ABSTRACT
ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of intracellular Ca2+ content have been suggested to impair SARS-CoV-2 replication. Here, we demonstrate that a nontoxic caloxin-derivative compound (PI-7) reduces intracellular Ca2+ levels and impairs SARS-CoV-2 infection. Furthermore, a rare homozygous intronic variant of ATP2B1 is shown to be associated with the severity of COVID-19. The mechanism of action during SARS-CoV-2 infection involves the PI3K/Akt signaling pathway activation, inactivation of FOXO3 transcription factor function, and subsequent transcriptional inhibition of the membrane and reticulum Ca2+ pumps ATP2B1 and ATP2A1, respectively. The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 lacks toxicity in vitro, its prophylactic use as a therapeutic agent against COVID-19 is envisioned here.
Subject(s)
COVID-19 , Calcium , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , SARS-CoV-2 , Signal Transduction , Virus Replication , Humans , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects , Proto-Oncogene Proteins c-akt/metabolism , COVID-19/virology , COVID-19/metabolism , Signal Transduction/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Calcium/metabolism , Animals , Forkhead Box Protein O3/metabolism , Forkhead Box Protein O3/genetics , Chlorocebus aethiops , COVID-19 Drug Treatment , Vero Cells , Female , Calcium-Transporting ATPases/metabolism , Calcium-Transporting ATPases/genetics , MaleABSTRACT
Medulloblastoma (MB) is a highly malignant childhood brain tumor. Group 3 MB (Gr3 MB) is considered to have the most metastatic potential, and tailored therapies for Gr3 MB are currently lacking. Gr3 MB is driven by PRUNE-1 amplification or overexpression. In this paper, we found that PRUNE-1 was transcriptionally regulated by lysine demethylase LSD1/KDM1A. This study aimed to investigate the therapeutic potential of inhibiting both PRUNE-1 and LSD1/KDM1A with the selective inhibitors AA7.1 and SP-2577, respectively. We found that the pharmacological inhibition had a substantial efficacy on targeting the metastatic axis driven by PRUNE-1 (PRUNE-1-OTX2-TGFß-PTEN) in Gr3 MB. Using RNA seq transcriptomic feature data in Gr3 MB primary cells, we provide evidence that the combination of AA7.1 and SP-2577 positively affects neuronal commitment, confirmed by glial fibrillary acidic protein (GFAP)-positive differentiation and the inhibition of the cytotoxic components of the tumor microenvironment and the epithelial-mesenchymal transition (EMT) by the down-regulation of N-Cadherin protein expression. We also identified an impairing action on the mitochondrial metabolism and, consequently, oxidative phosphorylation, thus depriving tumors cells of an important source of energy. Furthermore, by overlapping the genomic mutational signatures through WES sequence analyses with RNA seq transcriptomic feature data, we propose in this paper that the combination of these two small molecules can be used in a second-line treatment in advanced therapeutics against Gr3 MB. Our study demonstrates that the usage of PRUNE-1 and LSD1/KDM1A inhibitors in combination represents a novel therapeutic approach for these highly aggressive metastatic MB tumors.
