Ekspresija mRNA agutiju srodnog peptida i mRNA melanokortin-4 receptora u arkuatnoj jezgri za vrijeme gravidnosti i laktacije štakorica.

Pregnancy is associated with a range of physiological adjustments to adapt the body to the demands of the growth of the fetus and subsequent lactation. It has been observed that agouti-related peptide (AGRP) and melanocortin-4 receptor (MC4R) are involved in energy homeostasis. A randomized controlled experimental study was planned to investigate the expression of AGRP and MC4R mRNAs in the stages of pregnancy and lactation in rat arcuate nucleus (ARC) of the hypothalamus. Thirty-two adult female rats were randomly divided into six groups. Pregnant rats were assigned into three groups (n = 6) of 7, 14, and 21 days of pregnancy. Two more groups were also assigned of non-suckling rats (n = 5), immediately separated from their pups after parturition, and suckling rats (n = 5), allowed to suckle five pups until day 8 (increasing milk). The sixth group consisted of four ovariectomized rats, which were assigned two weeks after surgery and served as control. Using real-time PCR, the relative expressions (compared to controls) of MC4R and AGRP mRNAs in ARC were calculated in the pregnant, suckling and non-suckling rats. Expression of AGRP mRNAs in pregnant rats on days 14 and 21 was higher than that observed in suckling and non-suckling rats (P<0.05). Expression of MC4R mRNAs in pregnant rats on days 14 and 21 was lower than that observed in suckling rats, and was higher on day 7 than that observed in both suckling and non-suckling rats (P<0.05). In conclusion, expression of AGRP in pregnancy and MC4R in lactation in ARC of rats controls energy homeostasis.Gravidnost je povezana s nizom fizioloških prilagodbi kojima tijelo odgovara zahtjevima povezanima s rastom fetusa i kasnije laktacije. Uočeno je da su agutiju srodan peptid (AGRP) i melanokortin-4 receptor (MC4R) uključeni u energetsku homeostazu. Randomiziranim kontroliranim pokusom planirano je u arkuatnoj jezgri (ARC) hipotalamusa štakorica istražiti ekspresiju mRNA AGRP i mRNA MC4R tijekom gravidnosti i laktacije. Trideset dvije odrasle štakorice nasumično su bile podijeljene u šest skupina. Gravidne su štakorice podijeljene u tri skupine (n = 6), s obzirom na 7., 14. i 21. dan gravidnosti. Još dvije skupine (n = 5) činile su nedojne štakorice koje su odvojene od svoje mladunčadi odmah nakon porođaja te dojne štakorice kojima je dozvoljeno dojenje 5 mladunaca do osmog dana rastuće laktacije. Četiri ovarijektomizirane štakorice, dva tjedna nakon operacije, dodijeljene su u 6. kontrolnu skupinu. Koristeći PCR u stvarnom vremenu, relativna ekspresija (usporedba s kontrolama) mRNA MC4R i mRNA AGRP u ARC izračunata je za gravidne, nedojne i dojne štakorice. Ekspresija mRNA AGRP kod gravidnih štakorica 14. i 21. dan bila je veća od one opažene kod dojnih i nedojnih štakorica (P<0,05). Ekspresija MC4R mRNA u gravidnih štakorica 14. i 21. dan bila je manja u odnosu na dojne štakorica i veća 7. dan u odnosu na skupine dojnih i nedojnih štakorica (P<0,05). Zaključno, ekspresija AGRP u ARC tijekom gravidnosti i MC4R u ARC tijekom laktacije kontrolira energetsku homeostazu štakorica

Promjene u RF-amidu srodnom peptidu-3 hipotalamusa i ekspresijama gena Kiss1 tijekom spermatogeneze kod štakora u uvjetima kroničnog stresa.

