Replication Stage Research Articles

Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics insitu, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

Abstract 1318: A genome-wide association study of Waldenström macroglobulinemia/lymphoplasmacytic lymphoma demonstrates association with chromosome 6

Abstract Waldenström macroglobulinemia (WM) is a unique subset of lymphoplasmacytic lymphoma (LPL) that is defined by the presence of an LPL infiltrate in the bone marrow together with a monoclonal IgM protein in the serum. A somatic activating mutation, MYD88 L265P, occurs in 85+% of WM and in 25%-50% of patients with the precursor condition, IgM monoclonal gammopathy of undetermined significance (MGUS); however, germline MYD88 mutations have not been observed in WM patients, and the genetic basis for WM predisposition remains undefined. To identify novel WM susceptibility loci we conducted a two-stage genome-wide association study (GWAS) in over 450 WM cases and 4300 controls of European ancestry. Discovery (stage 1) included 217 WM cases (40% familial) and 3798 controls genotyped on the Illumina Omni Express or Illumina Omni2.5 platforms following standard quality control procedures. The genotyped data were imputed using the Haplotype Reference Consortium panel as a reference and analyzed using logistic regression. In stage 1, we identified three loci on chromosomes 6, 14 and 3 significantly associated (P<5.0x10-8) with risk of WM. Eleven promising SNPs in these and other suggestive loci (P<5.0x10-7) were selected for replication (stage 2) in 269 WM or LPL cases (4% familial) and 571 controls, and genotyping was conducted using standard methods on Taqman and Sequenom platforms or Sanger sequencing (1 SNP). Preliminary results confirm replication of the chromosome 6 locus. Stratification on familial status will illuminate the contribution of familial disease. These results will provide insight into the underlying genetic basis of WM susceptibility. Citation Format: Mary L. McMaster, Sonja I. Berndt, Shengchao A. Li, Susan Slager, Joseph Vijai, Charles C. Chung, Bin Zhu, Laurie Burdette, Brenda Birmann, Elizabeth E. Brown, James R. Cerhan, Karin Ekstrom-Smedby, Henrik Hjalgrim, Geffen Kleinstern, Brian K. Link, James McKay, Alain Monnereau, Lindsay M. Morton, Alexandra Nieters, Nathaniel Rothman, Christine F. Skibola, Alex Smith, Lauren R. Teras, Claire M. Vajdic, Roel Vermeulen, Belynda Hicks, Lynn R. Goldin, Neil E. Caporaso. A genome-wide association study of Waldenström macroglobulinemia/lymphoplasmacytic lymphoma demonstrates association with chromosome 6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1318. doi:10.1158/1538-7445.AM2017-1318

Identification of a flavonoid isolated from plum (Prunus domestica) as a potent inhibitor of Hepatitis C virus entry

Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases that often requires liver transplantation. The standard therapies are limited by severe side effects, resistance development, high expense and in a substantial proportion of cases, fail to clear the infection which bespeak the need for development of well-tolerated antivirals. Since most of the drug development strategies target the replication stage of viral lifecycle, the identification of entry inhibitors might be crucial especially in case of liver-transplant recipients. In the present study we have evaluated fruits which are known for their hepatoprotective effects in order to screen for entry inhibitors. We report the identification of a flavonoid, rutin, isolated from Prunus domestica as a new HCV entry inhibitor. Characterization and confirmation of the chemical structure was done by LC-ESI-MS, NMR and IR spectral analyses. Rutin significantly inhibited HCV-LP binding to hepatoma cells and inhibited cell-culture derived HCV (HCVcc) entry into hepatoma cells. Importantly, rutin was found to be non-toxic to hepatoma cells. Furthermore, rutin inhibits the early entry stage of HCV lifecycle possibly by directly acting on the viral particle. In conclusion, rutin is a promising candidate for development of anti-HCV therapeutics in the management of HCV infection.

