Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Beijing isolates from the Mediterranean area

Background  

The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases.     

Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.     

A rice phenomics study—phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net “houses” for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chyr-Guan Chern, Ming-Jen Fan, and Su-May Yu have contributed equally to this work.     

Penetrating living cells using semiconductor nanowires

A recent publication by Kim et al. on penetrating both human embryonic kidney cells and mouse embryonic stem cells with Si nanowires highlights the increasing interest in using proven semiconductor materials not only to detect specific biomolecules in solutions but also to deliver genetic material or potentially screen for the presence of particular molecules at the cell level. Many semiconductors are biocompatible and this recent work has shown that penetrating cells with large diameters compared with those of the semiconductor nanowire is not fatal to the cell and that the cells remain functional for a few days.     

Risk of chronic myeloid and acute leukemia mortality after exposure to ionizing radiation among workers at four U.S. nuclear weapons facilities and a nuclear naval shipyard

A nested case-control study was conducted among workers at five U.S. nuclear facilities to evaluate leukemia mortality risk (excluding chronic lymphocytic) from ionizing radiation using worksite doses and adjusting for potential confounding. Conditional logistic regression was used to estimate the relative risk (RR) of exposed workers and the excess relative risk (ERR) per unit of radiation among 206 cases and 823 age-matched controls. Adjusting for sex and benzene, the RR of leukemia for workers receiving more than 10 mSv was higher compared to those receiving lower or no dose; however, the risk increase was attenuated in the highest dose group. The ERR per 10 mSv was 1.44% (95% CI: < -1.03%, 7.59%) but was higher for workers born after 1921 compared to workers born earlier or when excluding leukemias of uncertain type. Excluding the 7% who were high-dose workers (> 100 mSv), the sex- and benzene-adjusted ERR per 10 mSv was 6.82% (95% CI: -2.87%, 24.1%). The results suggest that risks among these nuclear workers are comparable to those observed in high-dose populations, although no evidence was observed of a positive quadratic dose-response term in this study. This large study is among the first to jointly evaluate benzene and ionizing radiation risk.     

Effect of hypoxia on the release of vascular endothelial growth factor and testosterone in mouse TM3 Leydig cells

Hypoxia has been shown to stimulate the expression of vascular endothelial growth factor (VEGF), which is a major mediator for angiogenesis and vasculogenesis. During hypoxia, VEGF promotes angiogenesis in the testis. However, the effect of VEGF on the steroidogenesis of testosterone and the cell proliferation in Leydig cells is unclear. To assess the effects and the action mechanisms of hypoxia, a mouse TM3 Leydig cell line was employed in the present study. The Leydig cells were incubated in an incubator chamber (95% N2-5% CO2) for 1-24 h. The cultured media were collected and assayed by testosterone RIA and VEGF enzyme immunoassay. 3-(4,50-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide assay was used to detect the proliferation of Leydig cells. The present results showed that the proliferation of Leydig cells was enhanced significantly by hypoxia. The basal VEGF release was increased, and the response of VEGF production to human chorionic gonadotropin (hCG) was also enhanced in hypoxic condition. During hypoxia, administration of hCG or VEGF stimulated proliferation of Leydig cells, but the stimulatory effect was abolished by the administration of anti-VEGF antibody. Higher doses of VEGF stimulated testosterone release in a dose-dependent manner. Administration of anti-VEGF antibody abolished the stimulatory effect of VEGF on testosterone release. These data suggest that hypoxia stimulates cell proliferation and testosterone release in Leydig cells via an increase of VEGF production.     

Niche regulation of corneal epithelial stem cells at the limbus

Among all adult somatic stem cells,those of the corneal epithelium are unique in their exclusive location in a definedlimbai structure termed Palisades of Vogt.As a result,surgical engraftment oflimbal epithelial stem cells with or withoutex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency.Neverthe-less,compared to other stem cell examples,relatively little is known about the limbal niche,which is believed to play apivotal role in regulating self-renewal and fate decision of limbal epithelial stem cells.This review summarizes relevantliterature and formulates several key questions to guide future research into better understanding of the pathogenesis oflimbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing onthe limbal niche.     

Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system

BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.