markers in the Lithuanian athlete groups and general population. The indexes of physical development and functional capacity of the Lithuanian athletes correspond to the elite levels. Inherited qualities and adaptation to physical loads of the athletes can be assessed by statistical analysis of phenotypic indexes. Each group of athletes investigated had a typical genotype/allele combination. The genotypes of the gene variants... [to full text] / Žmonių populiacijų tyrimai atskleidžia jų fizinio pajėgumo genetinę įvairovę. Tokių ypatumų turi ir Lietuvos populiacija. Sportininkų individualaus genomo žinojimas ypač svarbus sporto teorijai, praktikai ir medicinai. Darbas skirtas svarbiausiems klausimams, susijusiems su genetinių veiksnių įtaka sportinio fizinio pajėgumo komponentams. Darbo metu buvo sukaupta Lietuvos didelio meistriškumo sportininkų imtis, kuri ištirta genetiškai ir pagal fenotipą. Sukurtoje Lietuvos didelio meistriškumo įvairių sporto šakų sportininkų DNR mėginių biobazėje sukaupta informacija apie sportininkų genotipus ir sportininkų fizinio išsivystymo bei funkcinio pajėgumo fenotipiniai duomenys. Parinkti stipriausi genai kandidatai, siejami su žmogaus fiziniu pajėgumu. Didelio meistriškumo sportininkai pirmą kartą Lietuvoje buvo tirti pagal 6 genų kandidatų DNR žymenų alelių, dažniausiai asocijuojamų su fiziniu pajėgumu, paplitimą. Tirtų genų kandidatų žymenų genotipų/alelių dažnių įvairovė išskirtose sportininkų grupėse ir bendroje Lietuvos populiacijoje turi savitumų. Visų tirtų Lietuvos sportininkų fizinio išsivystymo ir funkcinio pajėgumo rodikliai atitinka didelio meistriškumo sportininkų lygį. Fenotipinių rodiklių statistinė analizė parodė sportininkų organizmo įgimtus gebėjimus ir prisitaikymą prie fizinių krūvių. Kiekvienos išskirtos sporto šakų grupės sportininkams būdinga genotipų/alelių kombinacija. Tirtų genetinių variantų genotipai turi skirtingos įtakos vyrų bei moterų fiziniam... [toliau žr. visą tekstą]

Network biology has proven to be powerful tool for representing and analyzing complex molecular networks. It has also been successfully applied to biological field helping understand various biological processes. However, our current knowledge about the dynamics of gene networks during disease progression is rather limited. On the other hand, network construction is a prerequisite of network analysis. When the number of samples is limited, state-of-art computational methods for network construction are not robust in terms of low statistical power. In addition, molecular networks have been used extensively to improve the inference accuracy of causal coding variants, but this potential has not been investigated to the same extent for noncoding variants. To address those limitations, I first developed inference of multiple differential modules (iMDM) algorithm to study network dynamics. This method is able to identify both unique and shared modules from multiple gene networks, each of which denoting a different perturbation condition. Using iMDM algorithm, I identified different types of modules to understand heart failure progression and disease dynamics. Next, I developed a computational framework to construct condition specific transcriptional regulatory network. I also developed a computational method to rank transcription factors in the transcriptional regulatory network. Applying this framework to RNA-seq data for hematopoietic stem cell development, I successfully constructed corresponding transcriptional regulatory network and identified key transcriptional factors that play important roles. Finally, I developed Annotation of Regulatory Variants using Integrated Networks (ARVIN), a network-based algorithm, to identify causal genetic variants for diseases. By applying ARVIN to various diseases, we obtained a systems understanding of the gene circuitry that is affected by all enhancer mutations in a given disease.

Background: Hypertension affects 33% of Americans while type 2 diabetes and Alzheimer's disease (AD) affect 10% of Americans, respectively. Ryanodine receptor 3 gene (RYR3) codes for the RYR which functions to release stored endoplasmic reticulum calcium ions (Ca2+) to increase intracellular Ca2+ concentration. Increasing studies demonstrate that altered levels of intracellular Ca2+ affect cardiac contraction, insulin secretion, and neurodegeneration. In this study, we investigated associations of the RYR3 genetic variants with hypertension, AD, and diabetes. Methods: Family data sets were used to explore association of RYR3 polymorphisms with risk and age at onset (AAO) of hypertension, diabetes, and AD. Results: Family-based association tests using generalized estimating equations (FBAT-GEE) showed several unique or shared disease-1 associated variants in the RYR3 gene. Three single nuclear polymorphisms (SNPs; rs2033610, rs2596164, and rs2278317) are significantly associated with risk for hypertension, diabetes, and AD. Two SNPs (rs4780174 and rs7498093) are significantly associated with AAO of the 3 diseases. Conclusions: RYR3 variants are associated with hypertension, diabetes, and AD. Replication of these results of this gene in these 3 complex traits may help to better understand the genetic basis of calcium-signaling gene, RYR3 in association with risk and AAO of these diseases.