Genome-wide pathway analysis identifies VEGF pathway association with oral ulceration in systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is a genetically complex rheumatic disease characterized by heterogeneous clinical manifestations of unknown etiology. Recent studies have suggested the existence of a genetic basis for SLE heterogeneity. The objective of the present study was to identify new genetic variation associated with the clinically relevant phenotypes in SLE.MethodsA two-stage pathway-based approach was used to identify the genetic variation associated with the main clinical phenotypes in SLE. In the discovery stage, 482 SLE patients were genotyped using Illumina Human Quad610 microarrays. Association between 798 reference genetic pathways from the Molecular Signatures Database and 11 SLE phenotypes was tested using the set-based method implemented in PLINK software. Pathways significantly associated after multiple test correction were subsequently tested for replication in an independent cohort of 425 SLE patients. Using an in silico approach, we analyzed the functional effects of common SLE therapies on the replicated genetic pathways. The association of known SLE risk variants with the development of the clinical phenotypes was also analyzed.ResultsIn the discovery stage, we found a significant association between the vascular endothelial growth factor (VEGF) pathway and oral ulceration (P value for false discovery rate (PFDR) < 0.05), and between the negative regulation signaling pathway of retinoic acid inducible gene-I/melanoma differentiation associated gene 5 and the production of antinuclear antibodies (PFDR < 0.05). In the replication stage, we validated the association between the VEGF pathway and oral ulceration. Therapies commonly used to treat mucocutaneous phenotypes in SLE were found to strongly influence VEGF pathway gene expression (P = 4.60e-4 to 5.38e-14). Analysis of known SLE risk loci identified a strong association between PTPN22 and the risk of hematologic disorder and with the development of antinuclear antibodies.ConclusionsThe present study has identified VEGF genetic pathway association with the risk of oral ulceration in SLE. New therapies targeting the VEGF pathway could be more effective in reducing the severity of this phenotype. These findings represent a first step towards the understanding of the genetic basis of phenotype heterogeneity in SLE.

Geno- and phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line

BackgroundBabesia bovis is an intra-erythrocytic tick-transmitted apicomplexan protozoan parasite. It has a complex lifestyle including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed that certain B. bovis genes, including members of 6-Cys gene family, are differentially expressed during tick and mammalian stages of the parasite’s life cycle. Moreover, the 6-Cys E gene is differentially expressed in the T3Bo strain of B. bovis tick stages, and anti 6-Cys E antibodies were shown to be able to inhibit in vitro growth of the phenotypically distinct B. bovis Mo7clonal line.MethodsIn this study, the 6-Cys E gene of B. bovis T3Bo strain was disrupted by transfection using a plasmid containing 6-Cys gene E 5′ and 3′ regions to guide homologous recombination, and the egfp-bsd fusion gene under control of a ef-1α promoter, yielding a B. bovis clonal line designated 6-Cys EKO-cln. Full genome sequencing of 6-Cys EKO-cln parasites was performed and in vitro inhibition assays using anti 6-Cys E antibodies.ResultsFull genome sequencing of 6-Cys EKO-cln B. bovis demonstrated single insertion of egfp-bsd gene that disrupts the integrity of 6-Cys gene E. Undistinguishable growth rate of 6-Cys EKO-cln line compared to wild-type 6-Cys E intact T3Bo B. bovis strain in in vitro cultures indicates that expression of gene 6-Cys E is not essential for blood stage replication in this strain. In vitro inhibition assays confirmed the ability of anti-6 Cys E antibodies to inhibit the growth of the wild-type Mo7 and T3Bo B. bovis parasites, but no significant inhibition was found for 6-Cys EKO-cln line parasites.ConclusionsOverall, the data suggest that the anti-6 Cys E antibody neutralising effect on the wild type strains is likely due to mechanical hindrance, or cross-reactivity, rather than due to functional requirements of 6-Cys gene E product for survival and development of the erythrocyte stages. Further investigation is underway to determine if the 6-Cys E protein is required for replication and sexual stage development of B. bovis during tick stages.

