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Abstract Background Venous thromboembolism(VTE)is a common multifactorial disease. Anticoagulant protein deficiency is the most usual hereditary thrombophilia in the Chinese people, which includes protein C(PC), protein S and antithrombin deficiencies. Case presentation A retrospective analysis was conducted on clinical manifestations, laboratory tests, genetic information, and other relevant data of siblings diagnosed with VTE in 2020 at the Department of Pediatrics of Shenzhen Second People’s Hospital. The proband, a 12-year-old female, was admitted to the hospital in December 2020 with a complaint of pain in the left lower limb for four days. The examination found that the PC activity was 53%, and B-ultrasound showed bilateral thrombosis of the great saphenous vein in the thigh segment. The proband’s younger brother, a 10-year-old male, was admitted to the hospital in January 2021 due to right lower limb pain for two weeks. PC activity is 40%. B-ultrasound showed superficial venous thrombosis in the left lower limb and upper limb. Both siblings suffered from thalassemia and underwent splenectomy before recurrent thrombosis occurred. The proband’s mother was asymptomatic, and her PC activity was 45%. Both cases were treated with warfarin anticoagulation, and their symptoms improved. The proband’s mother was found to have a heterozygous mutation at this locus through Sanger sequencing. Conclusion Protein C deficiency should be considered for venous thromboembolism in childhood. The heterozygous mutation 1204 A > G in PROC exon 9 in this family is reported for the first time.

Prenatal air pollution, protein C (PROC) gene abnormal methylation, and genetic mutation can cause a series of neonatal diseases, but the complex relationship between them remains unclear. Here, we recruited 552 mothers and their own babies during January 2010–January 2012 in Zhengzhou to explore such association. The air pollutant data was obtained from the Environmental Monitoring Stations. The rs1799809 genotype and the methylation levels at the promoter region of PROC in genomic DNA samples were detected respectively by TaqMan probe and quantitative methylation specific PCR using real-time PCR system. The results show that the levels of neonatal PROC methylation were negatively associated with concentrations of NO2 during the entire pregnancy, particularly during the third trimester. Of particular significance, only in newborns carrying rs1799809 AA genotype, negatively significant associations between PROC methylation levels and exposure concentrations of air pollutants were observed. Further, we observed a significant interactive effect between neonatal rs1799809 genotype and SO2 exposure during the entire pregnancy on neonatal PROC methylation levels. Prenatal exposure to ambient air pollutants specifically was associated with the neonatal PROC promoter methylation level of newborns carrying the rs1799809 AA genotype. Taken together, these findings suggest that neonatal PROC methylation levels are associated with prenatal exposure to ambient air pollutants, and this association can be modified by rs1799809 genotype. [ABSTRACT FROM AUTHOR]

Edwardsiella tarda infection in fish results in economic losses in aquaculture industries worldwide. Besides, this pathogen is also a zoonotic agent causing gastrointestinal disease in humans. Detection of virulence factors expressed by E. tarda is a key for molecular diagnosis. This study aimed to design SYBR® Green-based quantitative PCR (qPCR) primer for E. tarda virulent protein C (EvpC) gene. A sequence of EvpC with accession number AY424360.4:6500-6991 obtained from GenBank NCBI was selected as the basis for qPCR primer design. qPCR primers were designed using Primer3 online software. Further analysis related to the secondary structure of each primer was carried out using Beacon Designer Free Edition. Five pairs of qPCR primers were successfully designed with Primer3. Based on the results of Primer3 and Beacon Designer analysis, F primer pair 1 (5'-GAATCCACCGACGATAAGCACAAA-3') and R primer pair 1 (5'-GACACGCAGCACCGACATCA-3') were the most favorable primer set since this primer met the criteria in terms of length, melting temperature, GC content, and self complementarity. In addition, this primer set had the highest (closest to zero) -ΔG value of cross dimer, self dimer, and hairpin. It can be concluded that SYBR® Green-based qPCR primer set for EvpC has been successfully designed. However, laboratory optimization experiments are essential for this designed primer set. [ABSTRACT FROM AUTHOR]

SQUAMOSA promoter-binding protein-like (SPL) is a class of plant-specific transcription factors that play critical roles in regulating plant growth and development. However, little systematic research on SPL genes has been conducted in strawberry. In this study, 14 SPL genes were identified in the genome of woodland strawberry (Fragaria vesca), one of the model plants of the family Rosaceae. Chromosome localization analysis indicated that the 14 FvSPL genes were unevenly distributed on six chromosomes. Phylogenetic analysis indicated that the FvSPL proteins could be clustered into six groups (G1 to G6). Genes with similar structure were classified into the same group, implying their functional redundancy. In addition, nine out of the 14 FvSPL genes, belonging to G1, G2, and G5, were found to be the putative targets of FvmiR156 genes. Expression analysis indicated FvSPL genes exhibited highly diverse expression patterns in the tissues and organs examined. The transcript levels of most FvmiR156-targeted FvSPL genes in fruit were lower than those non-miR156-targeted genes. In addition, the expression of the FvmiR156-targeted FvSPL genes decreased during fruit ripening, whereas the expression of FvmiR156 genes increased in fruit during this process. The results provide a foundation for future functional analysis of FvSPL genes in strawberry growth and development. [ABSTRACT FROM AUTHOR]

Reports of recurrent thromboembolism in thalassemia, particularly in hemoglobin H (HbH) disease associated with congenital thrombophilic mutations, are scarce. However, several mutations were detected in a 22-year-old woman with HbH disease. The patient experienced the first thrombotic event at the age of 20 years and had four recurrent thromboses in a short time interval, despite receiving anticoagulant treatment. The present study reports a case with six nucleotide substitutions, including a missense 565C>T (Arg189Trp) mutation and two synonymous mutations, 66T>C (Pro22Pro) and 423G>T (Ser141Ser), identified in the protein C gene. The other three mutations, 947G>A (Arg316His), 981A>G (Val327Val), and 775C>A (rs13146272), were identified in the protein S, antithrombin and cytochrome P450, family 4, subfamily V, polypeptide 2 genes, respectively. These findings suggest that if thrombotic events repeatedly occur in a patient with thalassemia, not only the risk factors associated with a hypercoagulable state, but the acquired and congenital thrombophilia should be screened for. [ABSTRACT FROM AUTHOR]