A method has been developed for the rapid, quantitative separation of normal and abnormal phosphoglucose isoemrase allozymes from individuals heterozygous for genetic variant forms of the enzyme. The method utilizes a substrate gradient elution of the enzyme from carboxymethyl Biogel and is far superior in terms of resolution and recovery to methods based on electrophoresis and isoelectric focusing. Four different genetic variant forms of the enzyme were isolated and subjected to a systematic comparison of their physical, catalytic and stability properties. The physical and catalytic properties of the variants were similar; however, clear differences in the stability of the allozymes were apparent.

The main challenge in studying genetics has evolved from identifying variations and their impact on traits to comprehending the molecular mechanisms through which genetic variations affect human biology, including disease susceptibility. Despite having identified a vast number of variants associated with human traits through large scale genome wide association studies (GWAS) a significant portion of them still lack detailed insights into their underlying mechanisms [1]. Addressing this uncertainty requires the development of precise and scalable approaches to discover how genetic variation precisely influences phenotypes at a molecular level. In this study, we developed a pipeline to automate the annotation of structural variant feature effects. We applied this pipeline to a dataset of 33,942 variants from the ClinVar and GnomAD databases, which included both pathogenic and benign associations. To bridge the gap between genetic variation data and molecular phenotypes, I implemented Non-negative Matrix Factorization (NMF) on this large-scale dataset. This algorithm revealed 6 distinct clusters of variants with similar feature profiles. Among these groups, two exhibited a predominant presence of benign variants (accounting for 70% and 85% of the clusters), while one showed an almost equal distribution of pathogenic and benign variants. The remaining three groups were predominantly composed of pathogenic variants, comprising 68%, 83%, and 77% of the respective clusters. These findings revealed valuable insights into the underlying mechanisms contributing to pathogenicity. Further analysis of this dataset and the exploration of disease-related genes can enhance the accuracy of genetic diagnosis and therapeutic development through the direct inference of variants that are likely to affect the functioning of essential genes.

Introdução: A anemia aplástica (AA) adquirida é uma doença grave, caracterizada por pancitopenia e medula óssea hipocelular sem que haja associação com aumento de reticulina ou infiltração anormal na medula. Embora o mecanismo fisiopatológico não esteja totalmente elucidado, atribui-se a uma resposta imunomediada dos linfócitos T no ambiente medular. A trombopoetina (codificada pelo gene THPO) é um hormônio glicoproteico produzido pelo fígado e responsável pelo estímulo de crescimento de megacariócitos, desenvolvimento plaquetário e de demais linhagens e, quando disfuncional, contribui para o desenvolvimento da AA adquirida. Objetivos: Investigar a presença de variantes genéticas no THPO em amostras de sangue periférico e medula óssea de pacientes com AA adquirida (grupo caso) e de indivíduos saudáveis (grupo controle) e verificar a presença de alterações no número de plaquetas durante o seguimento dos pacientes com AA adquirida. Métodos: O gene THPO foi sequenciado em amostras de DNA de medula óssea de 92 pacientes com AA adquirida e no DNA de sangue periférico de 92 controles, cujas amostras haviam sido previamente armazenado no Laboratório de Hematologia da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP). O sequenciamento foi realizado pelo método de Sanger. Realizou-se também a associação entre a presença (ou ausência) de variantes em THPO e o número de plaquetas em 83 pacientes utilizando o teste ANOVA Para outras análises estatísticas, foram utilizados os testes t e qui-quadrado com nível de significância de 5%. Resultados: Foram encontrados três polimorfismos de nucleotídeo único (SNPs) nos pacientes com AA adquirida (rs956732, rs6141 e rs3804618). Os mesmos três SNPs foram observados nos indivíduos do grupo controle (p>0,05). Não houve associação entre o número de plaquetas e a presença de SNPs nos pacientes (p>0,05). Conclusões: Três SNPs foram encontrados em frequências alélicas semelhantes tanto no grupo de pacientes quanto nos controles, sugerindo que a trombopoetina não apresenta alterações genéticas que possam ser associadas à fisiopatologia da AA adquirida nessa coorte. / Introduction: Acquired aplastic anemia (AA) is a severe illness, characterized by pancytopenia and hypocellular bone marrow without increased reticulin or abnormal infiltration of the bone marrow. Although the physiopathological mechanism has not been completely understood, an immune-mediated T-lymphocyte response has been attributed to the bone marrow environment. Thrombopoietin (encoded by THPO), a glycoprotein hormone produced by the liver and responsible for stimulating the growth of megakaryocytes, development of platelets and other lineages that when dysfunctional, contributes to the progress of acquired AA. Objectives: To screen the THPO gene for genetic variants in bone marrow of acquired AA patients and in the peripheral blood of controls, and to verify the correlation between the THPO status and platelet counts in the patients during the treatment. Method: Sanger sequencing of the THPO gene was carried out in 92 acquired AA patients (case group) and 92 controls, in DNA samples previously stored in the Hematology Laboratory of the Ribeirão Preto School of Medicine at the University of São Paulo. The association between the THPO status and the platelet counts was performed in 83 patients through the ANOVA test. The Chi-squared test and t-test were also applied for statistical analysis with a 5% significance level. Results: Three single nucleotide polymorphisms (SNPs) were found in the AA patients (rs956732, rs6141, and rs3804618), as well as in the healthy subjects (p>0,05). No association was verified between the platelet counts and the presence of SNPs in the AA patients (p>0,05). Conclusion: Three SNPs were found in both groups, suggesting that thrombopoietin does not harbor genetic variants that could be etiological for the acquired AA in our cohort.