Novel replicons and trans-encapsidation systems for Hepatitis C Virus proteins live imaging and virus-host interaction proteomics

Proteomics and imaging techniques are used more and more in tandem to investigate the virus-host interaction. Herein we present novel replicons, methods and trans-encapsidation systems suitable for determination of Hepatitis C Virus (HCV) proteins interactomes and live imaging of viral proteins dynamics in HCV cell culture (HCVcc) system. To identify endogenous factors involved in the HCV life cycle, we constructed full-length functional replicons with affinity purification (AP) tags fused to NS2 and NS5A proteins. Viral-host interactomes were determined and validated in HCVcc system. To investigate the dynamics of viral-host interactions, we developed a core-inducible packaging cell line which trans-encapsidates various subgenomic replicons suitable for AP in replication and assembly stages. Further, a transient trans-encapsidation system was developed for live imaging of the NS5A viral protein in replication and assembly steps, respectively. The NS5A dynamics was determined also in the full-length HCV replicon system. The analysis of NS5A dynamics showed a decreased mobility of the protein in assembly versus the replication step. The tools presented herein will allow the investigation of HCV-host interaction with improved biological relevance and biosafety.

A new locus regulating MICALL2 expression was identified for association with executive inhibition in children with attention deficit hyperactivity disorder.

Impaired executive inhibition is a core deficit of attention deficit hyperactivity disorder (ADHD), which is a common childhood-onset psychiatric disorder with high heritability. In this study, we performed a two-stage genome-wide association study of executive inhibition in ADHD in Han Chinese. We used the Stroop color-word interference test to evaluate executive inhibition. After quality control, 780 samples with phenotype and covariate data were included in the discovery stage, whereas 922 samples were included in the replication stage. We identified one new significant locus at 7p22.3 for the Stroop word interference time (rs11514810, P=3.42E-09 for discovery, P=0.01176 for replication and combined P=5.249E-09). Regulatory feature analysis and expression quantitative trait loci (eQTL) data showed that this locus contributes to MICALL2 expression in the human brain. Most genes in the network interacting with MICALL2 were associated with psychiatric disorders. Furthermore, hyperactive-impulsive-like behavior was induced by reducing the expression of the zebrafish gene that is homologous to MICALL2, which could be rescued by tomoxetine (atomoxetine), a clinical medication for ADHD. Our results suggested that MICALL2 is a new susceptibility gene for executive inhibition deficiency related to hyperactive-impulsive behavior in ADHD, further emphasizing the possible role of neurodevelopmental genes in the pathogenic mechanism of ADHD.

Genome-wide association analysis for chronic venous disease identifies EFEMP1 and KCNH8 as susceptibility loci

Chronic venous disease (CVD) is a multifactorial condition representing one of the most common disorders among populations of Western countries. The heritability of about 17% suggests genetic risk factors in CVD etiology. However, so far the genetic causes are unknown. We undertook the hitherto first genome-wide association study (GWAS) for CVD, analyzing more than 1.93 M SNPs in 4,942 German individuals, followed by replication in two independent German data sets. The combined analysis of discovery and replication stages (2,269 cases and 7,765 controls) yielded robust associations within the two genes EFEMP1 and KCNH8 (rs17278665, rs727139 with P < 5 × 10−8), and suggestive association within gene SKAP2 (rs2030136 with P < 5 × 10−7). Association signals of rs17278665 and rs727139 reside in regions of low linkage disequilibrium containing no other genes. Data from the ENCODE and Roadmap Epigenomics projects show that tissue specific marks overlap with the variants. SNPs rs17278665 and rs2030136 are known eQTLs. Our study demonstrates that GWAS are a valuable tool to study the genetic component of CVD. With our approach, we identified two novel genome-wide significant susceptibility loci for this common disease. Particularly, the extracellular matrix glycoprotein EFEMP1 is promising for future functional studies due to its antagonistic role in vessel development and angiogenesis.

Genome-wide association study identifies novel susceptibility loci for migraine in Han Chinese resided in Taiwan.