Subject(s)
Brain Neoplasms , Cerebellar Neoplasms , Medulloblastoma , Humans , Child , Medulloblastoma/drug therapy , Medulloblastoma/genetics , Histone Demethylases/genetics , Epigenesis, Genetic , Tumor MicroenvironmentABSTRACT
BACKGROUND: Neuroblastoma (NB) is the most common solid extracranial paediatric tumour. Genome-wide association studies have driven the discovery of common risk variants, but no large study has investigated the contribution of rare variants to NB susceptibility. Here, we conducted a whole-exome sequencing (WES) of 664 NB cases and 822 controls and used independent validation datasets to identify genes with rare risk variants and involved pathways. METHODS: WES was performed at 50× depth and variants were jointly called in cases and controls. We developed two models to identify mutations with high clinical impact (P/LP model) and to discover less penetrant risk mutations affecting non-canonical cancer pathways (RPV model). We performed a gene-level collapsing test using Firth's logistic regression in 242 selected cancer predisposition genes (CPGs) and a gene-sets burden analysis of biologically-informed pathways. FINDINGS: Twelve percent of patients carried P/LP variants in CPGs and showed a significant enrichment (P = 2.3 × 10-4) compared to controls (6%). We identified P/LP variants in 45 CPGs enriched in homologous recombination (HR) pathway. The most P/LP enriched genes in NB were BRCA1, ALK and RAD51C. Additionally, we found higher RPV burden in gene-sets of neuron differentiation, neural tube development and synapse assembly, and in gene-sets associated with neurodevelopmental disorders (NDD). INTERPRETATION: The high fraction of NB patients with P/LP variants indicates the need of genetic counselling. Furthermore, inherited rare variants predispose to NB development by affecting mechanisms related to HR and neurodevelopmental processes, and demonstrate that NDD genes are altered in NB at the germline level. FUNDING: Associazione Italiana per la Ricerca sul Cancro, Fondazione Italiana per la Lotta al Neuroblastoma, Associazione Oncologia Pediatrica e Neuroblastoma, Regione Campania, Associazione Giulio Adelfio onlus, and Italian Health Ministry.
Subject(s)
Genetic Predisposition to Disease , Neuroblastoma , Humans , Child , Genome-Wide Association Study , Mutation , Neuroblastoma/genetics , Homologous RecombinationABSTRACT
PURPOSE: Emerging evidence suggest that infection-dependent hyperactivation of complement system (CS) may worsen COVID-19 outcome. We investigated the role of predicted high impact rare variants - referred as qualifying variants (QVs) - of CS genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. METHODS: Exploiting exome sequencing data and 56 CS genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (aged ≥60 years) and 56,885 European individuals from the Genome Aggregation Database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized patients with COVID-19. RESULTS: We found an enrichment of QVs in 3 genes (MASP1, COLEC11, and COLEC10), which belong to the lectin pathway, in the asymptomatic cohort. Analyses of complement activity in serum showed decreased activity of lectin pathway in asymptomatic individuals with QVs. Finally, we found allelic variants associated with asymptomatic COVID-19 phenotype and with a decreased expression of MASP1, COLEC11, and COLEC10 in lung tissue. CONCLUSION: This study suggests that genetic rare variants can protect from severe COVID-19 by mitigating the activity of lectin pathway and prothrombin. The genetic data obtained through ES of 786 asymptomatic and 147 hospitalized individuals are publicly available at http://espocovid.ceinge.unina.it/.
Subject(s)
COVID-19 , Aged , COVID-19/genetics , Collectins/genetics , Collectins/metabolism , Germ Cells , Humans , Lectins/genetics , SARS-CoV-2 , Exome SequencingABSTRACT
Tracing the appearance and evolution of virus variants is essential in the management of the COVID-19 pandemic. Here, we focus on SARS-CoV-2 spread in Italian patients by using viral sequences deposited in public databases and a tracing procedure which is used to monitor the evolution of the pandemic and detect the spreading, within the infected population of emergent sub-clades with a potential positive selection. Analyses of a collection of monthly samples focused on Italy highlighted the appearance and evolution of all the main viral sub-trees emerging at the end of the first year of the pandemic. It also identified additional expanding subpopulations which spread during the second year (i.e., 2021). Three-dimensional (3D) modelling of the main amino acid changes in mutated viral proteins, including ORF1ab (nsp3, nsp4, 2'-o-ribose methyltransferase, nsp6, helicase, nsp12 [RdRp]), N, ORF3a, ORF8, and spike proteins, shows the potential of the analysed structural variations to result in epistatic modulation and positive/negative selection pressure. These analyzes will be of importance to the early identification of emerging clades, which can develop into new "variants of concern" (i.e., VOC). These analyses and settings will also help SARS-CoV-2 coronet genomic centers in other countries to trace emerging worldwide variants.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiology , Humans , Mutation , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.
Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/physiology , Virus Replication/physiology , Coronavirus Nucleocapsid Proteins/analysis , Giant Cells/drug effects , Giant Cells/virology , HEK293 Cells , Humans , Limit of Detection , Nasopharynx/virology , Phosphoproteins/analysis , Phosphoproteins/genetics , RNA, Antisense/pharmacology , RNA, Viral , Ribonuclease P/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Sensitivity and Specificity , Social Isolation , Viral Load , Viroporin Proteins/genetics , Virus Replication/drug effectsABSTRACT
Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Polyphosphates/pharmacology , SARS-CoV-2/drug effects , Administration, Inhalation , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , COVID-19/metabolism , COVID-19/virology , Caco-2 Cells , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cytokines/metabolism , HEK293 Cells , Host Microbial Interactions/drug effects , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Humans , In Vitro Techniques , Models, Biological , Molecular Docking Simulation , Nebulizers and Vaporizers , Polyphosphates/administration & dosage , Polyphosphates/chemistry , Proteasome Endopeptidase Complex/metabolism , Protein Interaction Domains and Motifs , Proteolysis/drug effects , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Homology, Amino Acid , Signal Transduction/drug effects , Vero Cells , Virus Replication/drug effectsABSTRACT
M2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC). Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-ß enhancement, IL-17F secretion, and extracellular vesicle protein content modulation. We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.
ABSTRACT
In contrast to the widely accepted consensus of the existence of a single RNA polymerase in bacteria, several actinomycetes have been recently shown to possess two forms of RNA polymerases due the to co-existence of two rpoB paralogs in their genome. However, the biological significance of the rpoB duplication is obscure. In this study we have determined the genome sequence of the lipoglycopeptide antibiotic A40926 producer Nonomuraea gerenzanensis ATCC 39727, an actinomycete with a large genome and two rpoB genes, i.e. rpoB(S) (the wild-type gene) and rpoB(R) (the mutant-type gene). We next analyzed the transcriptional and metabolite profiles in the wild-type gene and in two derivative strains over-expressing either rpoB(R) or a mutated form of this gene to explore the physiological role and biotechnological potential of the "mutant-type" RNA polymerase. We show that rpoB(R) controls antibiotic production and a wide range of metabolic adaptive behaviors in response to environmental pH. This may give interesting perspectives also with regard to biotechnological applications.
Subject(s)
Actinomycetales/genetics , Bacterial Proteins/genetics , DNA-Directed RNA Polymerases/genetics , Genome, Bacterial , Transcriptome , Actinomycetales/metabolism , Anti-Bacterial Agents/biosynthesis , Hydrogen-Ion Concentration , Mutation , Teicoplanin/analogs & derivatives , Teicoplanin/biosynthesisABSTRACT
Although previous research has identified cognitive styles that distinguish individuals with bipolar disorder (BD), individuals with major depressive disorder (MDD), and individuals without mood disorders from one another, findings have been inconsistent. The current study included 381 participants classified into a BD group, a MDD group, and a no mood disorder group. To differentiate between these groups, this study evaluated cognitive styles with a battery of traditional and more recently-developed measures. Receiver operating characteristics (ROC) analyses were used to determine the discriminate ability of variables with significant between group differences. Results supported that BD and MDD may be characterized by distinct cognitive styles. Given work showing that interventions for MDD may not be effective at treating BD, it is important to directly compare individuals with these disorders. By clarifying the overlapping and divergent cognitive styles characterizing BD and MDD, research can not only improve diagnostic validity, but also provide more efficacious and effective interventions.
