Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to 'rescue' a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.

Illness causal beliefs in Turkish immigrants

<p>Abstract</p> <p>Background</p> <p>People hold a wide variety of beliefs concerning the causes of illness. Such beliefs vary across cultures and, among immigrants, may be influenced by many factors, including level of acculturation, gender, level of education, and experience of illness and treatment. This study examines illness causal beliefs in Turkish-immigrants in Australia.</p> <p>Methods</p> <p>Causal beliefs about somatic and mental illness were examined in a sample of 444 members of the Turkish population of Melbourne. The socio-demographic characteristics of the sample were broadly similar to those of the Melbourne Turkish community. Five issues were examined: the structure of causal beliefs; the relative frequency of natural, supernatural and metaphysical beliefs; ascription of somatic, mental, or both somatic and mental conditions to the various causes; the correlations of belief types with socio-demographic, modernizing and acculturation variables; and the relationship between causal beliefs and current illness.</p> <p>Results</p> <p>Principal components analysis revealed two broad factors, accounting for 58 percent of the variation in scores on illness belief scales, distinctly interpretable as natural and supernatural beliefs. Second, beliefs in natural causes were more frequent than beliefs in supernatural causes. Third, some causal beliefs were commonly linked to both somatic and mental conditions while others were regarded as more specific to either somatic or mental disorders. Last, there was a range of correlations between endorsement of belief types and factors defining heterogeneity within the community, including with demographic factors, indicators of modernizing and acculturative processes, and the current presence of illness.</p> <p>Conclusion</p> <p>Results supported the classification of causal beliefs proposed by Murdock, Wilson & Frederick, with a division into natural and supernatural causes. While belief in natural causes is more common, belief in supernatural causes persists despite modernizing and acculturative influences. Different types of causal beliefs are held in relation to somatic or mental illness, and a variety of apparently logically incompatible beliefs may be concurrently held. Illness causal beliefs are dynamic and are related to demographic, modernizing, and acculturative factors, and to the current presence of illness. Any assumption of uniformity of illness causal beliefs within a community, even one that is relatively culturally homogeneous, is likely to be misleading. A better understanding of the diversity, and determinants, of illness causal beliefs can be of value in improving our understanding of illness experience, the clinical process, and in developing more effective health services and population health strategies.</p

Growth Differentiation Factor 15 Is Induced by Hepatitis C Virus Infection and Regulates Hepatocellular Carcinoma-Related Genes

Liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) are commonly induced by chronic hepatitis C virus (HCV) infection. We aimed to identify and characterize the involvement of previously screened cytokine GDF15 in HCV pathogenesis. We examined the GDF15 expression after HCV infection both in vitro and in vivo. Cultured JFH-1 HCV was used to determine the GDF15 function on virus propagation. GDF15 overexpression and RNA interference were employed to profile the GDF15-regulated genes, signaling pathways and cell biology phenotypes. The mRNA expression and protein secretion of GDF15 was dramatically increased in HCV-infected hepatoma cells, which maybe a host response to viral proteins or infection-induced cell stress. Patients infected with HCV had an average 15-fold higher blood GDF15 level than that of healthy volunteers. Three HCC individuals in the HCV cohort showed extremely high GDF15 concentrations. Transfection or exogenously supplied GDF15 enhanced HCV propagation, whereas knockdown of endogenous GDF15 resulted in inhibition of virus replication. Overexpressed GDF15 led to Akt activation and the phosphorylation of Akt downstream targeted GSK-3β and Raf. Several HCC-related molecules, such as E-cadherin, β-catenin, Cyclin A2/B1/D1, were up-regulated by GDF15 stimulation in vitro. Overexpression of GDF15 in hepatoma cells resulted in increased DNA synthesis, promoted cell proliferation, and importantly enhanced invasiveness of the cells. In conclusion, these results suggest that an elevated serum GDF15 level is a potential diagnostic marker for viral hepatitis, and GDF15 may contribute to HCV pathogenesis by altering the signaling and growth of host cells