The effects were evaluated of chronic stress and the glucocorticoid receptor antagonist (RU486) on mRNA expressions of RF-amide related peptide-3 (RFRP-3) in the dorsomedial hypothalamic nucleus (DMH) and Kiss1 in the arcuate nucleus (ARC) of male rats. Twenty-four male rats were allocated to four equal sized groups: the stress, RU486, stress/RU486, and control groups. In the stress group the rats were restrained 1 hour/day for 12 days. In the RU486 group, the rats were injected with RU486 for 12 days. In the stress/RU486 group, the rats were injected with RU486 1 hour before the stress process for 12 days. Relative expressions of RFRP-3 and Kiss1 mRNAs were determined using real-time PCR. The relative expression of RFRP-3 mRNA in the stress group was higher than that in the RU486 and control rats. The relative expression of RFRP-3 mRNA did not differ between the stress group and the stress/RU486 rats. Furthermore, the relative expressions of Kiss1 mRNA in the stress, RU486, and stress/RU486 groups were less than that of the control rats. The relative expression of Kiss1 mRNA did not differ between the stress, RU486, and stress/RU486 groups. In conclusion, dysfunction in male rat fertility caused by the chronic stress may be the result of the increase in REFP-3 and the decrease in Kiss1 mRNA expression.Istražen je učinak kroničnog stresa i antagonista glukokortikoidnog receptora (RU486) na ekspresiju mRNA RF-amidu srodnog peptida-3 (RFRP-3) u dorzomedijalnoj jezgri hipotalamusa (DMH), te na ekspresije gena Kiss1 u arkuatnom nukleusu (ARC) štakora. Dvadeset i četiri štakora bila su raspodijeljena u četiri jednake skupine: stresna skupina, RU486 skupina, stresna/RU486 skupina i kontrolna skupina. U stresnoj skupini štakori su 12 dana bili obuzdani tijekom jednog sata dnevno. U skupini RU486, štakorima je tijekom 12 dana bio primijenjivan RU486. U skupini stres/RU486, štakorima je tijekom 12 dana apliciran RU486 jedan sat prije postupka obuzdavanja. Relativne ekspresije RFRP-3 i Kiss1 mRNA određene su lančanom reakcijom polimerazom u stvarnom vremenu. Relativna ekspresija RFRP-3 mRNA u stresnoj skupini bila je veća nego u skupini RU486 i kontrolnoj skupini. Relativna ekspresija RFRP-3 mRNA nije bila različita između stresne skupine i stres/RU486 skupine. Nadalje, relativne ekspresije Kiss1 mRNA u stresnoj skupini, skupini RU486, i stresnoj skupini/RU486 bile su manje u odnosu na kontrolnu skupinu. Relativna ekspresija Kiss1 mRNA nije se razlikovala između stresne skupine, skupineRU486 i stresne skupine/RU486. Zaključno, disfunkcija plodnosti kod štakora izloženih kroničnom stresu može biti uzrokovana putem povećane ekspresije RFRP-3 i smanjene ekspresije Kiss1 mRNA

Oncogenes in KRAS Wild Type Pancreatic Cancer

© 2019 Somayeh AhmadlooPancreatic ductal adenocarcinoma (PDAC) is an invasive cancer, ranked the fourth most prevalent cause of cancer related death. Somatic genetic alterations are primary drivers of PDAC. 93% of patients have activating mutations in the master oncogene, KRAS. Several studies have investigated the mutational landscapes of pancreatic cancer. However, comprehensive studies of KRAS wild type pancreatic tumours are limited. Hence the process of initiation and progression of this cancer remains to be discovered and may be associated with genes that have not been identified. The current project aims to identify oncogenes in KRAS wild type Pancreatic cancer. It also aims to identify whether these oncogenes are from the MAPK pathway or independent of it. The genomic and transcriptomic landscapes of KRAS wild type PDAC were verified. In the absence of KRAS mutation, alternative oncogenes were found. In the genomic data analysis, two cohorts were analysed including 70 samples in the KRAS wild type cohort and 571 in the KRAS mutant cohort. In the absence of KRAS mutation, tumours were found to be rare as were (i) Oncogenic BRAF in-frame deletions, hotspot alterations and oncogenic fusions (known and novel) (frequency of 17%), oncogenic GNAS hotspot mutation (frequency of 12%) and somatic alterations in RET (7% frequency). Recurrent copy number gains (CNV >4) were observed in MYC (23% frequency), CDK6 (16% frequency), AKT2 (16% frequency), KDM6A (14% frequency), EGFR (12% frequency), RICTOR (12% frequency), MET (11% frequency), FGFR1(10% frequency), FGF3 (9% frequency) and FGF4 (9% frequency) in the KRAS wild type cohort. Other low frequency fusion events in the MAPK pathway include: RET-CCDC6; ROS1-SLC4A4; BRAF-SND1; BRAF-SDK1; TRIM24-BRAF; STK4-SLC13A3; ARHGAP24-MAPk10; BRAF-BRAF; STMN1-CDK5RAP3, and; SLC4A4-RASGRF1. Two independent differential expression analyses were performed on the RNA-seq of KRAS wild type versus KRAS mutant Pancreatic Adenocarcinomas, generated by the Australian and Canadian ICGC- pancreatic cancer Consortium consisting of RNA-seq from 88 and 224 bulk tumour samples respectively. Pathway analysis showed that the Calcium signalling pathway was over-expressed in both the Australian and Canadian wild type.This up-regulation of the Calcium signalling pathway in the whole cohort of KRAS wild type is consistent with GNAS mutation in the genomic analysis of the KRAS wild type cohort. Additionally, the MAPK signalling pathway shows no difference throughout the whole cohort of KRAS wild type. Together, these findings at the genomic level reveal that the MAPK signalling pathway is the dominant pathway in the KRAS wild type cohort. The results of comparing RNA expression in two groups of KRAS wild type and KRAS mutant analysis suggest that one oncogene has been substituted for another oncogene in the MAPK pathway, creating an interruption in the MAPK pathway. The lack of differences between KRAS mutant and KRAS wildtype in the MAPK pathway could suggest that the MAPK pathway is up-regulated in both sets, resulting in a lack of difference in the expression