ABSTRACT
Hypertrophic cardiomyopathy is a relatively frequent disease with a prevalence of 0.2% worldwide and a remarkable genetic heterogeneity, with more than 30 causative genes reported to date. Current PCR-based strategies are inadequate for genomic investigations involving many candidate genes. Here, we report a next-generation sequencing procedure associated with DNA sequence capture that is able to sequence 202 cardiomyopathy-related genes simultaneously. We developed a complementary data analysis pipeline to select and prioritize genetic variants. The overall procedure can screen a large number of target genes simultaneously, thereby potentially revealing new disease-causing and modifier genes. By using this procedure, we analyzed hypertrophic cardiomyopathy patients in a shorter time and at a lower cost than with current procedures. The specificity of the next-generation sequencing-based procedure is at least as good as other techniques routinely used for mutation searching, and the sensitivity is much better. Analysis of the results showed some novel variants potentially involved in the pathogenesis of hypertrophic cardiomyopathy: a missense mutation in MYH7 and a nonsense variant in INS-IGF2 (patient 1), a splicing variant in MYBPC3 and an indel/frameshift variant in KCNQ1 (patient 2), and two concomitant variations in CACNA1C (patient 3). Sequencing of DNA from the three patients within a pool allowed detection of most variants identified in each individual patient, indicating that this approach is a feasible and cost-effective procedure.
Subject(s)
Cardiomyopathy, Hypertrophic/diagnosis , Cardiomyopathy, Hypertrophic/genetics , Molecular Diagnostic Techniques , Adolescent , Base Sequence , Calcium Channels, L-Type/genetics , Cardiac Myosins/genetics , Carrier Proteins/genetics , Child , Female , High-Throughput Nucleotide Sequencing , Humans , KCNQ1 Potassium Channel/genetics , Male , Middle Aged , Molecular Sequence Data , Mutant Chimeric Proteins/genetics , Mutation , Myosin Heavy Chains/genetics , Sequence Alignment , Sequence Analysis, DNAABSTRACT
Recent work has identified behavioural approach system (BAS) sensitivity as a risk factor for the first onset and recurrence of mood episodes in bipolar disorder, but little work has evaluated risk factors for depression in individuals at risk for, but without a history of, bipolar disorder. The present study evaluated cognitive styles and the emotion-regulatory characteristics of emotional clarity and ruminative brooding as prospective predictors of depressive symptoms in individuals with high versus moderate BAS sensitivity. Three separate regressions indicated that the associations between dysfunctional attitudes, self-criticism, and neediness with prospective increases in depressive symptoms were moderated by emotional clarity and brooding. Whereas brooding interacted with these cognitive styles to exacerbate their impact on depressive symptoms, emotional clarity buffered against their negative impact. These interactions were specific to high-BAS individuals for dysfunctional attitudes, but were found across the full sample for self-criticism and neediness. These results indicate that emotion-regulatory characteristics and cognitive styles may work in conjunction to confer risk for and resilience against depression, and that some of these relationships may be specific to individuals at risk for bipolar disorder.
Subject(s)
Bipolar Disorder/diagnosis , Depression , Adolescent , Affect , Attitude , Bipolar Disorder/psychology , Cognition , Emotions , Female , Humans , Male , Models, Psychological , Personality , Philadelphia , Regression Analysis , Risk Assessment , Self-Assessment , Young AdultABSTRACT
We examined the concurrent associations between multiple cognitive vulnerabilities to depression featured in hopelessness theory, Beck's theory, and response styles theory and depressive symptoms and diagnoses in a sample of early adolescents. We also examined the specificity of these cognitive vulnerabilities to depression versus anxiety and externalizing psychopathology, controlling for co-occurring symptoms and diagnoses. Male and female, Caucasian and African American, 12- to 13-year-old adolescents were assessed in a cross-sectional design. Cognitive vulnerabilities of hopelessness, inferential style, rumination, and self-referent information processing were assessed with self-reports and behavioral tasks. Symptoms and diagnoses of depressive, anxiety, and externalizing disorders were assessed with self-report questionnaires and diagnostic interviews. Hopelessness exhibited the greatest specificity to depressive symptoms and diagnoses, whereas negative inferential styles, rumination, and negative self-referent information processing were associated with both depressive and anxiety symptoms and diagnoses and, in some cases, with externalizing disorders. Consistent with cognitive theories of depression, hopelessness, negative inferential styles, rumination, and negative self-referent information processing were associated with depressive symptoms and diagnoses. However, with the exception of hopelessness, most of the remaining cognitive vulnerabilities were not specific to depression. With further maturation of our sample, these cognitive vulnerabilities may become more specific to depression as cognitive styles further develop and consolidate, the rates of depression increase, and individuals' presentations of psychopathology become more differentiated.
