An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth.

The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.

Taking Action in Student Harassment Situations: Application of the Behaviour Change Wheel to Bystander Intervention.

This study applied the Behaviour Change Wheel (BCW) to understand barriers and facilitators to bystander behaviors in UK students. The BCW includes detailed examination of the capabilities, opportunities, and motivations involved in behaviors. Two surveys (n = 515; n = 201) and a focus group study (n = 12) were undertaken. Capability to intervene may be influenced by confidence and beliefs about physical ability and safety. Students appeared to have the physical opportunity to intervene, but social opportunity might be influenced by cultural norms. Motivations might be influenced by beliefs as well as inherent stereotypes about perpetrators and victims. Behavior change techniques (BCTs) such as instruction on how to perform the behavior, reattribution and creating a valued self-identity should be applied to overcome these barriers. A logic model to theorize the change processes underlying bystander behaviors in this population offers a new perspective on what needs to be addressed in interventions.

KCNJ10 mutations display differential sensitivity to heteromerisation with KCNJ16.

BACKGROUND/AIMS: Mutations in the inwardly-rectifying K(+)-channel KCNJ10/Kir4.1 cause autosomal recessive EAST syndrome (epilepsy, ataxia, sensorineural deafness and tubulopathy). KCNJ10 is expressed in the distal convoluted tubule of the kidney, stria vascularis of the inner ear and brain glial cells. Patients diagnosed clinically with EAST syndrome were genotyped and mutations in KCNJ10 were studied functionally. METHODS: Patient DNA was amplified and sequenced, and new mutations were identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K(+) currents were measured by 2-electrode voltage clamping and channel expression was analysed by Western blotting. RESULTS: We identified 3 homozygous mutations in KCNJ10 (p.F75C, p.A167V and p.V91fs197X), with mutation p.A167V previously reported in a compound heterozygous state. Oocytes expressing wild-type human KCNJ10 showed inwardly rectified currents, which were significantly reduced in all of the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba(2+) demonstrated a large residual function in p.A167V only, which was not compatible with causing disease. However, co-expression with KCNJ16 abolished function in these heteromeric channels almost completely. CONCLUSION: This study provides an explanation for the pathophysiology of the p.A167V KCNJ10 mutation, which had previously not been considered pathogenic on its own. These findings provide evidence for the functional cooperation of KCNJ10 and KCNJ16. Thus, in vitro ascertainment of KCNJ10 function may necessitate co-expression with KCNJ16.

Study of the effect of antiviral therapy on homocysteinemia in hepatitis C virus- infected patients.

BACKGROUND: Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease (CLD). About 80% of those exposed to the virus develop a chronic infection. Hyperhomocysteinemia, which is an independent risk factor for atherosclerotic vascular disease and thromboembolism, may develop in HCV-infected patients although altered alanine amino transferase (ALT) enzyme levels are generally associated with damage to liver cells. The gold standard therapy for chronic hepatitis C patients is pegylated interferon combined with an anti-viral drug (ribavirin). The current study aimed to investigate the effect of antiviral therapy on plasma homocysteine (Hcy) levels in HCV patients in addition to other parameters. METHODS: 532 HCV-infected patients and 70 healthy controls were recruited for the study. All patients were subjected to laboratory investigations including HCV-RNA levels, complete blood cell counts, serum levels of homocysteine, ALT, alkaline phosphatase (ALP), lipid profile and liver ultrasonographic examination. The outcome of treatment with pegylated interferon α plus ribavirin treatment and sustained virologic response (SVR) was determined 6-9 months post-therapy. RESULTS: Hyperhomocysteinemia was found in 91.35% of HCV-infected patients. The difference in plasma Hcy concentrations reached statistical significance between the patient and control groups. ALT, cholesterol and triglycerides (TGs) levels were found higher than normal in the patients group. After receiving a combined therapy for 24 weeks, 43.66% patients showed an SVR (responders); 30.98% patients were non-responders while 25.35% patients initially responded to therapy but again retrieved positive status of HCV infection six months post-therapy (relapse-cirrhotic patients). The mean levels of plasma Hcy, ALT and ALP were significantly reduced in responders within 10 weeks of therapy when compared with non-responders and relapse-cirrhotic patients. CONCLUSION: Elevated homocysteine levels in serum due to HCV infection can be reduced to normal range with the standard interferon α plus ribavirin treatment. This study highlights the significance of the measurement of serum homocysteine levels in the diagnosis and monitoring of HCV infection treatment in addition to other laboratory parameters.