Subcellular localization of Mitf in monocytic cells

Microphthalmia-associated transcription factor (Mitf) is a transcription factor that plays an important role in regulating the development of several cell lineages. The subcellular localization of Mitf is dynamic and is associated with its transcription activity. In this study, we examined factors that affect its subcellular localization in cells derived from the monocytic lineage since Mitf is present abundantly in these cells. We identified a domain encoded by Mitf exon 1B1b to be important for Mitf to commute between the cytoplasm and the nucleus. Deletion of this domain disrupts the shuttling of Mitf to the cytoplasm and results in its retention in the nucleus. M-CSF and RANKL both induce nuclear translocation of Mitf. We showed that Mitf nuclear transport is greatly influenced by ratio of M-CSF/Mitf protein expression. In addition, cell attachment to a solid surface also is needed for the nuclear transport of Mitf

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

[[abstract]]Background: Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. Principal Finding: In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10?5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). Conclusion:These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification

A group randomized trial of a complexity-based organizational intervention to improve risk factors for diabetes complications in primary care settings: study protocol

<p>Abstract</p> <p>Background</p> <p>Most patients with type 2 diabetes have suboptimal control of their glucose, blood pressure (BP), and lipids – three risk factors for diabetes complications. Although the chronic care model (CCM) provides a roadmap for improving these outcomes, developing theoretically sound implementation strategies that will work across diverse primary care settings has been challenging. One explanation for this difficulty may be that most strategies do not account for the complex adaptive system (CAS) characteristics of the primary care setting. A CAS is comprised of individuals who can learn, interconnect, self-organize, and interact with their environment in a way that demonstrates non-linear dynamic behavior. One implementation strategy that may be used to leverage these properties is practice facilitation (PF). PF creates time for learning and reflection by members of the team in each clinic, improves their communication, and promotes an individualized approach to implement a strategy to improve patient outcomes.</p> <p>Specific objectives</p> <p>The specific objectives of this protocol are to: evaluate the effectiveness and sustainability of PF to improve risk factor control in patients with type 2 diabetes across a variety of primary care settings; assess the implementation of the CCM in response to the intervention; examine the relationship between communication within the practice team and the implementation of the CCM; and determine the cost of the intervention both from the perspective of the organization conducting the PF intervention and from the perspective of the primary care practice.</p> <p>Intervention</p> <p>The study will be a group randomized trial conducted in 40 primary care clinics. Data will be collected on all clinics, with 60 patients in each clinic, using a multi-method assessment process at baseline, 12, and 24 months. The intervention, PF, will consist of a series of practice improvement team meetings led by trained facilitators over 12 months. Primary hypotheses will be tested with 12-month outcome data. Sustainability of the intervention will be tested using 24 month data. Insights gained will be included in a delayed intervention conducted in control practices and evaluated in a pre-post design.</p> <p>Primary and secondary outcomes</p> <p>To test hypotheses, the unit of randomization will be the clinic. The unit of analysis will be the repeated measure of each risk factor for each patient, nested within the clinic. The repeated measure of glycosylated hemoglobin A1c will be the primary outcome, with BP and Low Density Lipoprotein (LDL) cholesterol as secondary outcomes. To study change in risk factor level, a hierarchical or random effect model will be used to account for the nesting of repeated measurement of risk factor within patients and patients within clinics.</p> <p>This protocol follows the CONSORT guidelines and is registered per ICMJE guidelines:</p> <p>Clinical Trial Registration Number</p> <p>NCT00482768</p

Clinical symptoms and performance on the continuous performance test in children with attention deficit hyperactivity disorder between subtypes: a natural follow-up study for 6 months