Background Susceptibility genes for migraine, despite it being a highly prevalent and disabling neurological disorder, have not been analyzed in Asians by genome-wide association study (GWAS). Methods We conducted a two-stage case-control GWAS to identify susceptibility genes for migraine without aura in Han Chinese residing in Taiwan. In the discovery stage, we genotyped 1005 clinic-based Taiwanese migraine patients and 1053 population-based sex-matched controls using Axiom Genome-Wide CHB Array. In the replication stage, we genotyped 27 single-nucleotide polymorphisms with p < 10-4 in 1120 clinic-based migraine patients and 604 sex-matched normal controls by using Sequenom. Variants at LRP1, TRPM8, and PRDM, which have been replicated in Caucasians, were also genotyped. Results We identified a novel susceptibility locus (rs655484 in DLG2) that reached GWAS significance level for migraine risk in Han Chinese ( p = 1.45 × 10-12, odds ratio [OR] = 2.42), and also another locus (rs3781545in GFRA1) with suggestive significance ( p = 1.27 × 10-7, OR = 1.38). In addition, we observed positive association signals with a similar trend to the associations identified in Caucasian GWASs for rs10166942 in TRPM8 (OR = 1.33, 95% confidence interval [CI] = 1.14-1.54, Ppermutation = 9.99 × 10-5; risk allele: T) and rs1172113 in LRP1 (OR = 1.23, 95% CI = 1.04-1.45, Ppermutation = 2.9 × 10-2; risk allele: T). Conclusion The present study is the first migraine GWAS conducted in Han-Chinese and Asians. The newly identified susceptibility genes have potential implications in migraine pathogenesis. DLG2 is involved in glutamatergic neurotransmission, and GFRA1 encodes GDNF receptors that are abundant in CGRP-containing trigeminal neurons. Furthermore, positive association signals for TRPM8 and LRP1 suggest the possibility for common genetic contributions across ethnicities.

Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay.

Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting high-throughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers.

A functional SNP rs1892901 in FOSL1 is associated with gastric cancer in Chinese population

FOSL1 (FOS like antigen 1) is one kind of proto-oncogene, and may play a vital role in carcinogenesis of multiple cancers. However, studies about the relationship between SNPs in FOSL1 and gastric cancer are still lacking. Thus, we investigated the association of seven SNPs in FOSL1 with gastric cancer using case-control design in a two-stage strategy (Screening stage: 1,140 gastric cancer cases and 1,547 controls; Replication stage: 1,006 cases and 2,273 controls). We found that rs1892901 was significantly associated with increased risk of gastric cancer in additive model (adjusted OR = 1.25, 95%CI: 1.06–1.47, P = 0.008) in first stage. Following replication results revealed that the relationship between rs1892901 and gastric cancer risk was consistent with our primary results. In silico analysis showed that rs1892901 might alter multiple regulatory motifs, disturb protein binding, and affect the expression of FOSL1 and other important gastric cancer-related genes such as EGR1, CHD, EP300, FOS, JUN and FOSL2. Our findings indicated that functional SNP rs1892901 in FOSL1 might affect the expression of FOSL1, and ultimately increase the risk of gastric cancer. Further functional studies and large-scale population studies are warranted to confirm our findings.

A Genome-Wide Association Study Identifies Genetic Variants Associated with Mathematics Ability

Mathematics ability is a complex cognitive trait with polygenic heritability. Genome-wide association study (GWAS) has been an effective approach to investigate genetic components underlying mathematic ability. Although previous studies reported several candidate genetic variants, none of them exceeded genome-wide significant threshold in general populations. Herein, we performed GWAS in Chinese elementary school students to identify potential genetic variants associated with mathematics ability. The discovery stage included 494 and 504 individuals from two independent cohorts respectively. The replication stage included another cohort of 599 individuals. In total, 28 of 81 candidate SNPs that met validation criteria were further replicated. Combined meta-analysis of three cohorts identified four SNPs (rs1012694, rs11743006, rs17778739 and rs17777541) of SPOCK1 gene showing association with mathematics ability (minimum p value 5.67 × 10−10, maximum β −2.43). The SPOCK1 gene is located on chromosome 5q31.2 and encodes a highly conserved glycoprotein testican-1 which was associated with tumor progression and prognosis as well as neurogenesis. This is the first study to report genome-wide significant association of individual SNPs with mathematics ability in general populations. Our preliminary results further supported the role of SPOCK1 during neurodevelopment. The genetic complexities underlying mathematics ability might contribute to explain the basis of human cognition and intelligence at genetic level.