Protein C (PC) is a natural anticoagulant, which interacts with the endothelial PC receptor (EPCR). EPCR single-nucleotide polymorphism (SNP) 6936A/G results in high levels of a free soluble form of EPCR (sEPCR) and may affect the risk of coagulation. The objective of this study was to assess whether the 6936A/G SNP of the EPCR gene is involved in the procoagulant activity displayed by hematological malignancies. EPCR 6936A/G polymorphism analysis was performed in 205 patients with hematological malignancies and in 63 healthy controls. All the subjects were genotyped for the EPCR 6936A/G SNP (AA, AG and GG genotypes). The 6936A/G polymorphism distribution was similar between healthy donors and patients. The association between EPCR 6936A/G SNP and thrombosis was investigated in 110 patients. The disease-wise break-up revealed that 55 of the patients suffered from acute myeloid leukemia (AML). In AML patients, the incidence of thrombosis was 28.3% and significantly higher in the 6936AG compared with that in the 6936AA genotype (50 vs. 22%, respectively). In conclusion, this study revealed a significant association of the 6936AG genotype of EPCR with thrombotic events in AML. Therefore, the presence of the 6936AG genotype in AML patients may be considered as a risk indicator of thrombosis. [ABSTRACT FROM AUTHOR]

The article presents updates on the articles published in the "European Journal of Clinical Investigation" (EJCI) in the year 2012. It notes that obesity and insulin resistance affect oxidative stress development. Data that suggests that weight loss after bariatric surgery is connected with complement C3 and risks of metabolic and atherosclerotic processes is presented. It highlights relationship between cardiovascular diseases in peripheral arterial disease and myeloperoxidase levels.

The complex structure and function of the cerebral cortex critically depend on the balance of excitation and inhibition provided by the pyramidal projection neurons and GABAergic interneurons, respectively. The calretinin-expressing (CalR+) cell is a subtype of GABAergic cortical interneurons that is more prevalent in humans than in rodents. In rodents, CalR+ interneurons originate in the caudal ganglionic eminence (CGE) from Gsx2+ 34 progenitors, but in humans it has been suggested that a subpopulation of CalR+ 35 cells can also be generated in the cortical ventricular/subventricular zone (VZ/SVZ). The progenitors for cortically generated CalR+ subpopulation in primates are not yet characterized. Hence, the aim of this study was to identify patterns of expression of the transcription factors (TFs) that commit cortical stem cells to the CalR fate, with a focus on Gsx2. First, we studied the expression of Gsx2 and its downstream effectors, Ascl1 and Sp8 in the cortical regions of the fetal human forebrain at midgestation. Next, we established that a subpopulation of cells expressing these TFs are proliferating in the cortical SVZ, and can be co-labeled with CalR. The presence and proliferation of Gsx2+ cells, not only in the ventral telencephalon (GE) as previously reported, but also in the cerebral cortex suggests cortical origin of a subpopulation of CalR+ 44 neurons in humans. In vitro treatment of human cortical progenitors with Sonic hedgehog (Shh), an important morphogen in the specification of interneurons, decreased levels of Ascl1 and Sp8 proteins, but did not affect Gsx2 levels. Taken together, our ex-vivo and in vitro results on human fetal brain suggest complex endogenous and exogenous regulation of TFs implied in the specification of different subtypes of CalR+ cortical interneurons. [ABSTRACT FROM AUTHOR]

The expression of At4g34880 gene encoding amidase in Arabidopsis was characterized in this study. A promoter region of 1.5 kb on the upstream of the start codon of the gene (referred as AmidP) was fused with uidA (GUS) reporter gene, and transformed into Arabidopsis plant for determining its spatial expression. The results indicated that AmidP drived GUS expression in vascular system, predominately in leaves. Truncation analysis of AmidP demonstrated that VASCULAR VEIN ELEMENT (VVE) motif with a region of 176 bp sequence (−1500 to −1324) was necessary and sufficient to direct the vascular vein specific GUS expression in the transgenic plant. Tandem copy of VVE increased vascular system expression, and 5′- and 3′- deletions of VVE motif in combination with a truncated −65 CaMV 35S minimal promoter showed that 11bp cis-acting element, naming DOF2 domain, played an essential role for the vascular vein specific expression. Meanwhile, it was also observed that the other cis-acting elements among the VVE region are also associated with specificity or strength of GUS activities in vascular system. [ABSTRACT FROM AUTHOR]

A 34-year-old Japanese woman was admitted to our hospital complaining of developing bilateral pedal edema. Imaging studies led to a diagnosis of Budd-Chiari syndrome combined with internal jugular vein thrombus. We investigated the cause of thrombosis and found that the anticoagulant activity of protein C was decreased. Genetic analysis showed the presence of a c.l25C>A (Arg42Ser) substitution in the protein C gene (PROC) of the proband, which generates an Arg42Ser mutation that replaces the scissile bond Arg42-Ala43 normally cleaved by a furin-like processing protease. Her father and younger brother also carried this mutation, although they had no evidence of thrombosis. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Genetic variation in the Protein C gene (PROC) is associated with altered risk of adverse outcome for a number of diseases. Common single nucleotide polymorphisms (SNPs) in the promoter region and the adjacent 5’ region of PROC are associated with Protein C expression. We tested the hypothesis that common SNPs (minor allele frequency >10%) between the frequently studied promoter SNPs -1654 (rs1799808) and -1641 (rs1799809), and the end of PROC intron 2 alter nuclear transcription factor binding. Materials and Methods: We used electrophoretic mobility shift assays with 25-mer oligonucleotides centered on each of the 10 SNPs assessed in this potential regulatory region of the Protein C gene to test for differential binding to nuclear factors isolated from Hep-G2 cells. Results: We found that the G-allele oligo of the intron 2 SNP rs2069915[G/A] bound nuclear factors more avidly than the A-allele (p=1.9×10-9, n=24). Similarly, we found that the C-allele oligo of the intron 2 SNP rs2069916[C/T] bound nuclear factors more avidly than the T-allele, (p=3.7×10-6, n=19). Cold competition and supershift assays suggested that the protein differentially binding to the C-allele of rs2069916 was USF1. Notably, we observed minimal nuclear factor binding to oligos containing haplotypes of the previously reported -1654 and -1641 SNPs. Luciferase reporter assays that showed the A-T haplotype of rs2069915 and rs2069916 drives transcription significantly more than the C-G haplotype (t-test, P=0.015, n=12). Conclusion: Differential transcription factor binding occurs for common SNPs in the 5’ intronic regions of PROC which may contribute to PROC regulation and reported PROC SNP – phenotype associations. [Copyright &y& Elsevier]

