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We report the cloning and characterization of two melanocortin receptors (MCRs) from the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis shows that these shark receptors are orthologues of the MC3R and MC5R subtypes, sharing 65% and 70% overall amino acid identity with the human counterparts, respectively. The SacMC3R was expressed and pharmacologically characterized in HEK293 cells. The radioligand binding results show that this receptor has high affinity for adrenocorticotropic hormone (ACTH)-derived peptides while it has comparable affinity forα- andβ-melanocyte stimulating hormone (MSH), and slightly lower affinity forγ-MSH when compared with the human orthologue. ACTH(1–24)has high potency in a second-messenger cAMP assay whileα- andγ-MSH had slightly lower potency in cells expressing the SacMC3R. We used receptor–enhanced green fluorescence protein (EGFP) fusion to show the presence of SacMC3R in plasma membrane of Chinese hamster ovary and HEK293 cells but the SacMC5R was retained in intracellular compartments of these cells hindering pharmacological characterization. The anatomical distribution of the receptors were determined using reverse transcription PCR. The results showed that the SacMC3R is expressed in the hypothalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum of the spiny dogfish while the SacMC5R was found only in the same central regions. This report describes the first molecular characterization of a MC3R in fish. The study indicates that many of the important elements of the MC system existed before radiation of gnathostomes, early in vertebrate evolution, at least 450 million years ago. [ABSTRACT FROM AUTHOR]

Variation in skin color is the major host risk factor for melanoma and other forms of skin cancer. Individuals with red hair show an increased ratio of phaeomelanin to eumelanin in both hair and skin. This ratio is regulated by the melanocortin (MC) 1 receptor. There are several common point mutations in the human MC1 receptor that are overrepresented in North European red-heads, and in individuals with pale skin. In order to determine the functional significance of these mutations, we expressed the Asp84Glu, Val92Met, Arg163Gln, and Asp294His variants of the human MC1 receptors in eukaryotic cells and determined their ability to bind α-melanocyte stimulating hormone (MSH) peptides and increase intracellular cAMP. The mutants Asp84Glu and Asp294His showed a much lower response to α-MSH in cAMP and a slightly impaired ability to bind α-MSH, and the Val92Met mutant bound α-MSH with 100-fold lower affinity as compared with the wild-type. The Arg163Gln variant, widely found in some Asian populations, reached normal level of cAMP response but had just slightly lower potency for α-MSH in binding and second messenger studies. The results provide important pharmacological characterization of common MC1 receptor variants in various world populations. [ABSTRACT FROM AUTHOR]

Cites the formation of a human melanocortin 4 receptor molecular model. Introduction of His residues into the receptor protein to form metal ion binding sites; Insertion of micromolar affinity binding sites for zinc between transmembrane regions where the metal ion can activate the peptide; Impact of the activation on the binding of the G-protein coupled receptor; Prediction of the helicase orientation.

We report the cloning, expression, pharmacological characterization and tissue distribution of a melanocortin (MC) receptor gene in a shark, the spiny dogfish (Squalus acanthias ) (Sac). Phylogenetic analysis showed that this receptor is an ortholog of the MC4 subtype, sharing 71% overall amino acid identity with the human (Hsa) MC4 receptor. When expressed and characterized by radioligand binding assay for the natural MSH (melanocyte-stimulating hormone) peptides α-, β-, and γ-MSH, the SacMC4 receptor showed pharmacological properties very similar to the HsaMC4 receptor. Stimulation of SacMC4 receptor transfected cells with α-MSH caused a dose-dependent increase in intracellular cAMP levels. The SacMC4 receptor has Ala in position 59 where all other cloned MC receptors have Glu. We confirmed that this was not due to individual polymorphism and subsequently mutated the residue ‘back’ to Glu but the mutation did not affect the pharmacological properties of the receptor. SacMC4 receptor mRNA was detected by RT-PCR in the optic tectum, hypothalamus, brain stem, telencephalon and olfactory bulb but not in cerebellum or in peripheral tissues. This study describes the first characterization of an MC receptor in a cartilaginous fish, the most distant MC receptor gene cloned to date. Conservation of gene structure, pharmacological properties and tissue distribution suggests that this receptor may have similar roles in sharks as in mammals and that these were established more than 450 million years ago. [ABSTRACT FROM AUTHOR]

Background: There is limited data on the genetic characteristics of patients with familial hypercholesterolemia (FH) in Latvia. We aim to describe monogenic variants in patients from the Latvian Registry of FH (LRFH). Methods: Whole genome sequencing with 30× coverage was performed in unrelated index cases from the LRFH and the Genome Database of Latvian Population. LDLR, APOB, PCSK9, LDLRAP1, ABCG5, ABCG8, LIPA, LPA, CYP27A1, and APOE genes were analyzed. Only variants annotated as pathogenic (P) or likely pathogenic (LP) using the FH Variant Curation Expert Panel guidelines for LDLR and adaptations for APOB and PCSK9 were reported. Results: Among 163 patients, the mean highest documented LDL-cholesterol level was 7.47 ± 1.60 mmol/L, and 79.1% of patients had LDL-cholesterol ≥6.50 mmol/L. A total of 15 P/LP variants were found in 34 patients (diagnostic yield: 20.9%): 14 in the LDLR gene and 1 in the APOB gene. Additionally, 24, 54, and 13 VUS were detected in LDLR, APOB, and PCSK9, respectively. No P/LP variants were identified in the other tested genes. Conclusions: Despite the high clinical likelihood of FH, confirmed P/LP variants were detected in only 20.9% of patients in the Latvian cohort when assessed with genome-wide next generation sequencing. [ABSTRACT FROM AUTHOR]

Somatic genetic alterations in pituitary neuroendocrine tumors (PitNET) tissues have been identified in several studies, but detection of overlapping somatic PitNET candidate genes is rare. We sequenced and by employing multiple data analysis methods studied the exomes of 15 PitNET patients to improve discovery of novel factors involved in PitNET development. PitNET patients were recruited to the study before PitNET removal surgery. For each patient, two samples for DNA extraction were acquired: venous blood and PitNET tissue. Exome sequencing was performed using Illumina NexSeq 500 sequencer and data analyzed using two separate workflows and variant calling algorithms: GATK and Strelka2. A combination of two data analysis pipelines discovered 144 PitNET specific somatic variants (mean = 9.6, range 0–19 per PitNET) of which all were SNVs. Also, we detected previously known GNAS PitNET mutation and identified somatic variants in 11 genes, which have contained somatic variants in previous WES and WGS studies of PitNETs. Noteworthy, this is the third study detecting somatic variants in gene RYR1 in the exomes of PitNETs. In conclusion, we have identified two novel PitNET candidate genes (AC002519.6 and AHNAK) with recurrent somatic variants in our PitNET cohort and found 13 genes overlapping from previous PitNET studies that contain somatic variants. Our study demonstrated that the use of multiple sequencing data analysis pipelines can provide more accurate identification of somatic variants in PitNETs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Biobanks are an invaluable source of samples for large-scale metabolomics studies. • Representative serum and plasma samples were obtained from the BBMRI-LPC consortium. • Supervised multivariate analysis shows strong per-biobank discrimination. • The discrimination originates from metabolites affected by different pre-analytics. • Precautionary measures to monitor pre-examination flaws should be implemented. The development of metabolomics in clinical applications has been limited by the lack of validation in large multicenter studies. Large population cohorts and their biobanks are a valuable resource for acquiring insights into molecular disease mechanisms. Nevertheless, most of their collections are not tailored for metabolomics and have been created without specific attention to the pre-analytical requirements for high-quality metabolome assessment. Thus, comparing samples obtained by different pre-analytical procedures remains a major challenge. Here, 1H NMR-based analyses are used to demonstrate how human serum and plasma samples collected with different operating procedures within several large European cohort studies from the Biobanking and Biomolecular Resources Infrastructure – Large Prospective Cohorts (BBMRI-LPC) consortium can be easily revealed by supervised multivariate statistical analyses at the initial stages of the process, to avoid biases in the downstream analysis. The inter-biobank differences are discussed in terms of deviations from the validated CEN/TS 16945:2016 / ISO 23118:2021 norms. It clearly emerges that biobanks must adhere to the evidence-based guidelines in order to support wider-scale application of metabolomics in biomedicine, and that NMR spectroscopy is informative in comparing the quality of different sample sources in multi cohort/center studies. [ABSTRACT FROM AUTHOR]