Geminin deletion in pre-meiotic DNA replication stage causes spermatogenesis defect and infertility.

Geminin plays a critical role in cell cycle regulation by regulating DNA replication and serves as a transcriptional molecular switch that directs cell fate decisions. Spermatogonia lacking Geminin disappear during the initial wave of mitotic proliferation, while geminin is not required for meiotic progression of spermatocytes. It is unclear whether geminin plays a role in pre-meiotic DNA replication in later-stage spermatogonia and their subsequent differentiation. Here, we selectively disrupted Geminin in the male germline using the Stra8-Cre/loxP conditional knockout system. Geminin-deficient mice showed atrophic testes and infertility, concomitant with impaired spermatogenesis and reduced sperm motility. The number of undifferentiated spermatogonia and spermatocytes was significantly reduced; the pachytene stage was impaired most severely. Expression of cell proliferation-associated genes was reduced in Gmnnfl/Δ; Stra8-Cre testes compared to in controls. Increased DNA damage, decreased Cdt1, and increased phosphorylation of Chk1/Chk2 were observed in Geminin-deficient germ cells. These results suggest that geminin plays important roles in pre-meiotic DNA replication and subsequent spermatogenesis.

Long intergenic non-coding RNA expression signature in human breast cancer.

Breast cancer is a complex disease, characterized by gene deregulation. There is less systematic investigation of the capacity of long intergenic non-coding RNAs (lincRNAs) as biomarkers associated with breast cancer pathogenesis or several clinicopathological variables including receptor status and patient survival. We designed a two-stage study, including 1,000 breast tumor RNA-seq data from The Cancer Genome Atlas (TCGA) as the discovery stage, and RNA-seq data of matched tumor and adjacent normal tissue from 50 breast cancer patients as well as 23 normal breast tissue from healthy women as the replication stage. We identified 83 lincRNAs showing the significant expression changes in breast tumors with a false discovery rate (FDR) < 1% in the discovery dataset. Thirty-seven out of the 83 were validated in the replication dataset. Integrative genomic analyses suggested that the aberrant expression of these 37 lincRNAs was probably related with the expression alteration of several transcription factors (TFs). We observed a differential co-expression pattern between lincRNAs and their neighboring genes. We found that the expression levels of one lincRNA (RP5-1198O20 with Ensembl ID ENSG00000230615) were associated with breast cancer survival with P < 0.05. Our study identifies a set of aberrantly expressed lincRNAs in breast cancer.

Genome-wide association study in essential tremor identifies three new loci.

We conducted a genome-wide association study of essential tremor, a common movement disorder characterized mainly by a postural and kinetic tremor of the upper extremities. Twin and family history studies show a high heritability for essential tremor. The molecular genetic determinants of essential tremor are unknown. We included 2807 patients and 6441 controls of European descent in our two-stage genome-wide association study. The 59 most significantly disease-associated markers of the discovery stage were genotyped in the replication stage. After Bonferroni correction two markers, one (rs10937625) located in the serine/threonine kinase STK32B and one (rs17590046) in the transcriptional coactivator PPARGC1A were associated with essential tremor. Three markers (rs12764057, rs10822974, rs7903491) in the cell-adhesion molecule CTNNA3 were significant in the combined analysis of both stages. The expression of STK32B was increased in the cerebellar cortex of patients and expression quantitative trait loci database mining showed association between the protective minor allele of rs10937625 and reduced expression in cerebellar cortex. We found no expression differences related to disease status or marker genotype for the other two genes. Replication of two lead single nucleotide polymorphisms of previous small genome-wide association studies (rs3794087 in SLC1A2, rs9652490 in LINGO1) did not confirm the association with essential tremor.