Subject(s)
Anxiety/diagnosis , Depression/diagnosis , Depressive Disorder/diagnosis , Adaptation, Psychological , Adolescent , Black or African American , Child , Cognition , Cross-Sectional Studies , Female , Humans , Longitudinal Studies , Male , Personality , Prospective Studies , Surveys and Questionnaires , White PeopleABSTRACT
The increased risk of anxiety in children of parents with psychopathology is a significant public health problem, as early-onset is associated with a variety of difficulties later in life. The aim of this article is to determine if treating parents is associated with improvements in child anxiety through the review of both top-down (parent identified for treatment) and family-focused child anxiety treatment studies. The authors present conclusions based on the state of the current literature, discuss implications for research and clinical practice, and propose utilizing a family-based model for treating parental psychopathology, parental behavior, and child anxiety.
Subject(s)
Anxiety Disorders/therapy , Child of Impaired Parents/psychology , Depressive Disorder/therapy , Family Therapy/methods , Parent-Child Relations , Parents , Anxiety Disorders/psychology , Child , Cognitive Behavioral Therapy/methods , Depressive Disorder/psychology , Humans , Patient Acceptance of Health Care/statistics & numerical data , Randomized Controlled Trials as Topic , Risk Factors , Treatment OutcomeABSTRACT
OBJECTIVE: The study examined interest in treatment and treatment preferences and obstacles of low-income depressed parents. METHODS: A total of 273 primarily low-income, Hispanic parents of children aged seven to 17 attending an urban family medicine practice agreed to complete a survey by interview or self-report, including screening diagnoses and treatment history. Three groups were compared: major, subthreshold, and no depression. RESULTS: Nearly one-third had major (9%) or subthreshold depression (23%), and many in the depressed groups reported recent treatment (50% and 31%, respectively). Parents with any depression were significantly more likely than nondepressed parents to report interest in receiving help, endorse treatment obstacles, and report children's problems. CONCLUSIONS: High rates of personal and child problems, interest in treatment, and treatment obstacles among low-income, depressed parents highlight the need to develop acceptable mental health services for them and their children, even when parents do not meet full diagnostic criteria for depression.
Subject(s)
Depression , Family Practice , Health Services Needs and Demand , Parents/psychology , Adolescent , Adult , Child , Female , Health Care Surveys , Humans , Male , Middle Aged , Patient Acceptance of Health Care , Poverty , Urban Population , Young AdultABSTRACT
In the large intestine organic cation transporter type-2 (OCTN2) is recognized as a transporter of compounds such as carnitine and colony sporulation factor, promoting health of the colon intestinal epithelium. Recent reports suggest that OCTN2 expression in small intestine is under control of peroxisome proliferator-activated receptor-alpha (PPARalpha). However, PPARalpha contribution to colonic OCTN2 expression remains controversial. Here we examined the transcriptional regulation of colon OCTN2 gene by PPARgamma. To exclude any additional modulation of other PPAR to OCTN2 expression, we used both in vivo and in vitro PPAR-null models and specific PPAR inhibitors. The PPARgamma agonists thiazolidinediones increased both OCTN2 mRNA and protein expression in colonic epithelial cell lines independently by PPARalpha expression. The induction was blocked only by PPARgamma antagonists or by gammaORF4, a PPARgamma isoform with dominant negative activity, suggesting a PPARgamma-dependent mechanism. A conserved noncanonical PPAR-responsive element was found by computational analysis in the first intron of human OCTN2 gene and validated by EMSA assay. Promoter-reporter assays further confirmed transcriptional functionality of the putative PPAR response element, whereas selective mutation caused complete loss of responsiveness to PPARgamma activation. Finally, adenovirus-mediated overexpression of constitutively active PPARgamma mutant increased colon OCTN2 expression in PPARalpha(-/-) mice. Interestingly, animals overexpressing colon PPARgamma showed a significant increase in plasma carnitine, thus demonstrating the functional contribution of large intestine to systemic carnitine homeostasis. This study reveals a PPARgamma-dependent absorption machinery in colon that is likely involved in the health of colon epithelium, in the microbiota-host interactions and in the absorption of nutraceuticals and drugs.