Mutation in the tight-junction gene claudin 19 (CLDN19) and familial hypomagnesemia, hypercalciuria, nephrocalcinosis (FHHNC) and severe ocular disease.

BACKGROUND/AIMS: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare renal tubular disorder complicated by progressive renal failure during childhood or adolescence. Recently, causative mutations in the CLDN19 gene have been identified in FHHNC patients presenting with severe ocular involvement. The aim of the study was to investigate the molecular genetic defect underlying FHHNC in a consanguineous Pakistani family. METHODS: Clinical and biochemical parameters of the proband were studied during the follow-up period over 5 years. Genotyping of 7 members of the family was performed by amplifying microsatellite markers, tightly linked to the CLDN16 and CLDN19 genes. The two genes were sequenced directly in an automated sequencer. PCR-RFLP assay and bioinformatic analysis were performed to verify the identified mutation. RESULTS: Genotyping revealed that the proband was homozygous for the marker loci tightly linked to the CLDN19 gene. Sequence analysis in the proband revealed homozygosity for a novel missense mutation in exon 3 of the CLDN19 gene (389G>A) resulting in G130D amino acid substitution. Bioinformatic analysis supported the pathogenicity of the identified mutation. Family screening revealed nephrolithiasis in 3 of 6 (50%) heterozygous carriers of the pathogenic mutation. CONCLUSION: This study supports the fundamental role of claudin 19 for magnesium homeostasis, normal tubular structures in the kidney, and undisturbed organization and development of the retina.

Toes that look like toes: Cambodian children's perspectives on prosthetic legs.

When designing assistive devices for people with disabilities in developing countries, product developers tend to prioritize economic and technical requirements, ignoring aesthetic and cultural issues. This can result in devices that create or reinforce social barriers, creating negative impacts on users' self-esteem and sense of well-being. To understand the social implications of using prosthetic legs for Cambodian children, a pilot research study was conducted in Phnom Penh and surrounding provinces. I used innovative research methods to help children share their perspectives. The objective was to help product designers develop improved assistive devices that not only increase mobility for child prosthesis users but also support the sociocultural integration of these children and their families. A philosophical hermeneutics research approach was used to seek the views of three children. The findings are useful for nongovernmental organizations and product developers that focus on the needs of impoverished children in rural, predominantly Buddhist communities.

Molecular Diagnosis and Identification of Genetic Variants Underlying Distal Renal Tubular Acidosis in Pakistani Patients Using Whole Exome Sequencing.

Introduction: Primary distal renal tubular acidosis (dRTA) is a rare genetic disorder characterized by an impaired urinary acidification process in distal nephrons that results in the production of alkaline urine. Loss of function variants in any of the three genes, ATP6V0A4, ATP6V1B1, or SLC4A1, which all play a role in normal acidification of urine by kidneys, may lead to dRTA. Objective: This study was designed to identify genetic variants underlying dRTA in Pakistani patients using whole exome sequencing, followed by confirmatory Sanger sequencing. Materials and Methods: Patients were identified following presentation with characteristic clinical features of dRTA including vomiting, dehydration, and highly alkaline urine with metabolic acidosis during the first few days of life. Whole exome sequencing and Sanger sequencing were employed for genetic analyses of the patients. In silico analyses of the identified variants were performed using web-based bioinfomatics programs. Results: Through whole exome sequencing, we identified two splice site variants (c.2257 + 1G>A and c.722 + 5G>A) in the ATP6V0A4 gene that likely underly the disease phenotype in the two families. Multiple in silico tools predicted these variants to affect the respective splice sites supporting their likely role in pathogenesis. Conclusion: The study extends the spectrum of ATP6V0A4 variants associated with dRTA and should benefit the genetic counseling and prenatal diagnosis of the affected families.

Molecular Study of Nephronophthisis in 7 Unrelated Pakistani Families.