Marek’s disease herpesvirus vaccines integrate into chicken host chromosomes yet lack a virus-host phenotype associated with oncogenic transformation

Marek’s disease (MD) is a lymphotropic and oncogenic disease of chickens that can lead to death in susceptible and unvaccinated host birds. The causative pathogen, MD virus (MDV), a highly oncogenic alphaherpesvirus, integrates into host genome near the telomeres. MD occurrence is controlled across the globe by biosecurity, selective breeding for enhanced MD genetic resistance, and widespread vaccination of flocks using attenuated serotype 1 MDV or other serotypes. Despite over 40 years of usage, the specific mechanism(s) of MD vaccine-related immunity and anti-tumor effects are not known. Here we investigated the cytogenetic interactions of commonly used MD vaccine strains of all three serotypes (HVT, SB-1, and Rispens) with the host to determine if all were equally capable of host genome integration. We also studied the dynamic profiles of chromosomal association and integration of the three vaccine strains, a first for MD vaccine research. Our cytogenetic data provide evidence that all three MD vaccine strains tested integrate in the chicken host genome as early as 1 day after vaccination similar to oncogenic strains. However, a specific, transformation-associated virus-host phenotype observed for oncogenic viruses is not established. Our results collectively provide an updated model of MD vaccine-host genome interaction and an improved understanding of the possible mechanisms of vaccinal immunity. Physical integration of the oncogenic MDV genome into host chromosomes along with cessation of viral replication appears to have joint signification in MDV’s ability to induce oncogenic transformation. Whereas for MD vaccine serotypes, a sustained viral replication stage and lack of the chromosome-integrated only stage were shared traits during early infection.

Association of kidney structure-related gene variants with type 2 diabetes-attributed end-stage kidney disease in African Americans.

African Americans (AAs) are at higher risk for developing end-stage kidney disease (ESKD) compared to European Americans. Genome-wide association studies have identified variants associated with diabetic and non-diabetic kidney diseases. Nephropathy loci, including SLC7A9, UMOD, and SHROOM3, have been implicated in the maintenance of normal glomerular and renal tubular structure and function. Herein, 47 genes important in podocyte, glomerular basement membrane, mesangial cell, mesangial matrix, renal tubular cell, and renal interstitium structure were examined for association with type 2 diabetes (T2D)-attributed ESKD in AAs. Single-variant association analysis was performed in the discovery stage, including 2041 T2D-ESKD cases and 1140 controls (non-diabetic, non-nephropathy). Discrimination analyses in 667 T2D cases-lacking nephropathy excluded T2D-associated SNPs. Nominal associations were tested in an additional 483 T2D-ESKD cases and 554 controls in the replication stage. Meta-analysis of 4218 discovery and replication samples revealed three significant associations with T2D-ESKD at CD2AP and MMP2 (P corr < 0.05 corrected for effective number of SNPs in each locus). Removal of APOL1 renal-risk genotype carriers revealed additional association at five loci, TTC21B, COL4A3, NPHP3-ACAD11, CLDN8, and ARHGAP24 (P corr<0.05). Genetic variants at COL4A3, CLDN8, and ARHGAP24 were potentially pathogenic. Gene-based associations revealed suggestive significant aggregate effects of coding variants at four genes. Our findings suggest that genetic variation in kidney structure-related genes may contribute to T2D-attributed ESKD in the AA population.

Potent inhibition of Western Equine Encephalitis virus by a fraction rich in flavonoids and phenolic acids obtained from Achyrocline satureioides