Background: Metformin has been used for the treatment of type 2 diabetes for over 60 years; however, its mechanism of pharmacological action is not fully clear. Different hypotheses exist regarding metformin distribution and redistribution mechanisms between plasma and erythrocytes/red blood cells (RBCs). Objective: We aimed to test the hypothesis that the metformin distribution between plasma and RBC occurs via concentration difference-driven passive transport and estimated transport rate coefficient values based on metformin concentration time series in plasma and RBCs from in vivo studies. Methods: An ordinary differential equation (ODE) system with two compartments was used to describe diffusion-based passive transport between plasma and RBCs. Metformin concentration time series in plasma and RBCs of 35 individuals were used for metformin transport parametrization. Plasma concentration has been approximated by biexponential decline. Results: A single passive transport coefficient, k = 0.044 ± 0.014 (h–1), can be applied, describing the uptake and release transport rate versus the linear equation v = k × (Mpl − MRBC), where Mpl is the metformin concentration in plasma and MRBC is the metformin concentration in RBCs. Conclusions: Our research suggests that passive transport can explain metformin distribution dynamics between plasma and RBCs because transport speed is proportional to the metformin concentration difference and independent of the transport direction. Concentration difference-driven passive transport can explain the mechanism of faster metformin distribution to RBCs the first few hours after administration, and faster release and domination of the redistribution transport rate after metformin concentration in plasma becomes smaller than in RBCs. [ABSTRACT FROM AUTHOR]

Abstract: The recently deorphanized niacin receptor subtypes NIACR1 (GPR109A) and NIACR2 (GPR109B) play an essential role in the regulation of metabolic processes and immune reactions. Both receptors belong to the G-protein-coupled receptor (GPCR) family, whose members have traditionally been treated as monomeric entities, but now appear to exist and function as both homodimers and heterodimers. In this study, a close physical interaction is shown between the highly homologous niacin receptor subtypes, NIACR1 and NIACR2, using bioluminescence resonance energy transfer (BRET2) in living cells. The extent of homo- and hetero-dimerization of the niacin receptors did not vary after activation of the receptors with selective agonists, indicating that the dimerization state of NIACR1 and NIACR2 is not regulated by ligand binding. Moreover, detection of niacin receptor dimers in both plasma membrane- and endoplasmic reticulum-enriched fractions suggests that they are formed early in the biosynthetic pathway. Taken together, these results demonstrate that niacin receptor dimerization is a constitutive process occurring early during biosynthesis. [Copyright &y& Elsevier]

Both in mammalian tissues and in transfected cells, only low levels ofa1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of thea1D-adrenoceptor is responsible for the inefficient surface expression of the receptor.In the present study, we created gene constructs for six N-terminally truncated variants of the humana1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density ofa1D-adrenoceptors, observed by[3H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes with receptor translocation to the plasma membrane.The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-typea1D-adrenoceptor increased the density of receptor binding sites 10-fold in Neuro2A and COS-7 cells. This indicates that, after the addition of a signal peptide, the long N-terminal tail of thea1D-adrenoceptor does not interfere with proper translocation of the receptor to the plasma membrane. This, in turn, indicates that the N-terminal tail of the wild-typea1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor.Neither the wild-typea1D-adrenoceptor (for which the expression level of[3H]-prazosin binding sites is low) nor the truncateda1D-adrenoceptor variant (for which the expression level of[3H]-prazosin binding sites is high) showed any constitutive activity in stimulating inositol phosphate accumulation. This indicates that the low expression level of[3H]-prazosin binding sites, after transfection with the wild-typea1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.British Journal of Pharmacology (2005) 144, 651-659. doi:10.1038/sj.bjp.0706087 [ABSTRACT FROM AUTHOR]

Metformin is the primary drug for type 2 diabetes treatment and a promising candidate for other disease treatment. It has significant deviations between individuals in therapy efficiency and pharmacokinetics, leading to the administration of an unnecessary overdose or an insufficient dose. There is a lack of data regarding the concentration-time profiles in various human tissues that limits the understanding of pharmacokinetics and hinders the development of precision therapies for individual patients. The physiologically based pharmacokinetic (PBPK) model developed in this study is based on humans' known physiological parameters (blood flow, tissue volume, and others). The missing tissue-specific pharmacokinetics parameters are estimated by developing a PBPK model of metformin in mice where the concentration time series in various tissues have been measured. Some parameters are adapted from human intestine cell culture experiments. The resulting PBPK model for metformin in humans includes 21 tissues and body fluids compartments and can simulate metformin concentration in the stomach, small intestine, liver, kidney, heart, skeletal muscle adipose, and brain depending on the body weight, dose, and administration regimen. Simulations for humans with a bodyweight of 70kg have been analyzed for doses in the range of 500-1500mg. Most tissues have a half-life (T1/2) similar to plasma (3.7h) except for the liver and intestine with shorter T1/2 and muscle, kidney, and red blood cells that have longer T1/2. The highest maximal concentrations (Cmax) turned out to be in the intestine (absorption process) and kidney (excretion process), followed by the liver. The developed metformin PBPK model for mice does not have a compartment for red blood cells and consists of 20 compartments. The developed human model can be personalized by adapting measurable values (tissue volumes, blood flow) and measuring metformin concentration time-course in blood and urine after a single dose of metformin. The personalized model can be used as a decision support tool for precision therapy development for individuals. [ABSTRACT FROM AUTHOR]

Background: Type 2 diabetes complications cause a serious emotional and economical burden to patients and healthcare systems globally. Management of both acute and chronic complications of diabetes, which dramatically impair the quality of patients' life, is still an unsolved issue in diabetes care, suggesting a need for early identification of individuals with high risk for developing diabetes complications. Methods: We performed a genome-wide association study in 601 type 2 diabetes patients after stratifying them according to the presence or absence of four types of diabetes complications: diabetic neuropathy, diabetic nephropathy, macrovascular complications, and ophthalmic complications. Results: The analysis revealed ten novel associations showing genome-wide significance, including rs1132787 (GYPA, OR = 2.71; 95% CI = 2.02–3.64) and diabetic neuropathy, rs2477088 (PDE4DIP, OR = 2.50; 95% CI = 1.87–3.34), rs4852954 (NAT8, OR = 2.27; 95% CI = 2.71–3.01), rs6032 (F5, OR = 2.12; 95% CI = 1.63–2.77), rs6935464 (RPS6KA2, OR = 2.25; 95% CI = 6.69–3.01) and macrovascular complications, rs3095447 (CCDC146, OR = 2.18; 95% CI = 1.66–2.87) and ophthalmic complications. By applying the targeted approach of previously reported susceptibility loci we managed to replicate three associations: MAPK14 (rs3761980, rs80028505) and diabetic neuropathy, APOL1 (rs136161) and diabetic nephropathy. Conclusions: Together these results provide further evidence for the implication of genetic factors in the development of type 2 diabetes complications and highlight several potential key loci, able to modify the risk of developing these conditions. Moreover, the candidate variant approach proves a strong and consistent effect for multiple variants across different populations. [ABSTRACT FROM AUTHOR]