Subject(s)
Carnitine/metabolism , Colon/metabolism , Gene Expression Regulation/physiology , Homeostasis/physiology , Organic Cation Transport Proteins/biosynthesis , PPAR gamma/metabolism , Animals , Carnitine/genetics , Cell Line, Transformed , Gene Expression Regulation/drug effects , Homeostasis/drug effects , Humans , Hypoglycemic Agents/pharmacology , Intestinal Absorption/drug effects , Intestinal Absorption/physiology , Intestine, Small/metabolism , Mice , Mice, Knockout , Organ Specificity/physiology , Organic Cation Transport Proteins/genetics , PPAR alpha/genetics , PPAR alpha/metabolism , PPAR gamma/agonists , Protein Isoforms/agonists , Protein Isoforms/genetics , Protein Isoforms/metabolism , Response Elements/physiology , Solute Carrier Family 22 Member 5 , Thiazolidinediones/pharmacologyABSTRACT
BACKGROUND: Molecular diagnosis for cystic fibrosis (CF) is based on the direct identification of mutations in the CFTR gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] (detection rate about 90% with scanning procedures) and on segregation analysis of intragenic polymorphisms for carrier and prenatal diagnosis in about 20% of CF families in which 1 or both causal mutations are unknown. METHODS: We identified 3 novel intragenic polymorphic repeats (IVS3polyA, IVS4polyA, and IVS10CA repeats) in the CFTR gene and developed and validated a procedure based on the PCR followed by capillary electrophoresis for large-scale analysis of these polymorphisms and the 4 previously identified microsatellites (IVS1CA, IVS8CA, IVS17bTA, and IVS17bCA repeats) in a single run. We validated the procedure for both single- and 2-cell samples (for a possible use in preimplantation diagnosis), and on a large number of CF patients bearing different genotypes and non-CF controls. RESULTS: The allelic distribution and heterozygosity results suggest that the 3 novel polymorphisms strongly contribute to carrier and prenatal diagnosis of CF in families in which 1 or both causal mutations have not been identified. At least 1 of the 4 previously identified microsatellites was informative in 78 of 100 unrelated CF families; at least 1 of all 7 polymorphisms was informative in 98 of the families. Finally, the analysis of haplotypes for the 7 polymorphisms revealed that most CF mutations are associated with different haplotypes, suggesting multiple slippage events but a single origin for most CFTR mutations. CONCLUSIONS: The analysis of the 7 polymorphisms is a rapid and efficient tool for routine carrier, prenatal, and preimplantation diagnosis of CF.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Polymorphism, Genetic , Repetitive Sequences, Nucleic Acid , Cystic Fibrosis/diagnosis , Electrophoresis, Capillary , Female , Humans , Male , Pedigree , Polymerase Chain Reaction , Prenatal DiagnosisABSTRACT
Bioinformatic tools are often used by researchers through interactive Web interfaces, resulting in a strong demand for computational resources. The tools are of different kind and range from simple, quick tasks, to complex analyses requiring minutes to hours of processing time and often longer than that. Batteries of computational nodes, such as those found in parallel clusters, provide a platform of choice for this application, especially when a relatively large number of concurrent requests is expected. Here, we describe a scheduling architecture operating at the application level, able to distribute jobs over a large number of hierarchically organized nodes. While not contrasting and peacefully living together with low-level scheduling software, the system takes advantage of tools, such as SQL servers, commonly used in Web applications, to produce low latency and performance which compares well and often surpasses that of more traditional, dedicated schedulers. The system provides the basic functionality necessary to node selection, task execution and service management and monitoring, and may combine loosely linked computational resources, such as those located in geographically distinct sites.