Introduction: Trauma patients are at high risk of complications and death from coagulopathy and inflammatory organ failure. Recent evidence implicates protein C (PC) as a key mediator of this process. We hypothesized that a mutation in the PC gene would ameliorate the inflammatory and coagulopathic response to hemorrhagic shock (HS) and resuscitation. Methods: FHH wild type and PC mutant rats underwent controlled hemorrhage for 120 min with 70% of blood volume removed. Rats were resuscitated with Ringers lactate (2x shed blood volume) and shed blood. Animals were sacrificed 4 h post-HS. Controls were untreated naïve rats. Results: AST and NFkB lung protein levels were elevated similarly in both WT and mutants compared with naïve rats. Plasma fibrinogen levels decreased significantly with progression of HS compared with baseline (BL) levels and returned towards normal 4 h after resuscitation. PC activity was similar in both groups at BL (0.5 ± 0.08 versus 0.6 ± 0.14; P = 0.14) and decreased from BL by 53% ± 24% in WT (P =0.08), by 67% ± 11% in mutants (P = 0.03) at sacrifice, and was not different between groups (P = 0.29). Conclusions: Our model of HS and resuscitation produced a hypocoaguable, hyperinflammatory state with increased levels of NFkB and decreased levels of fibrinogen and PC levels. The mutated PC did not appear to alter these responses in our model of HS and resuscitation. [Copyright &y& Elsevier]

The elucidation of the genomes of a large number of mammalian species has produced a huge amount of data on which to base physiological studies. These endeavours have also produced surprises, not least of which has been the revelation that the number of protein coding genes needed to make a mammal is only 22 333 (give or take). However, this small number belies an unanticipated complexity that has only recently been revealed as a result of genomic studies. This complexity is evident at a number of levels: (i) cis-regulatory sequences; (ii) noncoding and antisense mRNAs, most of which have no known function; (iii) alternative splicing that results in the generation of multiple, subtly different mature mRNAs from the precursor transcript encoded by a single gene; and (iv) post-translational processing and modification. In this review, we examine the steps being taken to decipher genome complexity in the context of gene expression, regulation and function in the hypothalamic-neurohypophyseal system (HNS). Five unique stories explain: (i) the use of transcriptomics to identify genes involved in the response to physiological (dehydration) and pathological (hypertension) cues; (ii) the use of mass spectrometry for single-cell level identification of biological active peptides in the HNS, and to measure in vitro release; (iii) the use of transgenic lines that express fusion transgenes enabling (by cross-breeding) the generation of double transgenic lines that can be used to study vasopressin (AVP) and oxytocin (OXT) neurones in the HNS, as well as their neuroanatomy, electrophysiology and activation upon exposure to any given stimulus; (iv) the use of viral vectors to demonstrate that somato-dendritically released AVP plays an important role in cardiovascular homeostasis by binding to V1a receptors on local somata and dendrites; and (v) the use of virally-mediated optogenetics to dissect the role of OXT and AVP in the modulation of a wide variety of behaviours. [ABSTRACT FROM AUTHOR]

J. Neurochem (2012) 120, 699-709. Abstract L1 cell adhesion molecule (L1CAM), an adhesion/signaling protein encoded by a gene target of the transcription repressor RE-1-Silencing Transcription factor (REST), is expressed in two alternatively spliced isoforms. The full-length isoform, typical of low-REST neural cells, plays key roles in survival/migration, outgrowth/fasciculation/regeneration of axons, synaptic plasticity; the isoform missing two mini-exons, abundant in a few high-REST non-neural cells, maintains some effect on migration and proliferation. To investigate whether and how L1CAM alternative splicing depends on REST we used neural cell models expressing low or high levels of REST (PC12, SH-SY5Y, differentiated NT2/D1 and primary neurons transduced or not with REST). The short isoform was found to rise when the low-REST levels of neural cells were experimentally increased, while the full-length isoform increased in high-REST cells when the repressor tone was attenuated. These results were due to Nova2, a neural cell-specific splicing factor shown here to be repressed by REST. REST control of L1CAM occurs therefore by two mechanisms, transcription and alternative splicing. The splicing mechanism, affecting not only L1CAM but all Nova2 targets (∼7% of brain-specific splicing, including the mRNAs of other adhesion and synaptic proteins) is expected to be critical during development and important also for the structure and function of mature neural cells. [ABSTRACT FROM AUTHOR]

Abstract: REM2 is a member of the REM, RAD, and GEM/KIR (RGK) subfamily of RAS superfamily proteins and plays an important role in brain development and function. In this study, two Rem2 isoforms were isolated from the rainbow trout (Oncorhynchus mykiss). The two genes, designated O. mykiss rem2a and rem2b, both encode 304 amino acid proteins with 61% and 62% identities to zebrafish (Danio rerio) Rem2, respectively, and each with 43% identity to mammalian (human) REM2. To our knowledge, this is the first incidence of Rem2 isoforms in a species that are the result of gene duplication. Both isoforms possessed similar tissue expression profiles with the highest levels in the brain. The rem2a gene has significantly higher expression levels than rem2b in all tissues assayed except the brain and head kidney. In the central nervous system, both isoforms showed similar expression levels with the highest levels occurring in the olfactory bulb, cerebrum, and midbrain, though rem2a expression is significantly higher in the spinal cord. Based on known functional roles of Rem2 in synapse development and stem cell proliferation, the characterization of Rem2 in rainbow trout could shed light on its role in adult vertebrate neurogenesis and brain regeneration. [Copyright &y& Elsevier]

