CTCF-dependent chromatin bias constitutes transient epigenetic memory of the mother at the H19-Igf2 imprinting control region in prospermatogonia

Genomic imprints-parental allele-specific DNA methylation marks at the differentially methylated regions (DMRs) of imprinted genes-are erased and reestablished in germ cells according to the individual's sex. Imprint establishment at paternally methylated germ line DMRs occurs in fetal male germ cells. In prospermatogonia, the two unmethylated alleles exhibit different rates of de novo methylation at the H19/Igf2 imprinting control region (ICR) depending on parental origin. We investigated the nature of this epigenetic memory using bisulfite sequencing and allele-specific ChIP-SNuPE assays. We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele. We determined genetically that the chromatin bias, and also the delayed methylation establishment in the maternal allele, depended on functional CTCF insulator binding sites in the ICR. Our data suggest that, in primordial germ cells, maternally inherited allele-specific CTCF binding sets up allele-specific chromatin differences at the ICR. The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins. CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.     

The addition of a goal-based motivational interview to standardised treatment as usual to reduce dropouts in a service for patients with personality disorder: a feasibility study

Background

Verrucae are a common, infectious and sometimes painful problem. The optimal treatment for verrucae is unclear due to a lack of high quality randomised controlled trials. The primary objective of this study is to compare the clinical effectiveness of two common treatments for verrucae: cryotherapy using liquid nitrogen versus salicylic acid. Secondary objectives include a comparison of the cost-effectiveness of the treatments, and an investigation of time to clearance of verrucae, recurrence/clearance of verrucae at six months, patient satisfaction with treatment, pain associated with treatment, and use of painkillers for the treatments.

Methods/Design

This is an open, pragmatic, multicentre, randomised controlled trial with two parallel groups: cryotherapy using liquid nitrogen delivered by a healthcare professional for a maximum of 4 treatments (treatments 2-3 weeks apart) or daily self-treatment with 50% salicylic acid for a maximum of 8 weeks. Two hundred and sixty-six patients aged 12 years and over with a verruca are being enrolled into the study. The primary outcome is complete clearance of all verrucae as observed on digital photographs taken at 12 weeks compared with baseline and assessed by an independent healthcare professional. Secondary outcomes include self-reported time to clearance of verrucae, self-reported clearance of verrucae at 6 months, cost-effectiveness of the treatments compared to one another, and patient acceptability of both treatments including possible side effects such as pain. The primary analysis will be intention to treat. It is planned that recruitment will be completed by December 2009 and results will be available by June 2010.

Trial registration

Current Controlled Trials ISRCTN18994246.     

Establishment of an in vitro assay for assessing the effects of drugs on the liver stages of Plasmodium vivax malaria

Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages.     

Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma

Background

We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC).

Methodology/Principal Findings

Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (α-1,3) fucosylation. Increases in core (α-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific.

Conclusions/Significance

This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.     

A Crucial Role for Kupffer Cell-Derived Galectin-9 in Regulation of T Cell Immunity in Hepatitis C Infection

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (∼70–80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-γ, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4⁺CD25⁺FoxP3⁺CD127^low regulatory T cells, contraction of CD4⁺ effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.     

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.Members of the genus Crinivirus are emerging plant viruses in many parts of the world. An important factor contributing to the increase in the incidence of these viruses is their association with and transmission by whitefly vectors that have increased in distribution in the last several decades. Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus (family Closteroviridae), is specifically transmitted by the sweet potato whitefly, Bemisia tabaci biotype A, in a semipersistent, noncirculative manner (6). The virus is confined to phloem cells within infected plants and is not transmissible to plants by leaf rub inoculation. The bipartite single-stranded positive-sense LIYV genome components, consisting of RNA 1 (approximately 8.1 kb) and RNA 2 (approximately 7.2 kb), are separately encapsidated in flexuous filamentous particles that are characteristic of the family Closteroviridae (8, 11). These virions are comprised of four protein components: the major coat protein (CP), the minor coat protein (CPm), an Hsp70 homolog (Hsp70h), and a 59-kDa protein (P59). Like other viruses in the family Closteroviridae, LIYV has bipolar virions with a “body” composed mainly of the CP and a “head” that is formed by the assembly of CPm subunits (2, 4, 7, 22, 28). Hsp70h and P59 are detected in LIYV virions (22), but their locations have not been identified, as they are not readily detected by immunogold labeling and transmission electron microscopy (IGL-TEM). For two members of the family Closteroviridae, Citrus tristeza virus (CTV) and Beet yellows virus (BYV), the combination of Hsp70h, P61 (the homolog of LIYV P59 in CTV) or P64 (the homolog of LIYV P59 in BYV), and CPm encapsidates the 5′ end (∼630 to 650 nucleotides [nt]) of the RNA genome, demonstrating the complex interactions that exist among the capsid proteins and the genomic RNA (15, 21).In our previous studies, we demonstrated the transmission of LIYV using an in vitro acquisition and whitefly transmission system (13, 22). Results from previous work implicated a role for LIYV CPm in whitefly transmission. Antibodies to CPm blocked the in vitro acquisition/transmission of LIYV virion preparations by B. tabaci biotype A, while antibodies to CP, Hsp70h, and P59 did not (22). The in vitro whitefly membrane-feeding system had also been used to demonstrate B. tabaci biotype A transmission of virions that were derived from cloned infectious cDNAs of LIYV RNA 1 and RNA 2 of several genotypes, including pR6 (the first cloned wild-type [WT] infectious cDNA of LIYV RNA 2 [10]), establishing for the first time that these cloned constructs contained all of the information necessary for protoplast infection, virion formation, whitefly transmission, and infection in plants (12). In that study, the mutant p1-5b was among the cloned LIYV RNA 2 cDNAs derived from B. tabaci biotype A-transmitted virus maintained in Chenopodium murale plants.p1-5b contains a single-adenine-residue deletion in the CPm open reading frame (ORF) at nucleotide 592, a deletion that is predicted to result in a frameshift, 14 new amino acids, and premature termination of the protein (12). The predicted p1-5b CPm has 211 amino acids, compared to 453 amino acids in the wild-type (pR6 genotype) protein. The p1-5b genotype also contains three other nucleotide changes in the CPm ORF relative to the pR6 infectious clone sequence (27), all of which result in amino acid changes. In contrast, the p1-5b CP, Hsp70h, and P59 sequences are identical to that of pR6 (12). Possible polymorphisms throughout the rest of the p1-5b clone were not characterized. In a prior study, B. tabaci biotype A transmission of p1-5b virions was not observed, even though the mutation did not affect its infectivity in protoplasts (as determined by virion yields) and apparent particle morphology (12). However, those studies were disadvantaged by the necessity of propagation in protoplasts to obtain specific genotypes from infectious cloned cDNAs. Protoplasts yield low quantities of virion relative to plants, and virion concentration is a critical parameter in whitefly transmission (13). Although virion concentrations in those experiments were above typical thresholds for whitefly transmission (12, 13), low concentrations may still be limiting for transmission, making negative transmission results difficult to interpret. Obtaining adequate virion concentrations of specific genotypes for whitefly transmission to plants has therefore been a significant hurdle to LIYV transmission studies.The recently developed agroinoculation method for LIYV (24) permits the study of systemic plant infection by distinct LIYV genotypes, including those that are whitefly transmission deficient, and the recovery of higher virion yields than were possible using protoplasts. The objective of this study was to further examine the function of the LIVY CPm by extending our observations of p1-5b. We constructed mutants with the CPm frameshift restored to determine if engineered mutations that either restored or disrupted the formation of an intact CPm also affected systemic plant infection, virion formation, and B. tabaci biotype A transmission. Our study revealed that a mutant engineered with the restored CPm ORF produced a WT infection profile characterized by systemic virus movement within agroinoculated plants and the generation of CPm-containing virions that were whitefly transmissible. Intriguingly, systemic virus movement was also observed for a mutant engineered to express the 1-5b CPm, but the virions lacked an identifiable CPm and were defective in whitefly transmission. These results represent a significant advance in addressing challenging questions and hypotheses about Crinivirus whitefly transmission properties not testable using earlier systems.     

Population Status of the Critically Endangered Preuss’s Red Colobus Monkey (Piliocolobus preussi Matschie 1900) and Recommendations for Its Conservation

International Journal of Primatology - Overhunting and habitat loss from the expansion of agriculture and extractive industries are the primary threats to primate species, 65% of which are...     

The MRN complex promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 and MRE11 are required for RAD51-dependent homologous recombination and phosphorylation of histone H2A following a DNA double strand break (DSB), but neither MRE11 nor RAD50 substantially influence DSB resection at a chromosome-internal locus. In addition, we reveal intrinsic separation-of-function between T. brucei RAD50 and MRE11, with only RAD50 suppressing DSB repair using donors with short stretches of homology at a subtelomeric locus, and only MRE11 directing DSB resection at the same locus. Finally, we show that loss of either MRE11 or RAD50 causes a greater diversity of expressed VSG variants following DSB repair. We conclude that MRN promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.     

Discovery of Novel Herpes Simplexviruses in Wild Gorillas,Bonobos, and Chimpanzees Supports Zoonotic Origin of HSV-2

Viruses closely related to human pathogens can reveal the origins of human infectious diseases. Human herpes simplexvirus type 1 (HSV-1) and type 2 (HSV-2) are hypothesized to have arisen via host-virus codivergence and cross-species transmission. We report the discovery of novel herpes simplexviruses during a large-scale screening of fecal samples from wild gorillas, bonobos, and chimpanzees. Phylogenetic analysis indicates that, contrary to expectation, simplexviruses from these African apes are all more closely related to HSV-2 than to HSV-1. Molecular clock-based hypothesis testing suggests the divergence between HSV-1 and the African great ape simplexviruses likely represents a codivergence event between humans and gorillas. The simplexviruses infecting African great apes subsequently experienced multiple cross-species transmission events over the past 3 My, the most recent of which occurred between humans and bonobos around 1 Ma. These findings revise our understanding of the origins of human herpes simplexviruses and suggest that HSV-2 is one of the earliest zoonotic pathogens.