Background: The study was conducted to investigate the effects of metformin treatment on the human gut microbiome's taxonomic and functional profile in the Latvian population, and to evaluate the correlation of these changes with therapeutic efficacy and tolerance. Methods: In this longitudinal observational study, stool samples for shotgun metagenomic sequencing-based analysis were collected in two cohorts. The first cohort included 35 healthy nondiabetic individuals (metformin dose 2x850mg/day) at three time-points during metformin administration. The second cohort was composed of 50 newly-diagnosed type 2 diabetes patients (metformin dose–determined by an endocrinologist) at two concordant times. Patients were defined as Responders if their HbA1c levels during three months of metformin therapy had decreased by ≥12.6 mmol/mol (1%), while in Non-responders HbA1c were decreased by <12.6 mmol/mol (1%). Results: Metformin reduced the alpha diversity of microbiota in healthy controls (p = 0.02) but not in T2D patients. At the species level, reduction in the abundance of Clostridium bartlettii and Barnesiella intestinihominis, as well as an increase in the abundance of Parabacteroides distasonis and Oscillibacter unclassified overlapped between both study groups. A large number of group-specific changes in taxonomic and functional profiles was observed. We identified an increased abundance of Prevotella copri (FDR = 0.01) in the Non-Responders subgroup, and enrichment of Enterococcus faecium, Lactococcus lactis, Odoribacter, and Dialister at baseline in the Responders group. Various taxonomic units were associated with the observed incidence of side effects in both cohorts. Conclusions: Metformin effects are different in T2D patients and healthy individuals. Therapy induced changes in the composition of gut microbiome revealed possible mediators of observed short-term therapeutic effects. The baseline composition of the gut microbiome may influence metformin therapy efficacy and tolerance in T2D patients and could be used as a powerful prediction tool. [ABSTRACT FROM AUTHOR]

Metformin, a biguanide agent, is the first-line treatment for type 2 diabetes mellitus due to its glucose-lowering effect. Despite its wide application in the treatment of multiple health conditions, the glycemic response to metformin is highly variable, emphasizing the need for reliable biomarkers. We chose the RNA-Seq-based comparative transcriptomics approach to evaluate the systemic effect of metformin and highlight potential predictive biomarkers of metformin response in drug-naïve volunteers with type 2 diabetes in vivo. The longitudinal blood-derived transcriptome analysis revealed metformin-induced differential expression of novel and previously described genes involved in cholesterol homeostasis (SLC46A1 and LRP1), cancer development (CYP1B1, STAB1, CCR2, TMEM176B), and immune responses (CD14, CD163) after administration of metformin for three months. We demonstrate for the first time a transcriptome-based molecular discrimination between metformin responders (delta HbA1c ≥ 1% or 12.6 mmol/mol) and non-responders (delta HbA1c < 1% or 12.6 mmol/mol), that is determined by expression levels of 56 genes, explaining 13.9% of the variance in the therapeutic efficacy of the drug. Moreover, we found a significant upregulation of IRS2 gene (log2FC 0.89) in responders compared to non-responders before the use of metformin. Finally, we provide evidence for the mitochondrial respiratory complex I as one of the factors related to the high variability of the therapeutic response to metformin in patients with type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Background: Thiopurine methyltransferase (TPMT) plays a significant role in the metabolism of thiopurines, and, for patients with inflammatory bowel disease (IBD), it is useful to perform TPMT genotyping prior to azathioprine (AZA) treatment. In this study, we determined TPMT gene polymorphisms in a cohort of IBD patients in Latvia. Methods: DNA samples were obtained from 244 IBD patients, and qPCR was performed for detection of rs1800462, rs1800460, and rs1142345 single-nucleotide polymorphisms (SNPs). Three common, non-functional TPMT alleles (TPMT*2, *3B, and *3C) were identified (women, 51%; men, 49%). TPMT*2, *3A, *3B, and *3C allelic variants detected using qPCR were consistent with restriction fragment length polymorphism (RFLP) data. Results: Among patients, 78% had ulcerative colitis and 22% had Crohn's disease, with 93.9% of the former carrying a wild-type homozygous TPMT*1/*1 genotype and 6.1% carrying heterozygous genotypes. The most frequent polymorphisms were TPMT*1/*3A (5.3%: two variants: TPMT*3B and TPMT*3C), TPMT*1/*3C (0.4%), and TPMT*1/*2 (0.4%). None of the patients carried a TPMT*3B polymorphism and no patients were homozygous for any mutation. Conclusion: This is the first study to identify TPMT gene polymorphisms in adult IBD patients in Latvia. The results indicate that the frequency of common TPMT alleles is similar to that of other European populations. [ABSTRACT FROM AUTHOR]

Background: Pituitary adenomas (PA) have an increased potential for relapse in one to 5 years after resection. In this study, we investigated the genetic differences in genomic DNA of primary and rapidly recurrent tumours in the same patient to explain the causality mechanisms of PA recurrence. Case presentation: The patient was a 69-year-old female with non-functional pituitary macroadenoma with extension into the left cavernous sinus (Knosp grade 2) who underwent craniotomy and partial resection in August 2010. Two years later, the patient had prolonged tumour growth with an essential suprasellar extension (Knosp grade 2), and a second craniotomy with partial tumour resection was performed in September 2012. In both tumours, the KI-67 level was below 1.5%. Exome sequencing via semiconductor sequencing of patient germline DNA and somatic DNA from both tumours was performed. Tmap alignment and Platypus variant calling were performed followed by variant filtering and manual review with IGV software. We observed an increased load of missense variants in the recurrent PA tumour when compared to the original tumour. The number of detected variants increased from ten to 26 and potential clonal expansion of four variants was observed. Additionally, targeted SNP analysis revealed five rare missense SNPs with a potential impact on the function of the encoded proteins. Conclusions: In this case study, an SNP located in HRAS is the most likely candidate inducing rapid PA progression. The relapsed PA tumour had a higher variation load and fast tumour recurrence in this patient could be caused by clonal expansion of the leftover tumour tissue. [ABSTRACT FROM AUTHOR]

Background: Thiopurine methyltransferase (TPMT) plays a significant role in the metabolism of thiopurines, and, for patients with inflammatory bowel disease (IBD), it is useful to perform TPMT genotyping prior to azathioprine (AZA) treatment. In this study, we determined TPMT gene polymorphisms in a cohort of IBD patients in Latvia. Methods: DNA samples were obtained from 244 IBD patients, and qPCR was performed for detection of rs1800462, rs1800460, and rs1142345 single-nucleotide polymorphisms (SNPs). Three common, non-functional TPMT alleles (TPMT*2, *3B, and *3C) were identified (women, 51%; men, 49%). TPMT*2, *3A, *3B, and *3C allelic variants detected using qPCR were consistent with restriction fragment length polymorphism (RFLP) data. Results: Among patients, 78% had ulcerative colitis and 22% had Crohn's disease, with 93.9% of the former carrying a wild-type homozygous TPMT*1/*1 genotype and 6.1% carrying heterozygous genotypes. The most frequent polymorphisms were TPMT*1/*3A (5.3%: two variants: TPMT*3B and TPMT*3C), TPMT*1/*3C (0.4%), and TPMT*1/*2 (0.4%). None of the patients carried a TPMT*3B polymorphism and no patients were homozygous for any mutation. Conclusion: This is the first study to identify TPMT gene polymorphisms in adult IBD patients in Latvia. The results indicate that the frequency of common TPMT alleles is similar to that of other European populations. [ABSTRACT FROM AUTHOR]

Metformin is a commonly used antihyperglycaemic agent for the treatment of type 2 diabetes mellitus. Nevertheless, the exact mechanisms of action, underlying the various therapeutic effects of metformin, remain elusive. The goal of this study was to evaluate the alterations in longitudinal whole-blood transcriptome profiles of healthy individuals after a one-week metformin intervention in order to identify the novel molecular targets and further prompt the discovery of predictive biomarkers of metformin response. Next generation sequencing-based transcriptome analysis revealed metformin-induced differential expression of genes involved in intestinal immune network for IgA production and cytokine-cytokine receptor interaction pathways. Significantly elevated faecal sIgA levels during administration of metformin, and its correlation with the expression of genes associated with immune response (CXCR4, HLA-DQA1, MAP3K14, TNFRSF21, CCL4, ACVR1B, PF4, EPOR, CXCL8) supports a novel hypothesis of strong association between metformin and intestinal immune system, and for the first time provide evidence for altered RNA expression as a contributing mechanism of metformin's action. In addition to universal effects, 4 clusters of functionally related genes with a subject-specific differential expression were distinguished, including genes relevant to insulin production (HNF1B, HNF1A, HNF4A, GCK, INS, NEUROD1, PAX4, PDX1, ABCC8, KCNJ11) and cholesterol homeostasis (APOB, LDLR, PCSK9). This inter-individual variation of the metformin effect on the transcriptional regulation goes in line with well-known variability of the therapeutic response to the drug. [ABSTRACT FROM AUTHOR]

