Tetracycline inducible gene manipulation in serotonergic neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.     

First molecular epidemiological study of cutaneous leishmaniasis in Libya

Background

Cutaneous leishmaniasis (CL) is a major public health problem in Libya. The objective of this study was to investigate, for the first time, epidemiological features of CL outbreaks in Libya including molecular identification of parasites, the geographical distribution of cases and possible scenarios of parasite transmission.

Methodology/Principal Findings

We studied 450 patients that came from 49 areas distributed in 12 districts in north-west Libya. The patients'' ages ranged from 9 months to 87 years (median age 25 years); 54% of the cases were males. Skin scrapings spotted on glass slides were collected for molecular identification of causative agent. The ribosomal internal transcribed spacer 1 (ITS1) was amplified and subsequently characterized by restriction fragment length polymorphism (RFLP) analysis. In total, 195 samples were successfully identified of which 148 (75.9%) were Leishmania major, and 47 (24.1%) Leishmania tropica. CL cases infected with L. major were found in all CL areas whereas L. tropica cases came mainly from Al Jabal Al Gharbi (46.4%), Misrata (17.8%) and Tarhuna districts (10.7%). A trend of seasonality was noticed for the infections with L. major which showed a clear peak between November and January, but was less pronounced for infections by L. tropica.

Conclusion

The first molecular study on CL in Libya revealed that the disease is caused by L. major and L. tropica and the epidemiological patterns in the different foci were the same as in other Mediterranean foci of CL.     

Restoring Stem Cell Function in Aged Tissues by Direct Reprogramming?

Adult stem cells are responsible for the cellular turnover of many organs, and an impairment in their function leads to aging and disease. In efforts to reverse the process of tissue stem cell aging, we speculate on the promise and challenges of in vivo direct reprogramming strategies.     

Virulence of a Klebsiella pneumoniae strain carrying the New Delhi metallo-beta-lactamase-1 (NDM-1)

The aim of the study was to compare and evaluate virulence in five strains of Klebsiella pneumoniae, including an isolate carrying New Delhi metallo-beta-lactamase-1 (NDM-1). In vivo virulence was assessed using a murine sepsis model and using the nematode Caenorhabditis elegans killing model, and in vitro virulence by assessing various virulence factors. The NDM-1 carrying K. pneumoniae isolate was the most virulent in the murine sepsis model but there was no clear cut correlation to in vitro virulence factors or killing in C. elegans. It is concluded that K. pneumoniae carrying NDM-1 have an intrinsic virulence potential, which in coexistence with its multiresistance could promote and partly explain its epidemiological success.     

Lysozyme facilitates adherence of Enterococcus faecium to host cells and induction of necrotic cell death

The prevalence of infections with enterococci is increasing worldwide. However, little is known about the mechanisms which enable these opportunistic pathogens to cause infections of their host. Here we demonstrate that Enterococcus faecium in the presence of lysozyme induces necrosis in human and mouse cells after 4 h indicated by disrupted cellular membranes of epithelial (HeLa), myeloid (U937, J774A.1) and lymphoid (Jurkat J16, thymocytes), but not intestinal epithelial cells (CaCo-2, CMT-93). Using an appropriate mutant strain it was shown that the enterococcal surface-protein SgrA is involved in cell death induction in mouse cells (J774A.1, thymocytes). Microscopic analyses of epithelial cells 30 min post infection revealed that lysozyme increases adhesion of E. faecium to HeLa, but not CaCo-2 cells. At that time the phalloidin-FITC-stained cytoskeleton of infected cells was still intact, whereas 2 h post infection the F-actin network of HeLa, but not CaCo-2 cells was disrupted. Hence, the early, lysozyme-mediated increase of bacterial adherence plays an important role for cell death induction by E. faecium in HeLa cells. Moreover, bacterial extracellular hydrogen peroxide might contribute to necrosis induction, since the rate of propidium iodide-positive HeLa and J774A.1 cells was lowered after infection with a ROS-deficient E. faecium mutant.     

Estimating the Prevalence of Nonpaternity in Germany

The prevalence of nonpaternity in human societies is difficult to establish. To obtain a current and fairly unbiased estimate of the nonpaternity rate in Germany, we analysed a dataset consisting of 971 children and their parents in whom human leukocyte antigen (HLA) typing had been carried out in the context of bone marrow transplantation. In this sample, nine exclusions (0.93%) could be identified on the basis of more than 300 HLA-haplotypes defined by four HLA genes. Given this number of exclusions, a maximum likelihood estimate of the nonpaternity rate in the population of 0.94% was obtained with asymptotic 95% confidence limits of 0.33% and 1.55%, respectively. This result is in accordance with recent surveys as well as findings from Switzerland for a comparable sample, and it suggests that earlier estimates of the nonpaternity rate which were often in excess of 10% may have been largely exaggerated.     

Phylogeny, diversity, and species delimitation of the North American Round-Nosed Minnows (Teleostei: Dionda), as inferred from mitochondrial and nuclear DNA sequences

Accurate delimitation of species is a critical first step in protecting biodiversity. Detection of distinct species is especially important for groups of organisms that inhabit sensitive environments subject to recent degradation, such as creeks, springs, and rivers in arid or semi-desert regions. The genus Dionda currently includes six recognized and described species of minnows that live in clear springs and spring-fed creeks of Texas, New Mexico (USA), and northern Mexico, but the boundaries, delimitation, and characterization of species in this genus have not been examined rigorously. The habitats of some of the species in this genus are rapidly deteriorating, and many local populations of Dionda have been extirpated. Considering the increasing concerns over degradation of their habitat, and pending a more detailed morphological revision of the genus, we undertook a molecular survey based on four DNA regions to examine variation over the range of the genus, test species boundaries, and infer phylogenetic relationships within Dionda. Based on analyses of two mitochondrial (cytb and D-loop) and two nuclear (Rag1 and S7) DNA regions from specimens collected throughout the range of Dionda, we identified 12 distinct species in the genus. Formerly synonymized names are available for two of these species, and four other species remain undescribed. We also redefine the known range of six species. The limited distribution of several of the species, coupled with widespread habitat degradation, suggests that many of the species in this genus should be targets for conservation and recovery efforts.