Background: The receptor for activated C kinase 1 (RACK1) has been shown to be overexpressed in several types of cancers such as breast, colon, melanomas, and lung. RACK1 is linked to Ras-Raf-mediated signal transduction and transformed foci formation of 3T3 cells in vitro, and since this pathway is central in papillary thyroid carcinoma (PTC) oncogenesis, we hypothesized that RACK1 could play a role in the development or maintenance of PTC. No report on RACK1 expression in thyroid tissue is available; the present study was therefore aimed at identifying possible correlation of RACK1 expression at the mRNA or protein level in normal thyroid tissue compared to PTC. Methods: We used TaqMan quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry to study the RACK1 gene and protein expression in matched tumor and nontumor samples from 59 PTC patients. The tumor samples were divided into two main categories, low-risk (group 1-3) and high-risk (group 4-6), in accordance with both histological classification and clinical appearance. Results: RACK1 mRNA and protein levels were found highly overexpressed in tumor samples, whereas Ki-Ras mRNA was found to be relatively unchanged. B-Raf mRNA expression was low and detected only in tumor samples. Sequencing analysis detected no mutations in RACK1 or Ki-Ras, but 62.7% of the patients harbored the B-Raf single-nucleotide substitution T1799A (codon V600E). Phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry analysis demonstrated activation of the mitogen-activated protein kinase (MAPK) pathway in tumor cells. Poorly differentiated and undifferentiated PTCs expressed significantly higher RACK1 mRNA levels than well-differentiated PTCs ( p<0.017). Conclusions: Taken together, our findings point to an important role of RACK1 protein in PTC development and progression. Our data also emphasize the importance of assessing protein expression and not only mRNA levels. [ABSTRACT FROM AUTHOR]

The authors reflect on the characterization of mitral valve leaflets in hypertrophic cardiomyopathy (HCM) through cardiovascular magnetic resonance (CMR) imaging. They state that a thickened maximal left ventricular (LV) wall will not affect leaflet length. The authors note that mutation of the myosin-binding protein C gene has resulted from HCM with asymptomatic genotype.

Glucocorticoids regulate numerous physiological processes and are mainstays in the treatment of inflammation, autoimmune disease, and cancer, The traditional view that glucocorticoids act through a single glucocorticoid receptor (GR) protein has changed in recent years with the discovery of a large cohort of receptor subtypes arising from alternative processing of the GR gene. These isoforms differ in their expression, gene regulatory, and functional profiles. Post-translational modification of these proteins further expands GR diversity. Here, we discuss the origin and molecular properties of the GR isoforms and their contribution to the sensitivity and specificity of the glucocorticoid response. [ABSTRACT FROM AUTHOR]

Hypotheses about horizontal transfer of antifreeze protein genes to ice-living diatoms were addressed using two different statistical methods available in the program Prunier. The role of diversifying selection in driving the differentiation of a set of antifreeze protein genes in the diatom genus Fragilariopsis was also investigated. Four horizontal gene transfer events were identified. Two of these took place between two major eukaryote lineages, that is from the diatom Chaetoceros neogracile to the copepod Stephos longipes and from a basidiomycete clade to a monophyletic group, consisting of the diatom species Fragilariopsis curta and Fragilariopsis cylindrus. The remaining two events included transfers from an ascomycete lineage to the proteobacterium Stigmatella aurantiaca and from the proteobacterium Polaribacter irgensii to a group composed of 4 proteobacterium species. After the Fragilariopsis lineage acquired the antifreeze protein gene from the basidiomycetes, it duplicated and went through episodic evolution, characterized by strong positive selection acting on short segments of the branches in the tree. This selection pattern suggests that the paralogs differentiated functionally over relatively short time periods. Taken together, the results obtained here indicate that the group of antifreeze protein genes considered here have a complex evolutionary history. [ABSTRACT FROM AUTHOR]

The article presents a study on the protein encoded by the protein gene NCgll221 in corynebacterium glutamicum (C. glutamicum) functions as a mechanosensitive channel. The study involves an examination on small conductance (MscS) protein function and expressions of C. glutamicum NCgll221, which were induced in the neomycin-resistant gene Bacilus subtilis (B. subtilis). The results show that the protein encoded by NCgll221 functioned as a mechanosensitive channel.

far as protein-coding genes are concerned, there is a non-zero probability that at least one of the five possible overlapping sequences of any gene will contain an open-reading frame (ORF) of a length that may be suitable for coding a functional protein. It is, however, very difficult to determine whether or not such an ORF is functional. Recently, we proposed a method that predicts functionality of an overlapping ORF if it can be shown that it has been subject to purifying selection during its evolution. Here, we use simulation to test this method under several conditions and compare it with the method of Firth and Brown. We found that under most conditions, our method detects functional overlapping genes with higher sensitivity than Firth and Brown’s method, while maintaining high specificity. Further, we tested the hypothesis that the two aminoacyl tRNA synthetase classes have originated from a pair of overlapping genes. A central piece of evidence ostensibly supporting this hypothesis is the assertion that an overlapping ORF of a heat-shock protein-70 gene, which exhibits some similarity to class 2 aminoacyl tRNA synthetases, is functional. We found signature of purifying selection only in highly divergent sequences, suggesting that the method yields false-positives in high sequence divergence and that the overlapping ORF is not a functional gene. Finally, we examined three cases of overlap in the human genome. We find varying signatures of purifying selection acting on these overlaps, raising the possibility that two of the overlapping genes may not be functional. [ABSTRACT FROM AUTHOR]

Glucagon and the glucagon-like peptides (GLP-1 and GLP-2) share a common evolutionary origin and are triplication products of an ancestral glucagon exon. In mammals, a standard scenario is found where only a single proglucagon-derived peptide set exists. However, fish and amphibians have either multiple proglucagon genes or exons that are likely resultant of duplication events. Through phylogenetic analysis and examination of their respective functions, the proglucagon ligand-receptor pairs are believed to have evolved independently before acquiring specificity for one another. This review will provide a comprehensive overview of current knowledge of proglucagon-derived peptides and receptors, with particular focus on fish and amphibian species. [ABSTRACT FROM AUTHOR]