Background: Metformin is a widely used first-line drug for treatment of type 2 diabetes. Despite its advantages, metformin has variable therapeutic effects, contraindications, and side effects. Here, for the very first time, we investigate the short-term effect of metformin on the composition of healthy human gut microbiota. Methods: We used an exploratory longitudinal study design in which the first sample from an individual was the control for further samples. Eighteen healthy individuals were treated with metformin (2 × 850 mg) for 7 days. Stool samples were collected at three time points: prior to administration, 24 hours and 7 days after metformin administration. Taxonomic composition of the gut microbiome was analyzed by massive parallel sequencing of 16S rRNA gene (V3 region). Results: There was a significant reduction of inner diversity of gut microbiota observed already 24 hours after metformin administration. We observed an association between the severity of gastrointestinal side effects and the increase in relative abundance of common gut opportunistic pathogen Escherichia-Shigella spp. One week long treatment with metformin was associated with a significant decrease in the families Peptostreptococcaceae and Clostridiaceae_1 and four genera within these families. Conclusions: Our results are in line with previous findings on the capability of metformin to influence gut microbiota. However, for the first time we provide evidence that metformin has an immediate effect on the gut microbiome in humans. It is likely that this effect results from the increase in abundance of opportunistic pathogens and further triggers the occurrence of side effects associated with the observed dysbiosis. An additional randomized controlled trial would be required in order to reach definitive conclusions, as this is an exploratory study without a placebo control arm. Our findings may be further used to create approaches that improve the tolerability of metformin. [ABSTRACT FROM AUTHOR]

Novel series of compounds consisting of 2-amidocyclohex-1-ene carboxylate and phenyl parts which are connected by enyne (compounds 2a – f ), but-1-yne (compounds 4a – j ), and phenylethylene (compounds 5a – f ) linkers as HCA2 full agonists were designed and their functional activity using cAMP assay and binding affinity using radioligand ( 3 H-niacin) binding assay were evaluated. In general, compounds of all three series exhibit similar HCA2 binding and activation profile. However, the activity is strongly dependent on the substituent at the aromatic part of the structure. Among the structures evaluated, the highest affinity and potency in all series were exhibited by compounds containing 4-hydroxy and/or 2-chloro or 2-fluoro substituents. The most active compounds in the enyne and but-1-yne series in the cAMP assay are 2-fluoro,4-hydroxy and 2-chloro,4-hydroxy phenyl derivatives 2f , 4f , and 4g showing potency similar to the previously described 4-hydroxy-biphenyl analogue 5c . [ABSTRACT FROM AUTHOR]

Objective(s): High variability in clinical response to metformin is often observed in type 2 diabetes (T2D) patients, and it highlights the need for identification of genetic components affecting the efficiency of metformin therapy. Aim of this observational study is to evaluate the role of tagSNPs (tagging single nucleotide polymorphisms) from genomic regions coding for six metformin transporter genes with respect to the short-term efficiency. Design: 102 tagSNPs in 6 genes coding for metformin transporters were genotyped in the group of 102 T2D patients treated with metformin for 3 months. Methods: Most significant hits were analyzed in the group of 131 T2D patients from Slovakia. Pharmacokinetic study in 25 healthy nondiabetic volunteers was conducted to investigate the effects of identified polymorphisms. Results: In the discovery group of 102 patients, minor alleles of rs3119309, rs7757336 and rs2481030 were significantly nominally associated with metformin inefficiency (P = 1.9 × 10-6 to 8.1 × 10-6). Effects of rs2481030 and rs7757336 did not replicate in the group of 131 T2DM patients from Slovakia alone, whereas rs7757336 was significantly associated with a reduced metformin response in combined group. In pharmacokinetic study, group of individuals harboring risk alleles of rs7757336 and rs2481030 displayed significantly reduced AUC∞ of metformin in plasma. Conclusions: For the first time, we have identified an association between the lack of metformin response and SNPs rs3119309 and rs7757336 located in the 5' flanking region of the genes coding for Organic cation transporter 2 and rs2481030 located in the 5' flanking region of Organic cation transporter 3 that was supported by the results of a pharmacokinetic study on 25 healthy volunteers. [ABSTRACT FROM AUTHOR]

Objective: Although pituitary adenomas (PAs) affect a significant proportion of the population, only a fraction have the potential to become clinically relevant during an individual's lifetime, causing hormonal imbalance or complications due to mass effect. The overwhelming majority of cases are sporadic and without a clear familial history, and the genotype--phenotype correlation in PA patients is poorly understood. Our aim was to investigate the involvement of genes known for their role in familial cases on drug response and tumor suppression in the development and pathology of PAs in a patient group from Latvia. Design: The study included 143 cases and 354 controls, we investigated the role of single-nucleotide polymorphisms (SNPs) in seven genes (SSTR2, SSTR5, DRD2, MEN1, AIP, GNAS, and PRKAR1A) associated with pituitary tumor occurrence, phenotype, and clinical symptoms. Methods: Genotyping of 96 tag and nonsynonymous SNPs was performed in the genomic regions of interest. Results: We discovered a significant association (OR = 17.8, CI 0.95 = 2.18-145.5, P = 0.0002) between a rare MEN1 mutation (rs2959656) and clinically active adenoma in our patients. Additionally, rs7131056 at DRD2 was associated with a higher occurrence of extrasellar growth in patients with prolactinoma and somatotropinoma (OR = 2.79, CI 0.95 = 1.58-4.95, P = 0.0004). Conclusions: rs2959656, a nonsynonymous variant in MEN1, is associated with the development of clinically active PA. Furthermore, rs7131056 in DRD2 contributes to either faster growth of the adenoma or reduced symptomatic presentation, allowing PAs to become larger before detection. [ABSTRACT FROM AUTHOR]

Pituitary adenomas are one of the most common endocrine and intracranial neoplasms. Although they are theoretically monoclonal in origin, several studies have shown that they contain different multipotent cell types that are thought to play an important role in tumor initiation, maintenance, and recurrence after therapy. In the present study, we isolated and characterized cell populations from seven pituitary somatotroph, nonhormonal, and lactotroph adenomas. The obtained cells showed characteristics of multipotent mesenchymal stromal cells as observed by cell morphology, cell surface marker CD90, CD105, CD44, and vimentin expression, as well as differentiation to osteogenic and adipogenic lineages. They are capable of growth and passaging under standard laboratory cell culture conditions and do not manifest any hormonal cell characteristics. Multipotent mesenchymal stromal cells are present in pituitary adenomas regardless of their clinical manifestation and show no considerable expression of somatostatin 1–5 and dopamine 2 receptors. Most likely obtained cells are a part of tissue-supportive cells in pituitary adenoma microenvironment. [ABSTRACT FROM AUTHOR]