Nephronophthisis is an autosomal recessive cystic kidney disease characterized by tubular interstitial infiltration, periglomerular fibrosis, and cysts, and is the most frequent genetic cause of end-stage renal disease in children. Nephronophthisis is pleiotropic as almost all the causative genes are involved in primary cilium and centrosome function which are found in almost all human cells. Genetic heterogeneity in nephronophthisis makes the molecular and genetic diagnosis somewhat difficult. Homozygous deletions in the nephronophthisis 1 (NPHP1) gene are the major contributor of nephronophthisis cases, while other genes accounts for less than 3% each. Nephronophthisis-related ciliopathy is a term used for extrarenal symptoms in addition to nephronophthisis. Herein, we are reporting the molecular study of 7 children from independent families fulfilling the criteria of nephronophthisis. A deletion analysis of the NPHP1 gene was performed in each case, and NPHP5 mutation screening was performed in the absence of such deletion in patients with Senior Loken syndrome.

Tracing misalignment and unequal crossing over during evolution of the repetitive region of the SM50 gene in sea urchins.

The location of misalignment and unequal crossover involved in concerted evolution of tandemly repetitive sequences is difficult to document owing to the homogeneity of sequences that are subject to this process. However, the repetitive domain of the SM50 gene in sea urchins contains variation, within the gene itself, between alleles, and between species that has allowed us to determine where misalignment and unequal crossing over occurred during evolution of this gene. We have therefore analyzed the SM50 repeat regions in a variety of species to determine where recent changes in repeat numbers have occurred, and from this have deduced the mechanisms that lead to these changes. We next tried to determine whether recent misalignment and unequal crossover has produced allelic variation in current populations of sea urchins. We found SM50 alleles within three species that have different numbers of repeats. This marks the first reported documentation of allelic variation in the number of repeats in the SM50 gene. We also show how a single unequal crossover event could have produced the allelic variation. We have found that substitutions and small deletions in the sequences within the repeats can substantially affect how misalignment occurs, resulting in different patterns of repeats after concerted evolution.

Prognostic and diagnostic value of serum pseudocholinesterase, serum aspartate transaminase, and serum alinine transaminase in malignancies treated by radiotherapy.

BACKGROUND: There is substantial evidence that environmental factors cause or accelerate the onset of malignancy. Environmental factors, due to the presence of many pollutants and carcinogenic agents, alter cellular growth, which leads to biochemical changes in the blood. MATERIALS AND METHODS: In the present study, we estimated serum pseudocholinesterase (PCHE), serum aspartate transaminase (AST), and serum alanine transaminase (ALT) in 92 patients with head and neck cancer and 71 patients with cancer of the uterine cervix; all of them were patients attending our department for radiation therapy. We also estimated PCHE, AST, and ALT levels in 30 healthy normal individuals. The estimations in cancer patients were done before the start of radiotherapy, midway through radiotherapy (30 Gy dose), at the end of radiotherapy, and during subsequent monthly follow-up visits over a period of at least 6 months. RESULTS AND DISCUSSION: We have observed that the PCHE levels were lower (31-49% of normal value) in all patients with malignancies, except in those with stage II head and neck cancers. We also found that the levels start increasing as radiotherapy progresses. The patients with no detectable/visible disease activity at 6 months follow-up showed PCHE values in the normal range. Similarly, the AST and ALT values were much higher (138-229% of normal value) in all the malignant cases as compared to the normal healthy individuals. The values decrease and approach normal levels as radiotherapy progresses and, in 92% of head and neck cancer cases [stages IIA, IIB, and IIIA] with no disease activity, the PCHE, AST, and ALT were normal or near normal; the corresponding figure in cancer cervix cases was 89%. From the present study we conclude that PCHE, AST, and ALT can be used as good prognostic biochemical tumor markers in the management of malignancies of the head and neck and uterine cervix.

Distinct roles of different NF-kappa B subunits in regulating inflammatory and T cell stimulatory gene expression in dendritic cells.