Subject(s)
Computational Biology/methods , Database Management Systems , Databases, Factual , Information Storage and Retrieval/methods , Internet , Software , User-Computer InterfaceABSTRACT
BACKGROUND: Prediction of secondary structures in the expressed sequences of bacterial genomes allows to investigate spontaneous folding of the corresponding RNA. This is particularly relevant in untranslated mRNA regions, where base pairing is less affected by interactions with the translation machinery. Relatively large stem-loops significantly contribute to the formation of more complex secondary structures, often important for the activity of sequence elements controlling gene expression. RESULTS: Systematic analysis of the distribution of stem-loop structures (SLSs) in 40 wholly-sequenced bacterial genomes is presented. SLSs were searched as stems measuring at least 12 bp, bordering loops 5 to 100 nt in length. G-U pairing in the stems was allowed. SLSs found in natural genomes are constantly more numerous and stable than those expected to randomly form in sequences of comparable size and composition. The large majority of SLSs fall within protein-coding regions but enrichment of specific, non random, SLS sub-populations of higher stability was observed within the intergenic regions of the chromosomes of several species. In low-GC firmicutes, most higher stability intergenic SLSs resemble canonical rho-independent transcriptional terminators, but very frequently feature at the 5'-end an additional A-rich stretch complementary to the 3' uridines. In all species, a clearly biased SLS distribution was observed within the intergenic space, with most concentrating at the 3'-end side of flanking CDSs. Some intergenic SLS regions are members of novel repeated sequence families. CONCLUSION: In depth analysis of SLS features and distribution in 40 different bacterial genomes showed the presence of non random populations of such structures in all species. Many of these structures are plausibly transcribed, and might be involved in the control of transcription termination, or might serve as RNA elements which can enhance either the stability or the turnover of cotranscribed mRNAs. Three previously undescribed families of repeated sequences were found in Yersiniae, Bordetellae and Enterococci.
Subject(s)
DNA, Bacterial/ultrastructure , Genome, Bacterial , Nucleic Acid Conformation , Base Composition , DNA Shuffling , Repetitive Sequences, Nucleic Acid , Species SpecificityABSTRACT
BACKGROUND: Protein kinases are a well defined family of proteins, characterized by the presence of a common kinase catalytic domain and playing a significant role in many important cellular processes, such as proliferation, maintenance of cell shape, apoptosis. In many members of the family, additional non-kinase domains contribute further specialization, resulting in subcellular localization, protein binding and regulation of activity, among others. About 500 genes encode members of the kinase family in the human genome, and although many of them represent well known genes, a larger number of genes code for proteins of more recent identification, or for unknown proteins identified as kinase only after computational studies. RESULTS: A systematic in silico study performed on the human genome, led to the identification of 5 genes, on chromosome 1, 11, 13, 15 and 16 respectively, and 1 pseudogene on chromosome X; some of these genes are reported as kinases from NCBI but are absent in other databases, such as KinBase. Comparative analysis of 483 gene regions and subsequent computational analysis, aimed at identifying unannotated exons, indicates that a large number of kinase may code for alternately spliced forms or be incorrectly annotated. An InterProScan automated analysis was performed to study domain distribution and combination in the various families. At the same time, other structural features were also added to the annotation process, including the putative presence of transmembrane alpha helices, and the cystein propensity to participate into a disulfide bridge. CONCLUSION: The predicted human kinome was extended by identifying both additional genes and potential splice variants, resulting in a varied panorama where functionality may be searched at the gene and protein level. Structural analysis of kinase proteins domains as defined in multiple sources together with transmembrane alpha helices and signal peptide prediction provides hints to function assignment. The results of the human kinome analysis are collected in the KinWeb database, available for browsing and searching over the internet, where all results from the comparative analysis and the gene structure annotation are made available, alongside the domain information. Kinases may be searched by domain combinations and the relative genes may be viewed in a graphic browser at various level of magnification up to gene organization on the full chromosome set.