Functional Analysis of A Novel Splicing Mutation in The Mutase Gene of Two Unrelated Pedigrees

Objective: Methylmalonic acidura (MMA) is a rare autosomal recessive inborn error of metabolism. In this study we present a novel nucleotide change in the mutase (MUT) gene of two unrelated Iranian pedigrees and introduce the methods used for its functional analysis. Materials and Methods: Two probands with definite diagnosis of MMA and a common novel variant in the MUT were included in a descriptive study. Bioinformatic prediction of the splicing variant was done with different prediction servers. Reverse transcription- polymerase chain reaction (RT-PCR) was done for splicing analysis and the products were analyzed by sequencing. Results: The included index patients showed elevated levels of propionylcarnitine (C3). Urine organic acid analysis confirmed the diagnosis of MMA, and screening for mutations in the MUT revealed a novel C to G variation at the 3´ splice acceptor site in intron 12. In silico analysis suggested the change as a mutation in a conserved sequence. The splicing analysis showed that the C to G nucleotide change at position -3 in the acceptor splice site can lead to retention of the intron 12 sequence. Conclusion: This is the first report of a mutation at the position -3 in the MUT intron 12 (c.2125-3C>G). The results suggest that the identified variation can be associated with the typical clinical manifestations of MMA

An Efficient Trio-Based Mini-Haplotyping Method for Genetic Diagnosis of Phenylketonuria

Objective The phenylalanine hydroxylase (PAH) locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat (STR) genotyping. Materials and Methods One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat (VNTR) were labeled with three different non-overlapping dyes 5-carboxyfluorescein (FAM), 6-carboxy-N,N,N’,N’-tetramethylrhodamine (HEX) and 6-carboxy-N,N,N’,N’-tetramethylrhodamine (TAMRA). The polymerase chain reaction (PCR) products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software. Results Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift (migrated at a slower electrophoretic rate) relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability (P<0.001). Conclusion Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems

Ekspresija mRNA agutiju srodnog peptida i mRNA melanokortin-4 receptora u arkuatnoj jezgri za vrijeme gravidnosti i laktacije štakorica.