TLRs play a critical role in inducing inflammatory and immune responses against microbial agents. In this study, we have investigated the role of NF-kappaB transcription factors in regulating TLR-induced gene expression in dendritic cells, a key APC type. The p50 and cRel NF-kappaB subunits were found to be crucial for regulating genes important for dendritic cell-induced T cell responses (e.g., CD40, IL-12, and IL-18) but not for genes encoding inflammatory cytokines (e.g., TNF-alpha, IL-1alpha, and IL-6). In striking contrast, the RelA subunit was crucial for expression of inflammatory cytokine genes but not T cell stimulatory genes. These novel findings reveal a fundamentally important difference in biological function of genes regulated by different NF-kappaB subunits. Focusing on RelA target gene specificity mechanisms, we investigated whether the kappaB site and/or the unique composition of RelA played the most crucial role. Surprisingly, studies of IL-6 expression showed that the kappaB site is not a primary determinant of RelA target gene specificity. Instead, a major specificity mechanism is the unique ability of RelA to interact with the transcriptional coactivator CREB-binding protein, a function not shared with the closely related cRel subunit. Together, our findings indicate novel and critically important overall roles of NF-kappaB in TLR-induced gene expression that are mediated by unique functions of distinct subunits.

Lack of essential role of NF-kappa B p50, RelA, and cRel subunits in virus-induced type 1 IFN expression.

Type 1 IFNs (IFN-alphabeta) play pivotal roles in the host antiviral response and in TLR-induced signaling. IFN regulatory factor (IRF) and NF-kappaB transcription factors are thought to be crucial for virus-induced mRNA expression of IFN-beta. Although recent studies have demonstrated essential roles for IRF3 and IRF7, the definitive role of NF-kappaB factors in IFN-beta (or IFN-alpha) expression remains unknown. Using mice deficient in distinct members of the NF-kappaB family, we investigated NF-kappaB function in regulating type 1 IFN expression in response to Sendai virus and Newcastle disease virus infection. Surprisingly, IFN-beta and IFN-alpha expression was strongly induced following virus infection of mouse embryonic fibroblasts (MEFs) from p50(-/-), RelA/p65(-/-), cRel(-/-), p50(-/-)cRel(-/-), and p50(-/-)RelA(-/-) mice. Compared with wild-type MEFs, only RelA(-/-) and p50(-/-)RelA(-/-) MEFs showed a modest reduction in IFN-beta expression. To overcome functional redundancy between different NF-kappaB subunits, we expressed a dominant-negative IkappaBalpha protein in p50(-/-)RelA(-/-) MEFs to inhibit activation of remaining NF-kappaB subunits. Although viral infection of these cells failed to induce detectable NF-kappaB activity, both Sendai virus and Newcastle disease virus infection led to robust IFN-beta expression. Virus infection of dendritic cells or TLR9-ligand CpG-D19 treatment of plasmacytoid dendritic cells from RelA(-/-) or p50(-/-)cRel(-/-) mice also induced robust type 1 IFN expression. Our findings therefore indicate that NF-kappaB subunits p50, RelA, and cRel play a relatively minor role in virus-induced type 1 IFN expression.

Sea urchin metalloproteases: a genomic survey of the BMP-1/tolloid-like, MMP and ADAM families.

Analysis of the Strongylocentrotus purpuratus genome has revealed approximately 240 metalloprotease genes, and they represent all 23 families expressed in vertebrates. EST/cDNA sequencing and microarray analysis show that nearly 70% are represented in embryo RNA. Among them are many metalloproteases with demonstrated developmental roles in other systems-BMP-1/TLD (tolloid) (astacins), MMPs (matrix metalloproteases) and the ADAMs (disintegrin/metalloproteases). The developmental functions of these kinds of metalloproteases include modifying the extracellular matrix, regulating signaling pathways or modulating cellular adhesive properties. The unexpectedly large number of BMP-1/TLD-like protease genes (23) results primarily from expansion of a set encoding an unusual domain conserved in structure and primary sequence only in nematode astacins. Such proteases may have interesting developmental functions because the expression patterns of several are highly regulated along the primary axis at times when cell differentiation and morphogenesis begin. The size of the sea urchin MMP family and the clustered arrangement of many of its members are similar to vertebrates, but phylogenetic analyses suggest that different ancestral genes were independently amplified in sea urchins and vertebrates. One expansion appears to be genes encoding MMPs that have putative transmembrane domains and may be membrane-tethered (MT). Interestingly, the genes encoding TIMPs, inhibitors of MMPs, have also been amplified and the 10 genes are tandemly arranged in a single cluster. In contrast, there are fewer ADAM and ADAMTS genes in sea urchins, but they represent all but one of the chordate-specific groups. The genome sequence now opens the door to experimental manipulations designed to understand how modulation of the extracellular environment affects development.