Borrelia burdorferi genotype in the northeastern United States is associated with Lyme borreliosis severity. Analysis of DNA sequences of the outer surface protein C gene and rrs-rrlA intergenic spacer from extracts of Ixodes spp. ticks in 3 US regions showed linkage disequilibrium between the 2 loci within a reqion but not consistently between regions. [ABSTRACT FROM AUTHOR]

Activated protein C (APC) is an important anticoagulant which plays a role in pathophysiology of pregnancy, e.g. in maintenance of the uteroplacental circulation and developement of the fetus as well as in pathogenesis of preeclampsia. The study objective was to compare the levels of the respective components of the protein C system (protein C, PC; protein S, PS; thrombomodulin, TM) as well as thrombin activatable fibrinolysis inhibitor - TAFI in mother's blood, cord blood and amniotic fluid. The study group consisted of 136 healthy parturients at term, divided into subgroups of 30-35. The immunoenzymatic method (ELISA) was used to measure the antigens of the components studied. The concentrations of PC and PS antigens were the highest in the mother's blood plasma (135.11±1.05% and 92.0±13.24%, respectively), lower in cord blood plasma (57.60±10.32% and 33.19±4.96%, respectively) and the lowest in amniotic fluid (6.75±3.50% and 2.40±1.64%, respectively); the differences between the levels of that of mother, fetus and amniotic fluid were statistically significant (p≤0.0001). The TM and TAFI antigen concentrations were the highest in cord blood plasma (11.35±3.71 ng/ml and 91.50 (median; range: 71.76-160.77) ng/ml, respectively) and lower in maternal plasma (4.51±0.71 ng/ml and 55.46 - median; range: 39.77-68.54 ng/ml, respectively); the differences between the levels of that of cord blood plasma and maternal plasma were statistically significant (p≤0.0001). Of the three protein C system components, PC and PS occur in relatively high concentrations in maternal blood, being lower in fetal blood and the lowest in amniotic fluid. On the other hand, as an exception, the concentrations of TM and TAFI are the highest in fetus blood. [ABSTRACT FROM AUTHOR]

HDL has anti-atherogenic properties, and plasma levels of HDL cholesterol correlate inversely with risk of coronary artery disease. HDL reportedly functions as a cofactor to the anticoagulant activated protein C (APC) in the degradation of factor Va (FVa). The aim of the present study was to elucidate the mechanism by which HDL functions as cofactor to APC. Consistent with a previous report, HDL isolated from human plasma by ultracentrifugation was found to stimulate APC-mediated degradation of FVa. However, further purification of HDL by gel filtration revealed that the stimulating activity was not a property of HDL. Instead, the stimulating activity eluted completely separately from HDL in the high-molecular-weight void volume fractions. The active portion of these fractions stimulated FVa degradation by APC and supported the assembly of factor Xa and FVa into a functional prothrombinase complex. Both the procoagulant and anticoagulant activities were blocked by addition of annexin V, suggesting that the active portion was negatively charged phospholipid membranes. These results demonstrate that HDL does not stimulate the APC/protein S effect and that the activity previously reported to be a property of HDL is instead caused by contaminating negatively charged phospholipid membranes. [ABSTRACT FROM AUTHOR]

Abstract: The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HC•TSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2•bikunin. In addition, Gg pre-α-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein–glycosaminoglycan–protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2•bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-α-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of HC transfer is likely to predate the role of HC·HA complexes in female fertility and thus has evolved in the context of inflammation rather than fertility. [Copyright &y& Elsevier]

Centromeres are the attachment points between the genome and the cytoskeleton: centromeres bind to kinetochores, which in turn bind to spindles and move chromosomes. Paradoxically, the DNA sequence of centromeres has little or no role in perpetuating kinetochores. As such they are striking examples of genetic information being transmitted in a manner that is independent of DNA sequence (epigenetically). It has been found that RNA transcribed from centromeres remains bound within the kinetochore region, and this local population of RNA is thought to be part of the epigenetic marking system. Here we carried out a genetic and biochemical study of maize CENPC, a key inner kinetochore protein. We show that DNA binding is conferred by a localized region 122 amino acids long, and that the DNA-binding reaction is exquisitely sensitive to single-stranded RNA. Long, single-stranded nucleic acids strongly promote the binding of CENPC to DNA, and the types of RNAs that stabilize DNA binding match in size and character the RNAs present on kinetochores in vivo. Removal or replacement of the binding module with HIV integrase binding domain causes a partial delocalization of CENPC in vivo. The data suggest that centromeric RNA helps to recruit CENPC to the inner kinetochore by altering its DNA binding characteristics. [ABSTRACT FROM AUTHOR]

The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act in effector triggered susceptibility. Here, we report the molecular cloning and functional characterization of the SnTox3-encoding gene, designated SnTox3, as well as the initial characterization of the SnTox3 protein. SnTox3 is a 693 bp intron-free gene with little obvious homology to other known genes. The predicted immature SnTox3 protein is 25.8 kDa in size. A 20 amino acid signal sequence as well as a possible pro sequence are predicted. Six cysteine residues are predicted to form disulfide bonds and are shown to be important for SnTox3 activity. Using heterologous expression in Pichia pastoris and transformation into an avirulent S. nodorum isolate, we show that SnTox3 encodes the SnTox3 protein and that SnTox3 interacts with the wheat susceptibility gene Snn3. In addition, the avirulent S. nodorum isolate transformed with SnTox3 was virulent on host lines expressing the Snn3 gene. SnTox3-disrupted mutants were deficient in the production of SnTox3 and avirulent on the Snn3 differential wheat line BG220. An analysis of genetic diversity revealed that SnTox3 is present in 60.1% of a worldwide collection of 923 isolates and occurs as eleven nucleotide haplotypes resulting in four amino acid haplotypes. The cloning of SnTox3 provides a fundamental tool for the investigation of the S. nodorum-wheat interaction, as well as vital information for the general characterization of necrotroph-plant interactions. [ABSTRACT FROM AUTHOR]