Background: The mechanisms by which genetic variants, such as single nucleotide polymorphisms (SNPs), identified in genome-wide association studies act to influence body mass remain unknown for most of these SNPs, which continue to puzzle the scientific community. Recent evidence points to the epigenetic and chromatin states of the genome as having important roles. Methods: We genotyped 355 healthy young individuals for 52 known obesity-associated SNPs and obtained DNA methylation levels in their blood using the Illumina 450 K BeadChip. Associations between alleles and methylation at proximal cytosine residues were tested using a linear model adjusted for age, sex, weight category, and a proxy for blood cell type counts. For replication in other tissues, we used two open-access datasets (skin fibroblasts, n = 62; four brain regions, n =121-133) and an additional dataset in subcutaneous and visceral fat (n =149). Results: We found that alleles at 28 of these obesity-associated SNPs associate with methylation levels at 107 proximal CpG sites. Out of 107 CpG sites, 38 are located in gene promoters, including genes strongly implicated in obesity (MIR148A, BDNF, PTPMT1, NR1H3, MGAT1, SCGB3A1, HOXC12, PMAIP1, PSIP1, RPS10-NUDT3, RPS10, SKOR1, MAP2K5, SIX5, AGRN, IMMP1L, ELP4, ITIH4, SEMA3G, POMC, ADCY3, SSPN, LGR4, TUFM, MIR4721, SULT1A1, SULT1A2, APOBR, CLN3, SPNS1, SH2B1, ATXN2L,a n d IL27). Interestingly, the associated SNPs are in known eQTLs for some of these genes. We also found that the 107 CpGs are enriched in enhancers in peripheral blood mononuclear cells. Finally, our results indicate that some of these associations are not blood-specific as we successfully replicated four associations in skin fibroblasts. Conclusions: Our results strongly suggest that many obesity-associated SNPs are associated with proximal gene regulation, which was reflected by association of obesity risk allele genotypes with differential DNA methylation. This study highlights the importance of DNA methylation and other chromatin marks as a way to understand the molecular basis of genetic variants associated with human diseases and traits. [ABSTRACT FROM AUTHOR]

Background: The mechanisms by which genetic variants, such as single nucleotide polymorphisms (SNPs), identified in genome-wide association studies act to influence body mass remain unknown for most of these SNPs, which continue to puzzle the scientific community. Recent evidence points to the epigenetic and chromatin states of the genome as having important roles. Methods: We genotyped 355 healthy young individuals for 52 known obesity-associated SNPs and obtained DNA methylation levels in their blood using the Illumina 450 K BeadChip. Associations between alleles and methylation at proximal cytosine residues were tested using a linear model adjusted for age, sex, weight category, and a proxy for blood cell type counts. For replication in other tissues, we used two open-access datasets (skin fibroblasts, n = 62; four brain regions, n= 121-133) and an additional dataset in subcutaneous and visceral fat (n = 149). Results: We found that alleles at 28 of these obesity-associated SNPs associate with methylation levels at 107 proximal CpG sites. Out of 107 CpG sites, 38 are located in gene promoters, including genes strongly implicated in obesity (MIR148A, BDNF, PTPMT1, NR1H3, MGAT1, SCGB3A1, HOXC12, PMAIP1, PSIP1, RPS10-NUDT3, RPS10, SKOR1, MAP2K5, SIX5, AGRN, IMMP1L, ELP4, ITIH4, SEMA3G, POMC, ADCY3, SSPN, LGR4, TUFM, MIR4721, SULT1A1, SULT1A2, APOBR, CLN3, SPNS1, SH2B1, ATXN2L, and IL27). Interestingly, the associated SNPs are in known eQTLs for some of these genes. We also found that the 107 CpGs are enriched in enhancers in peripheral blood mononuclear cells. Finally, our results indicate that some of these associations are not blood-specific as we successfully replicated four associations in skin fibroblasts. Conclusions: Our results strongly suggest that many obesity-associated SNPs are associated with proximal gene regulation, which was reflected by association of obesity risk allele genotypes with differential DNA methylation. This study highlights the importance of DNA methylation and other chromatin marks as a way to understand the molecular basis of genetic variants associated with human diseases and traits. [ABSTRACT FROM AUTHOR]

Background: Familial hypercholesterolemia (FH) is one of the commonest monogenic disorders, predominantly inherited as an autosomal dominant trait. When untreated, it results in early coronary heart disease. The vast majority of FH remains undiagnosed in Latvia. The identification and early treatment of affected individuals remain a challenge worldwide. Most cases of FH are caused by mutations in one of four genes, APOB, LDLR, PCSK9, or LDLRAP1. The spectrum of disease-causing variants is very diverse and the variation detection panels usually used in its diagnosis cover only a minority of the disease-causing gene variants. However, DNA-based tests may provide an FH diagnosis for FH patients with no physical symptoms and with no known family history of the disease. Here, we evaluate the use of targeted next-generation sequencing (NGS) to identify cases of FH in a cohort of patients with coronary artery disease (CAD) and individuals with abnormal low-density lipoprotein-cholesterol (LDL–C) levels. Methods: We used targeted amplification of the coding regions of LDLR, APOB, PCSK9, and LDLRAP1, followed by NGS, in 42 CAD patients (LDL–C, 4.1–7.2 mmol/L) and 50 individuals from a population-based cohort (LDL–C, 5.1–9.7 mmol/L). Results: In total, 22 synonymous and 31 nonsynonymous variants, eight variants in close proximity (10 bp) to intron-exon boundaries, and 50 other variants were found. We identified four pathogenic mutations (p.(Arg3527Gln) in APOB, and p.(Gly20Arg), p.(Arg350*), and c.1706–10G > A in LDLR) in seven patients (7.6 %). Three possible pathogenic variants were also found in four patients. Conclusion: NGS-based methods can be used to detect FH in high-risk individuals when they do not meet the defined clinical criteria. [ABSTRACT FROM AUTHOR]

The proteolysis of the pro-opiomelanocortin precursor results in the formation of melanocortins (MCs), a group of peptides that share the conserved -H-F-R-W- sequence, which acts as a pharmacophore for five subtypes of MC receptors (MCRs). MC type 2 receptor (MC2R; also known as ACTHR) is the most specialized of all the MCRs. It is predominantly expressed in the adrenal cortex and specifically binds ACTH. Unlike other MCRs, it requires melanocortin receptor accessory protein 1 (MRAP) for formation of active receptor and for its transport to the cell membrane. The molecular mechanisms underlying this specificity remain poorly understood. In this study, we used directed mutagenesis to investigate the role of various short MC2R sequence segments in receptor membrane trafficking and specific activation upon stimulation with ligands. The strategy of the study was to replace two to five amino acid residues within one MC2R segment with the corresponding residues of MC4R. In total, 20 recombinant receptors C-terminally fused to enhanced green fluorescent protein were generated and their membrane trafficking efficiencies and cAMP response upon stimulation with α-MSH and ACTH(1-24) were estimated during their stand-alone expression and coexpression withMRAP.Our results indicate that both themotif that determines the ligandrecognition specificity and the intracellular retention signal are formed by a specific extracellular structure, which is supported by the correct alignment of the transmembrane domains. Our results also indicate that the aromatic-residue-rich segmentof the secondextracellular loopis involved in the effects mediated by the second ACTH pharmacophore (-K-K-R-R-). [ABSTRACT FROM AUTHOR]

The combination of the obesity epidemic and an aging population presents growing challenges for the healthcare system. Obesity and aging are major risk factors for a diverse number of diseases and it is of importance to understand their interaction and the underlying molecular mechanisms. Herein the authors examined the methylation levels of 27578 CpG sites in 46 samples from adult peripheral blood. The effect of obesity and aging was ascertained with general linear models. More than one hundred probes were correlated to aging, nine of which belonged to the KEGG group map04080. Additionally, 10 CpG sites had diverse methylation profiles in obese and lean individuals, one of which was the telomerase catalytic subunit (TERT). In eight of ten cases the methylation change was reverted between obese and lean individuals. One region proved to be differentially methylated with obesity (LINC00304) independent of age. This study provides evidence that obesity influences age driven epigenetic changes, which provides a molecular link between aging and obesity. This link and the identified markers may prove to be valuable biomarkers for the understanding of the molecular basis of aging, obesity and associated diseases. [ABSTRACT FROM AUTHOR]

Abstract: 2-(3-(Naphthalen-2-yl)propanamido)cyclohex-1-enecarboxylic acid and its 6-hydroxynaphthalen-2-yl analogue are well-known hydroxyl-carboxylic acid (HCA) receptor HCA2 agonists. A series of novel aryl derivatives of 2-amidocyclohex-1-ene carboxylic acid that contained rigidity elements, such as an E-double bond, triple bond, and trans or cis-substituted cyclopropane rings, instead of the saturated ethane linker in the amide part of the molecules were designed and synthesized, and the derivatives’ potency for the activation of HCA1, HCA2, and HCA3 receptors by 3′–5′-cyclic adenosine monophosphate (cAMP) assay were evaluated. The SAR studies revealed that the rigidifying of appropriate molecules enabled modulation of the potency and selectivity of the HCA2 receptor activation. [Copyright &y& Elsevier]