Pregnancy is associated with a range of physiological adjustments to adapt the body to the demands of the growth of the fetus and subsequent lactation. It has been observed that agouti-related peptide (AGRP) and melanocortin-4 receptor (MC4R) are involved in energy homeostasis. A randomized controlled experimental study was planned to investigate the expression of AGRP and MC4R mRNAs in the stages of pregnancy and lactation in rat arcuate nucleus (ARC) of the hypothalamus. Thirty-two adult female rats were randomly divided into six groups. Pregnant rats were assigned into three groups (n = 6) of 7, 14, and 21 days of pregnancy. Two more groups were also assigned of non-suckling rats (n = 5), immediately separated from their pups after parturition, and suckling rats (n = 5), allowed to suckle five pups until day 8 (increasing milk). The sixth group consisted of four ovariectomized rats, which were assigned two weeks after surgery and served as control. Using real-time PCR, the relative expressions (compared to controls) of MC4R and AGRP mRNAs in ARC were calculated in the pregnant, suckling and non-suckling rats. Expression of AGRP mRNAs in pregnant rats on days 14 and 21 was higher than that observed in suckling and non-suckling rats (P<0.05). Expression of MC4R mRNAs in pregnant rats on days 14 and 21 was lower than that observed in suckling rats, and was higher on day 7 than that observed in both suckling and non-suckling rats (P<0.05). In conclusion, expression of AGRP in pregnancy and MC4R in lactation in ARC of rats controls energy homeostasis.Gravidnost je povezana s nizom fizioloških prilagodbi kojima tijelo odgovara zahtjevima povezanima s rastom fetusa i kasnije laktacije. Uočeno je da su agutiju srodan peptid (AGRP) i melanokortin-4 receptor (MC4R) uključeni u energetsku homeostazu. Randomiziranim kontroliranim pokusom planirano je u arkuatnoj jezgri (ARC) hipotalamusa štakorica istražiti ekspresiju mRNA AGRP i mRNA MC4R tijekom gravidnosti i laktacije. Trideset dvije odrasle štakorice nasumično su bile podijeljene u šest skupina. Gravidne su štakorice podijeljene u tri skupine (n = 6), s obzirom na 7., 14. i 21. dan gravidnosti. Još dvije skupine (n = 5) činile su nedojne štakorice koje su odvojene od svoje mladunčadi odmah nakon porođaja te dojne štakorice kojima je dozvoljeno dojenje 5 mladunaca do osmog dana rastuće laktacije. Četiri ovarijektomizirane štakorice, dva tjedna nakon operacije, dodijeljene su u 6. kontrolnu skupinu. Koristeći PCR u stvarnom vremenu, relativna ekspresija (usporedba s kontrolama) mRNA MC4R i mRNA AGRP u ARC izračunata je za gravidne, nedojne i dojne štakorice. Ekspresija mRNA AGRP kod gravidnih štakorica 14. i 21. dan bila je veća od one opažene kod dojnih i nedojnih štakorica (P<0,05). Ekspresija MC4R mRNA u gravidnih štakorica 14. i 21. dan bila je manja u odnosu na dojne štakorica i veća 7. dan u odnosu na skupine dojnih i nedojnih štakorica (P<0,05). Zaključno, ekspresija AGRP u ARC tijekom gravidnosti i MC4R u ARC tijekom laktacije kontrolira energetsku homeostazu štakorica

Allelic Imbalance in Regulation of <i>ANRIL</i> through Chromatin Interaction at 9p21 Endometriosis Risk Locus

<div><p>Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging “allele-specific” functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with <i>ANRIL</i> promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a <i>cis</i>-regulatory variant where G allele was associated with increased <i>ANRIL</i> expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage.</p></div

Prioritization of candidate causal variants via target re-sequencing and DNase-seq data.

<p>A) Distribution of variants exhibiting strong LD (<i>r</i>² > 0.8) with two endometriosis-associated SNPs (rs10965235 and rs1537377). SNPs and indels are coded by diamonds and squares, respectively. The positions of rs10965235 and rs1537377 are shown as vertical lines. The intervals encompassing all the variants showing strong LD with rs10965235 and rs1537377 are highlighted by light green and pink shades, respectively. B) DHSs in endometrial carcinoma cell lines across the two intervals. The transcript structures of <i>ANRIL</i> and the sites of the variants that are in strong LD are depicted. The densities of aligned reads from DNase-seq estimated by F-Seq are plotted. DHSs in which the densities of aligned reads significantly surpassing the threshold are represented by dark blue. Locations in which aligned reads are depleted are depicted by light blue. The positions of the SNPs identified by GWASs (rs10965235 and rs1537377) and candidate causal SNPs (rs17834457 and rs17761446) are highlighted by blue and red arrows, respectively. C) DNase-seq signals at the variant sites showing strong LD. Signals are represented as relative values to the threshold determined by F-Seq. If the relative signal surpasses 1.0, the corresponding variant site coincides with significant DHS.</p