Many genes that are required at specific points in the cell cycle exhibit cell cycle-dependent expression. In the earlydiverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9-LIGKA, PNT1 and PNT2-were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3′ UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3′ UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we characterized a novel soybean gene encoding a neutral PR-5 protein and compared it to two acidic isoforms of soybean PR-5 protein. This gene, designated as Glycine max osmotin-like protein, b isoform (GmOLPb, accession no. AB370233), encoded a putative protein having the greatest similarity to chickpea PR-5b (89% identity). Unlike the two acidic PR-5, GmOLPa and P21, the protein had a C-terminal elongation responsible for possible vacuolar targeting and after maturation showed a calculated molecular mass of 21.9kDa with pI 6.0. The 3D models, predicted by the homology modeling, contained four α-helixes and 16 β-strands and formed three characteristic domains. The two acidic PR-5 proteins also showed a 3D structure very similar to GmOLPb, although the electrostatic potential on molecular surface of each PR-5 was significantly different. In the study of the gene expression under conditions of high-salt stress, GmOLPb was highly induced in the leaves of the soybean, particularly in the lower part of a leaf. The expression started at 2h after initiation of the stress and was highly induced between 18–72h. Gene expression of P21e (protein homologous to P21) was transiently induced by high-salt stress, but took place earlier than the gene expressions of GmOLPa and GmOLPb. Such differential expression was observed also under investigation with methyl jasmonate and salicylic acid. These results suggested that each soybean PR-5 might play a distinctive role in the defensive system protecting the soybean plant against high-salt stress, particularly in the leaves of the soybean. [Copyright &y& Elsevier]

BCL2 and BCL-xL facilitate G0 quiescence by decreasing RNA content and cell size and up-regulating p27 protein, but the precise mechanism is not understood. We investigated the relationship between cell cycle regulation and the anti-apoptosis function of BCL2 and BCL-xL. Neither caspase inhibition nor abrogation of mitochondria-dependent apoptosis by BAX and BAK deletion fully recapitulated the G0 effects of BCL2 or BCLxL, suggesting that mechanisms in addition to anti-apoptosis are involved in the cell cycle arrest function of BCL2 or BCL-xL. We found that BCL2 and BCL-xL expression in bax-/- bak-/- cells did not confer cell cycle effects, consistent with the G0 function of BCL2 and BCL-xL being mediated through BAX or BAK. Stabilization of p27 in G0 in BCL2 or BCL-xL cells was due to phosphorylation of p27 at Ser10 by the kinase Mirk. In bax-/- bak-/- cells, total p27 and p27 phosphorylated at Ser10 were elevated. Re-expression of BAX in bax-/- bak-/- cells and silencing of BAX and BAK in wild type cells confirmed that endogenous BAX and BAK modulated p27. These data revealed a novel role for BAX and BAK in the regulation of G0 quiescence. [ABSTRACT FROM AUTHOR]

Macaca thibetana is a threatened primate species endemic to China. Genetic diversities based on a 476-bp fragment at the 5'-end of the mitochondrial DNA control region HVSI were assessed. Haplotype diversity is high (0.8521), but nucleotide diversity among all haplotypes is only 0.0574. No haplotype was shared between Sichuan (SC) and Huangshan Mountain (HS) populations. Phylogenetic trees, analysis of molecular variance and network analysis consistently indicated that the SC and HS populations are significantly different. They should therefore be conserved as different units, with priority and more attention given to the HS populations. [ABSTRACT FROM AUTHOR]

We determined the complete mitochondrial genomes of Pseudolabrus sieboldi and P. eoethinus, and analyzed the genome organization, codon usage, and transition/transversion mutation ratio of the mitochondrial genome. The mitochondrial genomes of P. sieboldi and P. eoethinus are 16,507 and 16,508 bp in length, respectively, and consisted of 37 genes (13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs), which is typical for vertebrate mitochondrial DNA. All protein-coding genes of two species used the initiation codon ATG except the cytochrome c oxidase subunit (CO) 1, which began with GTG as an initiation codon. However, the termination codon for the NADH dehydrogenase subunit (ND) 6 gene encoded with TAA in P. sieboldi, and TAG in P. eoethinus. The 12S and 16S rRNA genes were 949 and 1694 bp, respectively, in P sieboldi, and were 948 and 1693 bp in P. eoethinus. The A+T content of the two rRNA genes were 52.9% in P. sieboldi and 52.5% in P. eoethinus, which is slightly lower than that of other labrid species. The identity of the 13 protein-coding genes ranged between 67% (ND6) and 94% (CO2 and ATP8). The G+C contents of all of the protein-coding genes of P. sieboldi were slightly higher than those of P. eoethinus. Our data contribute to the identification, and further our understanding, of the comparative genetics of Pseudolabrus species distributed in East Asia. [ABSTRACT FROM AUTHOR]

Using PCR, the complete mitochondrial genome was sequenced in three frillneck lizards (Chlamydosaurus kingii). The mitochondria spanned over 16,761 bp. As in other vertebrates, two rRNA genes, 22 tRNA genes and 13 protein coding genes were identified. However, similar to some other squamate reptiles, two control regions (CRI and CRII) were identified, spanning 801 and 812 bp, respectively. Our results were compared with another Australian member of the family Agamidae, the bearded dragon (Pogana vitticeps). The overall base composition of the light-strand sequence largely mirrored that observed in P. vitticeps. Furthermore, similar to P. vitticeps, we observed an insertion 801 bp long between the ND5 and ND6 genes. However, in contrast to P. vitticeps we did not observe a conserved sequence block III region. Based on a comparison among the three frillneck lizards, we also present data on the proportion of variable sites within the major mitochondrial regions. [ABSTRACT FROM AUTHOR]

AbstractHbUEP, an ubiquitin extension protein gene from latex of the rubber tree (Hevea brasiliensis) was cloned and sequenced using a differentially ethphon-induced expressed cDNA subtraction library. The cDNA had 771 bp nucleotides, comprising a 226 bp 3? untranslated region (UTR), 77 bp 5?UTR and a 468 bp open reading frame encoding a 156 amino acid peptide. Southern blotting analysis showed that this gene was a low copy number gene in the H. brasiliensisgenome. Within 24 h after application of ethphon, the gene was expressed weakly in both control and latex sampled at 6 h, and strongly in latex sampled at 12 h, showing that this gene expression could be regulated by ethphon. Ethphon could increase the latex yield in H. brasiliensis. It is suggested that the HbUEPgene may be involved in the regulation of ethphon-induced high latex yield in H. brasiliensis. [ABSTRACT FROM AUTHOR]