Abstract: Variations in the FTO gene and near the TMEM18 gene are risk factors for common form of obesity, but have also been linked with type 2 diabetes (T2D). Our aim was to investigate the contribution of these variants to risk of T2D in a population in Latvia. Four single nucleotide polymorphisms (SNP) in the first and fourth intronic regions of FTO and one close to TMEM18 were genotyped in 987 patients with T2D and 1080 controls selected from the Latvian Genome Data Base (LGDB). We confirmed association of SNPs in the first intron (rs11642015, rs62048402 and rs9939609) of FTO and rs7561317 representing the TMEM18 locus with T2D. Association between SNP in FTO and T2D remained significant after correction for body mass index (BMI). The rs57103849 located in the fourth intron of FTO and rs7561317 in TMEM18 showed BMI independent association with younger age at diagnosis of T2D. Our results add to the evidence that BMI related variants in and near FTO and TMEM18 may increase the risk for T2D not only through secondary effects of obesity. The influence of variants in the fourth intron of the FTO gene on development of T2D may be mediated by mechanisms other than those manifested by SNPs in the first intron of the same gene. [Copyright &y& Elsevier]

The heritability of high-density lipoprotein cholesterol (HDL-C) level is estimated at approximately 50%. Recent genome-wide association studies have identified genes involved in regulation of high-density lipoprotein cholesterol (HDL-C) levels. The precise genetic profile determining heritability of HDL-C however are far from complete and there is substantial room for further characterization of genetic profiles influencing blood lipid levels. Here we report an association study comparing the distribution of 139 SNPs from more than 30 genes between groups that represent extreme ends of HDL-C distribution. We genotyped 704 individuals that were selected from Genome Database of Latvian Population. 10 SNPs from CETP gene showed convincing association with low HDL-C levels (rs1800775, rs3764261, rs173539, rs9939224, rs711752, rs708272, rs7203984, rs7205804, rs11076175 and rs9929488) while 34 SNPs from 10 genes were nominally associated (p<0.05) with HDL-C levels. We have also identified haplotypes from CETP with distinct effects on determination of HDL-C levels. Our conclusion: So far the SNPs in CETP gene are identified as the most common genetic factor influencing HDL-C levels in the representative sample from Latvian population. [ABSTRACT FROM AUTHOR]

Several studies examined the fine-scale structure of human genetic variation in Europe. However, the European sets analyzed represent mainly northern, western, central, and southern Europe. Here, we report an analysis of approximately 166,000 single nucleotide polymorphisms in populations from eastern (northeastern) Europe: four Russian populations from European Russia, and three populations from the northernmost Finno-Ugric ethnicities (Veps and two contrast groups of Komi people). These were compared with several reference European samples, including Finns, Estonians, Latvians, Poles, Czechs, Germans, and Italians. The results obtained demonstrated genetic heterogeneity of populations living in the region studied. Russians from the central part of European Russia (Tver, Murom, and Kursk) exhibited similarities with populations from central–eastern Europe, and were distant from Russian sample from the northern Russia (Mezen district, Archangelsk region). Komi samples, especially Izhemski Komi, were significantly different from all other populations studied. These can be considered as a second pole of genetic diversity in northern Europe (in addition to the pole, occupied by Finns), as they had a distinct ancestry component. Russians from Mezen and the Finnic-speaking Veps were positioned between the two poles, but differed from each other in the proportions of Komi and Finnic ancestries. In general, our data provides a more complete genetic map of Europe accounting for the diversity in its most eastern (northeastern) populations. [ABSTRACT FROM AUTHOR]

Abstract: The glyoxalase system and its main enzyme, glyoxalase 1 (GLO1), protect cells from advanced glycation end products (AGEs), such as methylglyoxal (MG) and other reactive dicarbonyls, the formation of which is increased in diabetes patients as a result of excessive glycolysis. MG is partly responsible for harmful protein alterations in living cells, notably in neurons, leading to their dysfunction, and recent studies have shown a negative correlation between GLO1 expression and tissue damage. Neuronal dysfunction is a common diabetes complication due to elevated blood sugar levels, leading to high levels of AGEs. The aim of our study was to determine whether single nucleotide polymorphisms (SNPs) in the GLO1 gene influence activity of the enzyme. In total, 125 healthy controls, 101 type 1 diabetes, and 100 type 2 diabetes patients were genotyped for three common SNPs, rs2736654 (A111E), rs1130534 (G124G), and rs1049346 (5′-UTR), in GLO1. GLO1 activity was determined in whole blood lysates for all participants of the study. Our results showed a significant association between the minor alleles rs1130534 and rs1049346 and decreased enzyme activity (P =0.001 and P =2.61×10−5, respectively). Increased allelic counts of the risk alleles were strongly associated with decreased GLO1 activity (standardised β =−0.24, P =2.15×10−5), indicating independent actions of these variants on GLO1 activity, as supported by the haplotype analysis. We showed for the first time an association between genetic variants with GLO1 enzyme activity in humans. SNPs in GLO1 can be used to predict enzyme activity and detoxifying capabilities, but further studies are needed to link these SNPs with common complications in diabetes. [Copyright &y& Elsevier]

Metformin is the most widely used oral antidiabetic drug for the treatment of type 2 diabetes (T2D). So far, the number of polymorphisms in SLC22A1, SLC22A2, and SLC47A1 genes coding for organic cation transporter 1 (OCT1), OCT2, and multidrug and toxin extrusion transporter 1 (MATE1) metformin transporters have been described in association with the efficacy of metformin. However, there is no information on the influence of genetic variations within these genes on the side effects of metformin. In this study, we assessed whether five single-nucleotide polymorphisms and two indel polymorphisms are associated with the side effects of metformin in patients with T2D.Seven polymorphisms in OCT1, OCT2, and MATE1 genes were compared between 53 T2D patients with side effects of metformin and 193 metformin users without symptoms of metformin intolerance.We found a statistically significant association between the A allele of the rs628031 (P=0.012, odds ratio=0.389, confidence interval 95% [0.186-0.815]) as well as 8 bp insertion (rs36056065) in the OCT1 gene (P=0.002, odds ratio=0.405, confidence interval 95% [0.226-0.724]) and the presence of the side effects of metformin.Two genetic variations in OCT1 that are in strong linkage disequilibrium may predispose toward an increased prevalence of the side effects of metformin in patients with T2D. [ABSTRACT FROM AUTHOR]

The purinergic 12 receptor (P2Y12) is a major drug target for anticoagulant therapies, but little is known about the regions involved in ligand binding and activation of this receptor. We generated four randomized P2Y12 libraries and investigated their ligand binding characteristics. P2Y12 was expressed in a Saccharomyces cerevisiae model system. Four libraries were generated with randomized amino acids at positions 181, 256, 265 and 280. Mutant variants were screened for functional activity in yeast using the natural P2Y12 ligand ADP. Activation results were investigated using quantitative structure-activity relationship (QSAR) models and ligand-receptor docking. We screened four positions in P2Y12 for functional activity by substitution with amino acids with diverse physiochemical properties. This analysis revealed that positions E181, R256 and R265 alter the functional activity of P2Y12 in a specific manner. QSAR models for E181 and R256 mutant libraries strongly supported the experimental data. All substitutions of amino acid K280 were completely inactive, highlighting the crucial role of this residue in P2Y12 function. Ligand-receptor docking revealed that K280 is likely to be a key element in the ligand-binding pocket of P2Y12. The results of this study demonstrate that positions 181, 256, 265 and 280 of P2Y12 are important for the functional integrity of the receptor. Moreover, K280 appears to be a crucial feature of the P2Y12 ligand-binding pocket. These results are important for rational design of novel antiplatelet agents. [ABSTRACT FROM AUTHOR]