Achyrocline satureioides (Lam.) DC. Asteraceae, ‘marcela del campo’, possess several pharmacological properties. Previously we reported antiviral activity of an aqueous extract of A. satureioides against an alphavirus, Western Equine Encephalitis virus. Alphaviruses are highly virulent pathogens which cause encephalitis in humans and equines. There are no effective antiviral to treat its infections. The aim of this study was to evaluate in vitro cytotoxic and antiviral activities against Western Equine Encephalitis virus of five water extract chromatographic fractions from A. satureioides and identify the main compounds of the bioactive fraction. Also, it was to assess in vivo cytogenotoxic ability of the active fraction. Cytotoxicity studies revealed low toxicity of the most of fractions in Vero and in equine peripheral blood mononuclear cells. Antiviral studies showed that the water crude extract – Sephadex LH 20 – fraction 3 MeOH–H2O (Fraction 3) was active against Western Equine Encephalitis virus with Effective Concentration 50%=5μg/ml. Selectivity Indices were 126.0 on Vero and 133.6 on peripheral blood mononuclear cells, four times higher than aqueous extract selectivity index. Regarding the mechanism of action we demonstrated that F3 exerted its action in intracellular replication stages. Further, fraction 3 showed important virucidal action. Fraction 3 contains, in order of highest to lowest: chlorogenic acid, luteolin, 5,7,8-trimethoxyflavone, 3-O-methylquercetin and caffeic acid. Fraction 3 did not induce in vivo toxic nor mutagenic effect. Therefore, it is safe its application as antiviral potential. Further studies of antiviral activity in vivo will be developed using a murine model.

The cell cycle-related genes as biomarkers for schizophrenia

BackgroundRecent studies suggest that genomic abnormalities such as single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) may elevate the risk of schizophrenia. Such genomic abnormalities often occur during chromosomal DNA replication in the S phase of cell cycle. In addition, several studies showed that abnormal expressions of several cell cycle-related genes are associated with schizophrenia. Therefore, here we compared mRNA expression levels of cell cycle-related genes in peripheral blood cells between patients with schizophrenia and healthy controls. MethodmRNA expression levels of cell cycle-related genes in peripheral blood cells from patients with schizophrenia and healthy controls were measured with quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). The discovery, replication and intervention studies with Q-RT-PCR were performed as follows: discovery (40 cases and 20 controls), replication (82 cases and 74 controls) and intervention (22 cases and 18 controls). ResultNine genes were identified in the discovery and replication stages as schizophrenia-associated genes. Moreover, the combination of mRNA expression levels of CDK4, MCM7 and POLD4 was identified as a potential biomarker for schizophrenia with multivariate logistic regression analysis. The intervention stage revealed that the mRNA expression levels of these three genes were significantly decreased in the acute state of schizophrenia, and CDK4 was significantly recovered in the remission state of schizophrenia. ConclusionThe combination of mRNA expression levels of three cell cycle-related genes such as CDK4, MCM7 and POLD4 is expected to be a candidate for useful biomarkers for schizophrenia. Especially, the mRNA expression changes of CDK4 may be potential as both trait and state markers for schizophrenia.

Genetic Variant in ACVR2B Is Associated with Lean Mass.

Low lean mass (LM) is a risk factor for chronic disease, a major cause of disability and diminished quality of life, and is a heritable trait. However, relatively few specific genetic factors have been identified as potentially influencing this trait. In this study, we selected 1493 single-nucleotide polymorphisms (SNP) in 155 candidate genes involved in anabolic, catabolic, growth hormone, and other related pathways and examined their association with LM, assessed by dual-energy x-ray absorptiometry, in a sample of 2760 non-Hispanic and Hispanic white postmenopausal women from the Women's Health Initiative (WHI) Observational Study. We assessed the replication of our top findings in a meta-analysis of 20 genome-wide association studies (n = 38,292) conducted by the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium Musculoskeletal Working Group. We identified 32 SNPs that had nominally significant associations with LM in the WHI cohort. In the replication stage, we find that SNP rs2276541 in the activin A receptor, type IIB (ACVR2B), was significantly associated with LM (β = 0.15, P = 2.17 × 10). ACVR2B codes for a receptor for a negative regulator of skeletal muscle, myostatin, and has previously been identified in a candidate gene study as a determinant of skeletal muscle mass. Our findings support a previously proposed role of ACVR2B allelic variation as a determinant of muscle mass and extend prior findings in men and women. Additional large-scale studies will be needed to confirm our findings in different populations.