The tufted deer Elaphodus cephalophus are endangered animals in the world and little is understood about their mitochondrial (mt) genome. In our study, the mt genome of the tufted deer is identified—which is about 16 kb in length and contains 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a non-coding sequence (control region). The distinguishing feature is that GTG is the start codon of the NADH4L gene and the cyt b gene has a subterminal AAA followed by the stop codon TAG. According to 12 H strand protein-coding genes and phylogenetic analysis, Elaphodus may have a sister relationship with another deer group Muntiacus. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genome of the yellowfin seabream Acanthopagrus latus was determined in the present study. The genome was 16,609 bp in length and contained 37 genes (2 ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) and the control region (CR), with the content and order of genes being similar to those in typical teleosts. Comparisons of the 37 genes and CR among species indicate the CR was the highest divergent (0.3341), but tRNAGly possesses the lowest genetic variation (0.0542). Much greater p-genetic distances [mean = 0.1559, standard deviation (SD) = 0.0235; n = 1653] for the interspecies level with high frequency (99.4%) than those of the intraspecies level (mean = 0.0098, SD = 0.0090; n = 20) were inferred from 212 Cyt b sequence data, suggesting the Cyt b gene is conserved within Sparidae species and supporting the barcoding validity of Cyt b sequence data for Sparidae species identification. Phylogenetic analysis using amino acid sequences of 13 protein-coding genes supported that the genus Pagrus was not monophyletic, showing the need to re-evaluate the morphological characteristics of Pagrus fishes. [ABSTRACT FROM AUTHOR]

The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counter-selected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-β-D-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes. [ABSTRACT FROM AUTHOR]

Objective: The exposure of mammalian cells to extracellular stress induces the expression of immediate early genes such as c-fos and c-jun and activates transcription factor activator protein-1 (AP-1). The purpose of the current study was to investigate the role of c-Fos and JunD in stress-induced cell death. Materials and methods: We exposed cultured primary mouse embryonic fibroblasts (MEF) to ultraviolet light (UV-C) or hydrogen peroxide (H2O2). Induction of c-Fos and JunD and activation of MAPK/ERK1/2 signalling in the presence or absence of a MAPK inhibitor were analyzed by western blotting. Activation of AP-1 transcription factors was detected by the electrophoretic mobility shift assay and immunoprecipitation. Cell death was measured by changes in caspase 3 activities and nuclear morphology. Effects of c-Fos and JunD expression on cell death were investigated by transfection. Results: We found that the exposure of cultured primary MEF cells to UV or H2O2 caused a significant increase in c-Fos and JunD protein levels. In addition, these two proteins formed complexes with each other and contributed to activation of AP-1 transcription complexes. More importantly, under both stress conditions, overexpression of JunD alone or overexpression of both c-Fos and JunD reduced caspase 3 activity and cell death. At the same time, UV irradiation activated the MAPK/ERK1/2 signalling pathway. The suppression of MEK1/ERK1/2 activation inhibited UV-induced expression of c-Fos and JunD and increased caspase 3 activity and cell death. Conclusion: Our results suggest that both UV and H2O2 induce the activation of c-Fos/JunD AP-1 complexes resulting in the prevention of cell death. Moreover, UV irradiation-induced increases in c-Fos/JunD expression in primary MEF cells are mediated through the activation of the MAPK/ERK1/2 signalling pathway. [ABSTRACT FROM AUTHOR]

A genome wide scan in a consanguineous family of Indian origin with autosomal recessive developmental cataracts was performed by two-point linkage analysis with 382 microsatellite markers. It showed linkage to markers on chromosome 20q, between D20S852 and D20S912, with a maximum lod score of 5.4 obtained with D20S860. This region encompasses the beaded filament structural protein 1 ( BFSP1) gene. Direct sequencing revealed a 3343 bp deletion including exon 6 (c.736-1384_c.957-66 del) predicted to result in a shift of the open reading frame. This mutation was absent in 50 control individuals from south India. This is the first report of a mutation in the BFSP1 gene associated with human inherited cataracts. This further increases the genetic heterogeneity of inherited cataracts and provides clues as to the importance of BFSP1 in the cell biology of intermediate filaments and their role in the eye lens. [ABSTRACT FROM AUTHOR]

Abstract: The recent resurgence of Plasmodium vivax malaria requires close epidemiological surveillance and monitoring of the circulating parasite populations. In this study, we developed a combination of polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) method to investigate the genetic diversity of the P. vivax merozoite surface protein 3β (PvMSP3β) gene among four Asian parasite populations representing both tropical and temperate strains with dramatic divergent relapse patterns (N =143). Using P. vivax field isolates from symptomatic patients, we have validated the feasibility of this protocol in distinguishing parasite genotypes. We have shown that PCR alone could detect three major size polymorphisms of the PvMSP3β gene, and restriction analysis detected a total of 12 alleles within these Asian samples. Samples from different geographical areas differed dramatically in their PvMSP3β allele composition and frequency, indicating that complex, yet different parasite genotypes were circulating in different endemic areas. This protocol allowed easy detections of multiple infections, which reached 20.5% in the samples from Thailand. It is interesting to note that samples from one temperate site in China collected during a recent outbreak of the disease also showed a high level of genetic diversity with multiple infections accounting for 5.6% of the samples. When combined with the PvMSP3α locus, this method provides better capability in distinguishing P. vivax genotypes and detecting mixed genotype infections. [Copyright &y& Elsevier]