Lace B, Kempa I, Piekuse L, Grinfelde I, Klovins J, Pliss L, Krumina A, Vieira AR. Association studies of candidate genes and cleft lip and palate taking into consideration geographical origin. Eur J Oral Sci 2011; 119: 413-417. © 2011 Eur J Oral Sci Isolated cleft lip and/or palate (CL/CLP) is a complex congenital anomaly with many contributing factors. There are several genes involved in the aetiology of CL/CLP, they are different in selected populations. In a previous study, the mitochondrial haplotypes of Latvian subjects with CL/CLP were characterized. Latvian subjects with CL/CLP have mostly mitochondrial haplogroups U4/U5 compared with the ethnic population of Latvia. The aim of this study was to stratify the results of genotyping based on European mitochondrial DNA (mtDNA) haplotypes. DNA samples from 108 patients with CL/CLP and from 182 unrelated and unaffected individuals selected randomly in Latvia (used as controls) were obtained for investigation. In this study, we analysed the data taking into consideration mitochondrial haplogroups and found that gene associations depended on the genetic origin of the population. The phenotype of patients with non-U haplotypes was associated with markers in wingless-type MMTV integration site family, member 3 ( WNT3), collagen, type XI, alpha 2 ( COL11A2), and fibroblast growth factor receptor 1 ( FGFR1), whereas patients with U4 and U5 haplotypes showed significant association with WNT3 and COL11A2. It is unlikely that mtDNA variants play a direct role in the development of CL/CLP; rather, they may be a surrogate for population substructure and provide a tool to increase homogeneity and statistical power. [ABSTRACT FROM AUTHOR]

Adenosine plays an important part in the cardiac response to ischemia and reperfusion. The human adenosine receptor A3 (A3R), along with other adenosine receptors, is involved in mediation of those effects. The aim of the study was to ascertain whether the nonsynonymous single-nucleotide polymorphism (SNP) I248L (reference SNP ID: rs35511654) located in the A3R gene is associated with coronary heart disease (CHD). DNA samples from 683 individuals with CHD and from 826 control subjects selected from the Latvian Genome Database were successfully screened for rs35511654 using the TaqMan SNP Genotyping Assay. We observed a significantly decreased frequency of the rs35511654 C allele in a group of CHD patients compared with that in controls ( p=0.009). The association remained significant after adjustment for age, sex, and other nongenetic factors ( p=0.02). These results suggest that A allele of rs35511654 may predispose to CHD. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to characterize the genetic variance of somatostatin receptor 5 (SSTR5) and investigate the possible correlation of such variants with acromegaly risk and different disease characteristics. Design and methods: The SSTR5 gene coding region and 2000 bp upstream region was sequenced in 48 patients with acromegaly and 96 control subjects. Further, three single nucleotide polymorphisms (SNPs) were analyzed in the same group of acromegaly patients and in an additional group of 475 age- and sex-matched controls. Results: In total, 19 SNPs were identified in the SSTR5 gene locus by direct sequencing. Three SNPs (rs34037914, rs169068, and rs642249) were significantly associated with the presence of acromegaly using the initial controls. The allele frequencies were significantly (P<0.01) different between the acromegaly patients and the additional large control group. rs34037914 and rs642249 remained significantly associated with acromegaly after Bonferroni correction and permutation tests (odds ratio (OR)Z3.38; 95% confidence interval (CI), 1.78-6.42; PZ0.00016 and ORZ2.41; 95% CI, 1.41-4.13; PZ0.0014 respectively). Haplotype reconstruction revealed two possible risk haplotypes determined by rs34037914 (633T) and rs642249 (1044A) alleles. Both haplotypes were found in significantly higher frequency in acromegaly patients compared with controls (P<0.001). In addition, the 663T allele was significantly associated with a younger age of acromegaly diagnosis (unstandardized regression coefficient β= -10.4; P=0.002), increased body mass index (&beta=4.1; P=0.004), higher number of adenoma resection (P<0.001) and lack of observable tumor shrinkage after somatostatin analog treatment (P=0.014). Conclusions: Our results demonstrate a previously undetected strong association of two SSTR5 SNPs with acromegaly. The data also suggest a possible involvement of SSTR5 variants in decreased suppression of GH production and increased tumor proliferation. [ABSTRACT FROM AUTHOR]

Abstract: The adrenocorticotropic hormone (ACTH) receptor has highly specific membrane expression that is limited to adrenal cells; in other cell types the polypeptide fails to be transported to the cell surface. Unlike other evolutionarily related members of the melanocortin receptor family (MC1R–MC5R) that recognize different melanocortin peptides, ACTHR (MC2R) binds only ACTH. We used a mutagenesis approach involving systematic construction of chimeric ACTHR/MC4R receptors to identify the domains determining the selectivity of ACTHR membrane transport and ACTH binding. In total 15 chimeric receptors were created by replacement of selected domains of human ACTHR with the corresponding regions of human MC4R. We developed an analytical method to accurately quantify cell-membrane localization of recombinant receptors fused with enhanced green fluorescent protein by confocal fluorescence microscopy. The chimeric receptors were also tested for their ability to bind ACTH (1–24) and the melanocyte-stimulating hormone (MSH) analog, Nle4, DPhe7-α-MSH, and to induce a cAMP response. Our results indicate that substitution of the MC4R N-terminal segment with the homologous segment of ACTHR significantly decreased membrane transport. We also identified another signal localized in the third and fourth transmembrane regions as the main determinant of ACTHR intracellular retention. In addition, we found that the fourth and fifth transmembrane domains of the ACTHR are involved in ACTH binding selectivity. We discuss the mechanisms involved in bypassing these arrest signals via an interaction with melanocortin 2 receptor accessory protein (MRAP) and the possible mechanisms that determine the high ligand-binding specificity of ACTHR. [Copyright &y& Elsevier]

The paradigm of ligand-receptor interactions postulated as "one compound—one target" has been evolving; a multi-target, pleiotropic approach is now considered to be realistic. Novel series of 1,4,5,6,7,8-hexahydro-5-oxoquinolines, pyranopyrimidines and S-alkyl derivatives of pyranopyrimidines have been synthesized in order to characterise their pleiotropic, multitarget activity on the FFA3/GPR41, FFA2/GPR43, and HCA2/GPR109A receptors. Hexahydroquinoline derivatives have been known to exhibit characteristic activity as FFA3/GPR41 ligands, but during this study we observed their impact on FFA2/GPR43 and HCA2/GPR109A receptors as well as their electron-donating activity. Oxopyranopyrimidine and thioxopyranopyrimidine type compounds have been studied as ligands of the HCA2/GPR109A receptor; nevertheless, they exhibited equal or higher activity towards FFA3/GPR41 and FFA2/GPR43 receptors. S-Alkyl derivatives of pyranopyrimidines that have not yet been studied as ligands of GPCRs were more active towards HCA2/GPR109A and FFA3/GPR41 receptors than towards FFA2/GPR43. Representative compounds from each synthesized series were able to decrease the lipopolysaccharide-induced gene expression and secretion of proinflammatory cytokines (IL-6, TNF-α) and of a chemokine (MCP-1) in THP-1 macrophages, resembling the effect of HCA2/GPR109A ligand niacin and the endogenous ligand propionate. This study revealed groups of compounds possessing multitarget activity towards several receptors. The obtained data could be useful for further development of multitarget ligands. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have shown that SNPs in the FTO gene predispose to childhood and adult obesity. In this study, we examined the association between variants in FTO and KIAA1005, a gene that maps closely to FTO, and obesity, as well as obesity related traits among 450 well characterised severely obese children and 512 normal weight controls. FTO showed significant association with several obesity related traits while SNPs in KIAA1005 did not. When stratified by gender, the FTO variant rs9939609 showed association with obesity and BMI among girls (P =0.006 and 0.004, respectively) but not among boys. Gender differences were also found in the associations of the FTO rs9939609 with obesity related traits such as insulin sensitivity and plasma glucose. This study suggests that FTO may have an important role for gender specific development of severe obesity and insulin resistance in children. [Copyright &y& Elsevier]