Introdução: A anemia aplástica (AA) adquirida é uma doença grave, caracterizada por pancitopenia e medula óssea hipocelular sem que haja associação com aumento de reticulina ou infiltração anormal na medula. Embora o mecanismo fisiopatológico não esteja totalmente elucidado, atribui-se a uma resposta imunomediada dos linfócitos T no ambiente medular. A trombopoetina (codificada pelo gene THPO) é um hormônio glicoproteico produzido pelo fígado e responsável pelo estímulo de crescimento de megacariócitos, desenvolvimento plaquetário e de demais linhagens e, quando disfuncional, contribui para o desenvolvimento da AA adquirida. Objetivos: Investigar a presença de variantes genéticas no THPO em amostras de sangue periférico e medula óssea de pacientes com AA adquirida (grupo caso) e de indivíduos saudáveis (grupo controle) e verificar a presença de alterações no número de plaquetas durante o seguimento dos pacientes com AA adquirida. Métodos: O gene THPO foi sequenciado em amostras de DNA de medula óssea de 92 pacientes com AA adquirida e no DNA de sangue periférico de 92 controles, cujas amostras haviam sido previamente armazenado no Laboratório de Hematologia da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP). O sequenciamento foi realizado pelo método de Sanger. Realizou-se também a associação entre a presença (ou ausência) de variantes em THPO e o número de plaquetas em 83 pacientes utilizando o teste ANOVA Para outras análises estatísticas, foram utilizados os testes t e qui-quadrado com nível de significância de 5%. Resultados: Foram encontrados três polimorfismos de nucleotídeo único (SNPs) nos pacientes com AA adquirida (rs956732, rs6141 e rs3804618). Os mesmos três SNPs foram observados nos indivíduos do grupo controle (p>0,05). Não houve associação entre o número de plaquetas e a presença de SNPs nos pacientes (p>0,05). Conclusões: Três SNPs foram encontrados em frequências alélicas semelhantes tanto no grupo de pacientes quanto nos controles, sugerindo que a trombopoetina não apresenta alterações genéticas que possam ser associadas à fisiopatologia da AA adquirida nessa coorte. / Introduction: Acquired aplastic anemia (AA) is a severe illness, characterized by pancytopenia and hypocellular bone marrow without increased reticulin or abnormal infiltration of the bone marrow. Although the physiopathological mechanism has not been completely understood, an immune-mediated T-lymphocyte response has been attributed to the bone marrow environment. Thrombopoietin (encoded by THPO), a glycoprotein hormone produced by the liver and responsible for stimulating the growth of megakaryocytes, development of platelets and other lineages that when dysfunctional, contributes to the progress of acquired AA. Objectives: To screen the THPO gene for genetic variants in bone marrow of acquired AA patients and in the peripheral blood of controls, and to verify the correlation between the THPO status and platelet counts in the patients during the treatment. Method: Sanger sequencing of the THPO gene was carried out in 92 acquired AA patients (case group) and 92 controls, in DNA samples previously stored in the Hematology Laboratory of the Ribeirão Preto School of Medicine at the University of São Paulo. The association between the THPO status and the platelet counts was performed in 83 patients through the ANOVA test. The Chi-squared test and t-test were also applied for statistical analysis with a 5% significance level. Results: Three single nucleotide polymorphisms (SNPs) were found in the AA patients (rs956732, rs6141, and rs3804618), as well as in the healthy subjects (p>0,05). No association was verified between the platelet counts and the presence of SNPs in the AA patients (p>0,05). Conclusion: Three SNPs were found in both groups, suggesting that thrombopoietin does not harbor genetic variants that could be etiological for the acquired AA in our cohort.

Background: The human Aldoketoreductase 1B10 gene (AKR1B10) encodes one of the enzymes belonging to the family of aldoketoreductases and may be involved in detoxification of nutrients during digestion. Further, AKR1B10 mRNA (messenger ribonucleic acid) expression was diminished in brain regions potentially involved in the regulation of eating behavior in rats which are more sensitive to cocaine and alcohol. We hypothesized that the human AKR1B10 gene may also play a role in the regulation of human eating behavior.