<p>Abstract</p> <p>Background</p> <p>The aims of this study were to determine the time course of improvements in attention deficit hyperactivity disorder (ADHD) clinical symptoms and neurocognitive function in a realistic clinical setting, and the differences in ADHD symptom improvement using different classifications of ADHD subtypes.</p> <p>Methods</p> <p>The Child Behavior Checklist (CBCL) was completed by parents of ADHD children at the initial visit. The computerized Continuous Performance Test (CPT), Swanson, Nolan, and Pelham, and Version IV Scale for ADHD (SNAP-IV), and ADHD Rating Scale (ADHD-RS) were performed at baseline, one month, three months, and six months later, respectively. Patient care including drug therapy was performed at the discretion of the psychiatrist. The ADHD patients were divided into DSM-IV subtypes (Inattentive, Hyperactive-impulsive and Combined type), and were additionally categorized into aggressive and non-aggressive subtypes by aggression scale in CBCL for comparisons.</p> <p>Results</p> <p>There were 50 ADHD patients with a mean age of 7.84 ± 1.64 years; 15 of them were inattentive type, 11 were hyperactive-impulsive type, and 24 were combined type. In addition, 28 of the ADHD patients were grouped into aggressive and 22 into non-aggressive subtypes. There were significant improvements in clinical symptoms of hyperactivity and inattention, and impulsivity performance in CPT during the 6-month treatment. The clinical hyperactive symptoms were significantly different between ADHD patients sub-grouping both by DSM-IV and aggression. Non-aggressive patients had significantly greater changes in distraction and impulsivity performances in CPT from baseline to month 6 than aggressive patients.</p> <p>Conclusions</p> <p>We found that ADHD symptoms, which included impulsive performances in CPT and clinical inattention and hyperactivity dimensions, had improved significantly over 6 months under pragmatic treatments. The non-aggressive ADHD patients might have a higher potential for improving in CPT performance than aggressive ones. However, it warrant further investigation whether the different classifications of ADHD patients could be valid for predicting the improvements in ADHD patients' clinical symptoms and neurocognitive performance.</p

Loss of LMO4 in the Retina Leads to Reduction of GABAergic Amacrine Cells and Functional Deficits

BACKGROUND: LMO4 is a transcription cofactor expressed during retinal development and in amacrine neurons at birth. A previous study in zebrafish reported that morpholino RNA ablation of one of two related genes, LMO4b, increases the size of eyes in embryos. However, the significance of LMO4 in mammalian eye development and function remained unknown since LMO4 null mice die prior to birth. METHODOLOGY/PRINCIPAL FINDINGS: We observed the presence of a smaller eye and/or coloboma in ∼40% LMO4 null mouse embryos. To investigate the postnatal role of LMO4 in retinal development and function, LMO4 was conditionally ablated in retinal progenitor cells using the Pax6 alpha-enhancer Cre/LMO4flox mice. We found that these mice have fewer Bhlhb5-positive GABAergic amacrine and OFF-cone bipolar cells. The deficit appears to affect the postnatal wave of Bhlhb5+ neurons, suggesting a temporal requirement for LMO4 in retinal neuron development. In contrast, cholinergic and dopaminergic amacrine, rod bipolar and photoreceptor cell numbers were not affected. The selective reduction in these interneurons was accompanied by a functional deficit revealed by electroretinography, with reduced amplitude of b-waves, indicating deficits in the inner nuclear layer of the retina. CONCLUSIONS/SIGNIFICANCE: Inhibitory GABAergic interneurons play a critical function in controlling retinal image processing, and are important for neural networks in the central nervous system. Our finding of an essential postnatal function of LMO4 in the differentiation of Bhlhb5-expressing inhibitory interneurons in the retina may be a general mechanism whereby LMO4 controls the production of inhibitory interneurons in the nervous system

Isolation and Characterization of Novel Murine Epiphysis Derived Mesenchymal Stem Cells

BACKGROUND: While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. CONCLUSIONS/SIGNIFICANCES: These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities