Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA

A systematic review of the use of an expertise-based randomised controlled trial design

Acknowledgements JAC held a Medical Research Council UK methodology (G1002292) fellowship, which supported this research. The Health Services Research Unit, Institute of Applied Health Sciences (University of Aberdeen), is core-funded by the Chief Scientist Office of the Scottish Government Health and Social Care Directorates. Views express are those of the authors and do not necessarily reflect the views of the funders.Peer reviewedPublisher PD

Nitrogen and sulphur management: challenges for organic sources in temperate agricultural systems

A current global trend towards intensification or specialization of agricultural enterprises has been accompanied by increasing public awareness of associated environmental consequences. Air and water pollution from losses of nutrients, such as nitrogen (N) and sulphur (S), are a major concern. Governments have initiated extensive regulatory frameworks, including various land use policies, in an attempt to control or reduce the losses. This paper presents an overview of critical input and loss processes affecting N and S for temperate climates, and provides some background to the discussion in subsequent papers evaluating specific farming systems. Management effects on potential gaseous and leaching losses, the lack of synchrony between supply of nutrients and plant demand, and options for optimizing the efficiency of N and S use are reviewed. Integration of inorganic and organic fertilizer inputs and the equitable re-distribution of nutrients from manure are discussed. The paper concludes by highlighting a need for innovative research that is also targeted to practical approaches for reducing N and S losses, and improving the overall synchrony between supply and demand

Pesticide exposure and lymphohaematopoietic cancers: a case-control study in an agricultural region (Larissa, Thessaly, Greece)

<p>Abstract</p> <p>Background</p> <p>The causality of lymphohaematopoietic cancers (LHC) is multifactorial and studies investigating the association between chemical exposure and LHC have produced variable results. The aim of this study was to investigate the relationships between exposure to pesticides and LHC in an agricultural region of Greece.</p> <p>Methods</p> <p>A structured questionnaire was employed in a hospital-based case control study to gather information on demographics, occupation, exposure to pesticides, agricultural practices, family and medical history and smoking. To control for confounders, backward conditional and multinomial logistic regression analyses were used. To assess the dose-response relationship between exposure and disease, the chi-square test for trend was used.</p> <p>Results</p> <p>Three hundred and fifty-four (354) histologically confirmed LHC cases diagnosed from 2004 to 2006 and 455 sex- and age-matched controls were included in the study. Pesticide exposure was associated with total LHC cases (OR 1.46, 95% CI 1.05-2.04), myelodysplastic syndrome (MDS) (OR 1.87, 95% CI 1.00-3.51) and leukaemia (OR 2.14, 95% CI 1.09-4.20). A dose-response pattern was observed for total LHC cases (P = 0.004), MDS (P = 0.024) and leukaemia (P = 0.002). Pesticide exposure was independently associated with total LHC cases (OR 1.41, 95% CI 1.00 - 2.00) and leukaemia (OR 2.05, 95% CI 1.02-4.12) after controlling for age, smoking and family history (cancers, LHC and immunological disorders). Smoking during application of pesticides was strongly associated with total LHC cases (OR 3.29, 95% CI 1.81-5.98), MDS (OR 3.67, 95% CI 1.18-12.11), leukaemia (OR 10.15, 95% CI 2.15-65.69) and lymphoma (OR 2.72, 95% CI 1.02-8.00). This association was even stronger for total LHC cases (OR 18.18, 95% CI 2.38-381.17) when eating simultaneously with pesticide application.</p> <p>Conclusions</p> <p>Lymphohaematopoietic cancers were associated with pesticide exposure after controlling for confounders. Smoking and eating during pesticide application were identified as modifying factors increasing the risk for LHC. The poor pesticide work practices identified during this study underline the need for educational campaigns for farmers.</p

Pliocene-Quaternary crustal melting in central and northern Tibet and insights into crustal flow

There is considerable controversy over the nature of geophysically recognized low-velocity-high-conductivity zones (LV-HCZs) within the Tibetan crust, and their role in models for the development of the Tibetan Plateau. Here we report petrological and geochemical data on magmas erupted 4.7-0.3 Myr ago in central and northern Tibet, demonstrating that they were generated by partial melting of crustal rocks at temperatures of 700-1,050°C and pressures of 0.5-1.5 GPa. Thus Pliocene-Quaternary melting of crustal rocks occurred at depths of 15-50 km in areas where the LV-HCZs have been recognized. This provides new petrological evidence that the LV-HCZs are sources of partial melt. It is inferred that crustal melting played a key role in triggering crustal weakening and outward crustal flow in the expansion of the Tibetan Plateau

Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

Acetylation Regulates WRN Catalytic Activities and Affects Base Excision DNA Repair

Background: The Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone acetyltransferases is of key interest because of its potential importance in aging, DNA repair and transcription. Methodology/Principal Findings: Here, we have investigated the p300 acetylation mediated changes on the function of WRN in base excision DNA repair (BER). We show that acetylation of WRN increases in cells treated with methyl methanesulfonate (MMS), suggesting that acetylation of WRN may play a role in response to DNA damage. This hypothesis is consistent with our findings that acetylation of WRN stimulates its catalytic activities in vitro and in vivo, and that acetylated WRN enhances pol b-mediated strand displacement DNA synthesis more than unacetylated WRN. Furthermore, we show that cellular exposure to the histone deacetylase inhibitor sodium butyrate stimulates long patch BER in wild type cells but not in WRN depleted cells, suggesting that acetylated WRN participates significantly in this process. Conclusion/Significance: Collectively, these results provide the first evidence for a specific role of p300 mediated WRN acetylation in regulating its function during BER

A Gammaherpesvirus Cooperates with Interferon-alpha/beta-Induced IRF2 to Halt Viral Replication, Control Reactivation, and Minimize Host Lethality

The gammaherpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish latency in memory B lymphocytes and promote lymphoproliferative disease in immunocompromised individuals. The precise immune mechanisms that prevent gammaherpesvirus reactivation and tumorigenesis are poorly defined. Murine gammaherpesvirus 68 (MHV68) is closely related to EBV and KSHV, and type I (alpha/beta) interferons (IFNαβ) regulate MHV68 reactivation from both B cells and macrophages by unknown mechanisms. Here we demonstrate that IFNβ is highly upregulated during latent infection, in the absence of detectable MHV68 replication. We identify an interferon-stimulated response element (ISRE) in the MHV68 M2 gene promoter that is bound by the IFNαβ-induced transcriptional repressor IRF2 during latency in vivo. The M2 protein regulates B cell signaling to promote establishment of latency and reactivation. Virus lacking the M2 ISRE (ISREΔ) overexpresses M2 mRNA and displays uncontrolled acute replication in vivo, higher latent viral load, and aberrantly high reactivation from latency. These phenotypes of the ISREΔ mutant are B-cell-specific, require IRF2, and correlate with a significant increase in virulence in a model of acute viral pneumonia. We therefore identify a mechanism by which a gammaherpesvirus subverts host IFNαβ signaling in a surprisingly cooperative manner, to directly repress viral replication and reactivation and enforce latency, thereby minimizing acute host disease. Since we find ISREs 5′ to the major lymphocyte latency genes of multiple rodent, primate, and human gammaherpesviruses, we propose that cooperative subversion of IFNαβ-induced IRFs to promote latent infection is an ancient strategy that ensures a stable, minimally-pathogenic virus-host relationship

Estimating Contact Process Saturation in Sylvatic Transmission of Trypanosoma cruzi in the United States

Although it has been known for nearly a century that strains of Trypanosoma cruzi, the etiological agent for Chagas' disease, are enzootic in the southern U.S., much remains unknown about the dynamics of its transmission in the sylvatic cycles that maintain it, including the relative importance of different transmission routes. Mathematical models can fill in gaps where field and lab data are difficult to collect, but they need as inputs the values of certain key demographic and epidemiological quantities which parametrize the models. In particular, they determine whether saturation occurs in the contact processes that communicate the infection between the two populations. Concentrating on raccoons, opossums, and woodrats as hosts in Texas and the southeastern U.S., and the vectors Triatoma sanguisuga and Triatoma gerstaeckeri, we use an exhaustive literature review to derive estimates for fundamental parameters, and use simple mathematical models to illustrate a method for estimating infection rates indirectly based on prevalence data. Results are used to draw conclusions about saturation and which population density drives each of the two contact-based infection processes (stercorarian/bloodborne and oral). Analysis suggests that the vector feeding process associated with stercorarian transmission to hosts and bloodborne transmission to vectors is limited by the population density of vectors when dealing with woodrats, but by that of hosts when dealing with raccoons and opossums, while the predation of hosts on vectors which drives oral transmission to hosts is limited by the population density of hosts. Confidence in these conclusions is limited by a severe paucity of data underlying associated parameter estimates, but the approaches developed here can also be applied to the study of other vector-borne infections

Triadin/Junctin Double Null Mouse Reveals a Differential Role for Triadin and Junctin in Anchoring CASQ to the jSR and Regulating Ca2+ Homeostasis

Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca2+ imaging and Ca2+ selective microelectrodes we found that changes in e-c coupling, SR Ca2+content and resting [Ca2+] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca2+ regulation than Jct/CASQ association