The complete sequence (27,184 bp) of the mitochondrial (mt) genome of the phytopathogenic fungus Verticillium dahliae has been determined. It contains 14 protein-coding genes related to oxidative phosphorylation, two rRNA genes and a set of 25 tRNA genes. A single intron, that harbors an intronic ORF coding for a putative ribosomal protein ( rps), is located within the large rRNA gene ( rnl). Gene order comparisons of V. dahliae mtDNA and complete mt genomes of Pezizomycotina revealed four units of synteny for Sordariomycetes, namely rnl- trn (11–12)- nad2- nad3, nad4L- nad5- cob- cox1, nad1- nad4- atp8- atp6 and rns- trn (1–5)- cox3- trn (1–5)- nad6- trn (2–5). These four units, in different combinations, merged to single continuous unit in the orders of Hypocreales and Sordariales. V. dahliae (Phyllachorales) and all members of the genus showed a unique feature which is the translocation of the nad1- nad4- atp8- atp6- rns- cox3- nad6 region in between genes nad3 and atp9 of the Hypocreales mtDNA gene order. Analysis of mt intergenic sequences of Verticillium species permitted the design of a species-specific primer allowing the discrimination of V. longisporum against V. dahliae and V. albo-atrum. By considering the protein-coding gene sequences as one unit, a phylogenetic comparison with representatives of Ascomycota complete mtDNA was performed. [ABSTRACT FROM AUTHOR]

Regulatory DNA elements responsible for human protein C (PROC) gene expression have previously been identified in the upstream promoter region and first (untranslated) exon of the gene. Here we show that an additional sequence element located more than 500 bp downstream of the core promoter within intron 1 further enhances PROC promoter-driven reporter gene expression in human hepatoma cells. In common with core promoter constructs used in previous studies, the activity of this 3'-extended regulatory region is diminished by a naturally occurring promoter mutation. However, in contrast to constructs lacking intronic sequence, the promoter/intron regulatory region is repressed rather than activated by the transcription factor HNF-1. Using both conventional alignment procedures and complexity analysis to study the human and canine PROC sequences, we identified two conserved intronic regions, which were tested for their involvement in gene regulation. High-level gene expression from the intron-coupled promoter was dependent upon the integrity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for gene regulation and serve to illustrate that the results of PROC promoter/reporter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functional evaluation of regulatory mutations and polymorphisms. [ABSTRACT FROM AUTHOR]

Fifteen patients with selective IgG1 deficiency were screened for immunoglobulin H chain C region locus (IGHC) gene deletions and three deletion haptotypes were found: delG1, delG1-G4 and del G4. These haplotypes. together with four deletion haplotypes described by us previously (del G1(NY), del GI (VIT) del G1-G2 (NY) and del G2-G4 (HJE)), were further characterized using pulsed-field get electrophoresis (PFGE) to determine the physical extent of the deletions. The Mlu1 fragment sizes confirmed the deletions, although the deduced sizes of the most extensive deletions indicated that material had been inserted into the locus. [ABSTRACT FROM AUTHOR]

Abstract: The present study investigated cDNA chimeras using two closely related members of the rice secretory protein gene family as an example. The chimeras detected in initial cDNA products that were amplified using LA Taq polymerase involved two categories: single-site type and multiple-site type with the frequency being about 20% and 3%, respectively. Further investigation revealed that PCR buffer additives and type of DNA polymerase had a major effect on the formation of chimeras in mixed-template amplification. Heteroduplex repair by microbial DNA repair systems in cDNA cloning was confirmed to produce the chimeras too, but it was not the major source. [Copyright &y& Elsevier]

The aim of this study was to determine whether total fertilization failure in human IVF can be partially explained by alterations in the gene that codes for protein C inhibitor. Forty-six men had IVF total fertilization failure and 51 controls with normal fertilization were screened for mutations in the protein C inhibitor gene by direct sequencing. The main finding was that in men involved in total fertilization failure, a heterozygous adenosine/guanine (A/G) base combination in position 1389 (rs2069990) (exon 6) in the protein C inhibitor gene was significantly more common compared with controls (10.9% vs. 0). [Copyright &y& Elsevier]

Background Melanoma is the most aggressive skin cancer due to its high metastatic propensity and resistance to most traditional chemotherapeutic drugs [1,2]. At early stage melanoma can be cured by surgical excision, whereas metastatic melanoma is a highly lethal condition. To understand melanoma progression is crucial identify mutations that are involved in making an individual melanoma competent for metastatic spread. The most frequent known oncogenic mutation in melanoma is BRAF-V600E and several whole exome sequencing studies have revealed numerous other alterations [3-6]. It is well established that the aggressive behavior of melanoma is highly correlated with histological features, such as the thickness of the primary tumor and the mitotic index. Here we performed whole exome sequencing of 5 thin (<1mm in thickness) and 5 thick (>4mm in thickness) primary melanomas compared to matchednormal DNA. Materials and methods We have collected 10 fresh primary melanomas from 10 untreated patients: DNA samples from melanoma tissues and peripheral blood (normal DNA) were available from all of the recruited patients. Genomic DNA was extracted from tumor and peripheral blood samples using the QIAamp DNA Minikit, (Qiagen, Hilden, Germany). Extracted DNA was used for Next-Generation Sequencing analysis by Illumina. Results We confirmed recurrent somatic mutations in known melanoma-related genes, including BRAF, c-KIT, EGFR, PPP6C, MLL3 and several components of the glutamate signaling. In addition, we discovered mutations in genes not previously linked to this tumor, such as CSMD1, FGFR4 and components of the Hedgehog (HH) signaling pathway. In particular, in a thick melanoma we found a novel activating mutation in the transcription factor GLI1, one of the final effectors of the HH signaling. Additionally, we identified candidate metastasisdriving mutations such as ADAMTS6, ADAMTS7, CHD9, MLL3, NALCN and TSC2 in the 3 thick melanomas that produced metastasis. Interestingly, we identified several regions of focal somatic copy-number alterations (SCNAs) that were altered at significantly higher frequency in thick compared to thin melanomas. Several gene families are comprised among these regions of focal SCNAs, including components of Notch, HH and Wnt/β-catenin signaling pathways, BRAF, c-MYC and its cofactor PIM1, several ADAMs, EGFR and the HOX genes. Conclusion Our data identify potential drivers of melanoma progression, enhancing our understanding of the genomic complexity underlying melanoma. [ABSTRACT FROM AUTHOR]