Abstract: Mammalian G protein-coupled receptor (GPCR) genes are characterised by a large proportion of intronless genes or a lower density of introns when compared with GPCRs of invertebrates. It is unclear which mechanisms have influenced intron density in this protein family, which is one of the largest in the mammalian genomes. We used a combination of Hidden Markov Models (HMM) and BLAST searches to establish the comprehensive repertoire of Rhodopsin GPCRs from seven species and performed overall alignments and phylogenetic analysis using the maximum parsimony method for over 1400 receptors in 12 subgroups. We identified 14 different Ancestral Receptor Groups (ARGs) that have members in both vertebrate and invertebrate species. We found that there exists a remarkable difference in the intron density among ancestral and new Rhodopsin GPCRs. The intron density among ARGs members was more than 3.5-fold higher than that within non-ARG members and more than 2-fold higher when considering only the 7TM region. This suggests that the new GPCR genes have been predominantly formed intronless while the ancestral receptors likely accumulated introns during their evolution. Many of the intron positions found in mammalian ARG receptor sequences were found to be present in orthologue invertebrate receptors suggesting that these intron positions are ancient. This analysis also revealed that one intron position is much more frequent than any other position and it is common for a number of phylogenetically different Rhodopsin GPCR groups. This intron position lies within a functionally important, conserved, DRY motif which may form a proto-splice site that could contribute to positional intron insertion. Moreover, we have found that other receptor motifs, similar to DRY, also contain introns between the second and third nucleotide of the arginine codon which also forms a proto-splice site. Our analysis presents compelling evidence that there was not a major loss of introns in mammalian GPCRs and formation of new GPCRs among mammals explains why these have fewer introns compared to invertebrate GPCRs. We also discuss and speculate about the possible role of different RNA- and DNA-based mechanisms of intron insertion and loss. [Copyright &y& Elsevier]

The Adhesion family of G protein-coupled receptors (GPCRs) includes 33 receptors and is the second largest GPCR family. Most of these proteins are still orphans and fairly little is known of their tissue distribution and evolutionary context. We report the evolutionary history of the Adhesion family protein GPR123 as well as mapping of GPR123 mRNA expression in mouse and rat using in situ hybridization and real-time PCR, respectively. GPR123 was found to be well conserved within the vertebrate lineage, especially within the transmembrane regions and in the distal part of the cytoplasmic tail, containing a potential PDZ binding domain. The real-time PCR data indicates that GPR123 is predominantly expressed in CNS. The in situ data show high expression in thalamic nuclei and regions containing large pyramidal cells like cortex layers 5 and 6 and subiculum. Moreover, we found distinct expression in amygdala, hypothalamus, inferior olive and spinal cord. The CNS specific expression, together with the high sequence conservation between the vertebrate sequences investigated, indicate that GPR123 may have an important role in the regulation of neuronal signal transduction. [ABSTRACT FROM AUTHOR]

Abstract: We have cloned melanocortin receptors (MCRs) from several species of fish. The MC4R and MC5R subtypes arose early in vertebrate evolution and their primary structure is remarkably conserved. Expression and pharmacological characterization of the MCRs in fish has revealed that they bind and respond to melanocortin peptides with high potency. Detailed characterization of the binding properties of the different subtypes suggests that MCRs in early vertebrates had preference for adrenocorticotropic hormone (ACTH) peptides, while the high sensitivity for the shorter proopiomelanocortin (POMC) products, such as the α-, β-, and γ-melanocyte-stimulating hormone (MSH), has appeared later, perhaps as the MCR subtypes gained more specialized functions. The MCR repertoire shows in general high similarities in their primary structures, while they are however not similar in terms of functional roles. The MCRs serve therefore as an interesting model family to understand the molecular mechanisms of how functions of the genes can diverge during evolution. In this review, we provide an overview of our recent studies on the cloning, expression, pharmacology, 3D modeling, and genomic studies of the MCRs in non-mammalian species. [Copyright &y& Elsevier]

Abstract: We present seven new vertebrate homologs of the prolactin-releasing hormone receptor (PRLHR) and show that these are found as two separate subtypes, PRLHR1 and PRLHR2. Analysis of a number of vertebrate sequences using phylogeny, pharmacology, and paralogon analysis indicates that the PRLHRs are likely to share a common ancestry with the neuropeptide Y (NPY) receptors. Moreover, a micromolar level of NPY was able to bind and inhibit completely the PRLH-evoked response in PRLHR1-expressing cells. We suggest that an ancestral PRLH peptide started coevolving with a redundant NPY binding receptor, which then became PRLHR, approximately 500 million years ago. The PRLHR1 subtype was shown to have a relatively high evolutionary rate compared to receptors with fixed peptide preference, which could indicate a drastic change in binding preference, thus supporting this hypothesis. This report suggests how gene duplication events can lead to novel peptide ligand/receptor interactions and hence spur the evolution of new physiological functions. [Copyright &y& Elsevier]

Introduction. Evaluation of the first trimester uterine artery flow can predict the development of obstetrical complications. A genotype, making women prone to microthrombi. constitutes the main known susceptibility factor for anomalous development of placenta. Our aim was to study whether polymorphisms of 10 genes leading to blood clotting abnormalities are related to abnormal uterine artery blood flow in the first trimester, and may predict placenta-related diseases. Material and methods. In primary analyses we included 19 singleton pregnancies with abnormal blood flow in the uterine arteries during the first trimester of gestation, and 24 matched control with normal flow patterns. All patients were genotyped for sequence variations in F5, F2, F11, MTHFR, SERPINE-1, CYP4V2, SELE, GP6, angiotensinogen (AGT) and fibrinogen gamma (FGG) genes and followed up until delivery. Results. There were no differences between groups regarding selected sequence variations in any of these genes. The co-occurrence of several polymorphisms in the same patient was also not related to the blood flow patterns in the uterine arteries. Conclusions. Although we found certain trends of genetic polymorphisms being related to preeclampsia and fetal growth, we failed to find an association between clotting gene polymorphisms, single or in combination, with the abnormal uterine flow in the first trimester. [ABSTRACT FROM AUTHOR]

Background: Metformin is a widely prescribed antihyperglycemic agent that has been also associated with multiple therapeutic effects in various diseases, including several types of malignancies. There is growing evidence regarding the contribution of the epigenetic mechanisms in reaching metformin's therapeutic goals; however, the effect of metformin on human cells in vivo is not comprehensively studied. The aim of our study was to examine metformin-induced alterations of DNA methylation profiles in white blood cells of healthy volunteers, employing a longitudinal study design. Results: Twelve healthy metformin-naïve individuals where enrolled in the study. Genome-wide DNA methylation pattern was estimated at baseline, 10 h and 7 days after the start of metformin administration. The whole-genome DNA methylation analysis in total revealed 125 differentially methylated CpGs, of which 11 CpGs and their associated genes with the most consistent changes in the DNA methylation profile were selected: POFUT2, CAMKK1, EML3, KIAA1614, UPF1, MUC4, LOC727982, SIX3, ADAM8, SNORD12B, VPS8, and several differentially methylated regions as novel potential epigenetic targets of metformin. The main functions of the majority of top-ranked differentially methylated loci and their representative cell signaling pathways were linked to the well-known metformin therapy targets: regulatory processes of energy homeostasis, inflammatory responses, tumorigenesis, and neurodegenerative diseases. Conclusions: Here we demonstrate for the first time the immediate effect of short-term metformin administration at therapeutic doses on epigenetic regulation in human white blood cells. These findings suggest the DNA methylation process as one of the mechanisms involved in the action of metformin, thereby revealing novel targets and directions of the molecular mechanisms underlying the various beneficial effects of metformin. Trial registration: EU Clinical Trials Register, 2016-001092-74. Registered 23 March 2017, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001092-74/LV. [ABSTRACT FROM AUTHOR]

The article presents a study on HFE-related hemochromatosis (HH) which is an autosomal recessive, multisystemic disease caused by regulated uptake and accumulation of iron. Topics discussed include causes of HH due to mutations in HFE gene C282Y, use of TaqMan (single nucleotide polymorphism) SNP for carrying genotyping on polymerase chain reaction (PCR) system, and aim of the study to provide frequency of HH risk mutation for Ukraine and Belarus population.