Spatiotemporal analysis of purkinje cell degeneration relative to parasagittal expression domains in a model of neonatal viral infection

Infection of newborn Lewis rats with Borna disease virus (neonatal Borna disease [NBD]) results in cerebellar damage without the cellular inflammation associated with infections in later life. Purkinje cell (PC) damage has been reported for several models of early-life viral infection, including NBD; however, the time course and distribution of PC pathology have not been investigated rigorously. This study examined the spatiotemporal relationship between PC death and zonal organization in NBD cerebella. Real-time PCR at postnatal day 28 (PND28) revealed decreased cerebellar levels of mRNAs encoding the glycolytic enzymes aldolase C (AldoC, also known as zebrin II) and phosphofructokinase C and the excitatory amino acid transporter 4 (EAAT4). Zebrin II and EAAT4 immunofluorescence analysis in PND21, PND28, PND42, and PND84 NBD rat cerebella revealed a complex pattern of PC degeneration. Early cell loss (PND28) was characterized by preferential apoptotic loss of zebrin II/EAAT4-negative PC subsets in the anterior vermis. Consistent with early preferential loss of zebrin II/EAAT4-negative PCs in the vermis, the densities of microglia and the Bergmann glial expression of metallothionein I/II and the hyaluronan receptor CD44 were higher in zebrin II/EAAT4-negative zones. In contrast, early loss in lateral cerebellar lobules did not reflect a similar discrimination between PC phenotypes. Patterns of vermal PC loss became more heterogeneous at PND42, with the loss of both zebrin II/EAAT4-negative and zebrin II/EAAT4-positive neurons. At PND84, zebrin II/EAAT4 patterning was abolished in the anterior cerebellum, with preferential PC survival in lobule X. Our investigation reveals regional discrimination between patterns of PC subset loss, defined by zebrin II/EAAT4 expression domains, following neonatal viral infection. These findings suggest a differential vulnerability of PC subsets during the early stages of virus-induced neurodegeneration.     

Productive varicella-zoster virus infection of cultured intact human ganglia

Varicella-zoster virus (VZV) is a species-specific herpesvirus which infects sensory ganglia. We have developed a model of infection of human intact explant dorsal root ganglia (DRG). Following exposure of DRG to VZV, viral antigens were detected in neurons and nonneuronal cells. Enveloped virions were visualized by transmission electron microscopy in neurons and nonneuronal cells and within the extracellular space. Moreover, rather than remaining highly cell associated during infection of cultured cells, such as fibroblasts, cell-free VZV was released from infected DRG. This model enables VZV infection of ganglionic cells to be studied in the context of intact DRG.     

Expression of conserved signalling pathway genes during spontaneous vascular differentiation of R1 embryonic stem cells and in Py-4-1 endothelial cells

Embryonic stem (ES) cells are an invaluable model for identifying subtle phenotypes as well as severe outcomes of perturbing gene function that may otherwise result in lethality. However, though ES cells of different origins are regarded as equally pluripotent, their in vitro differentiation potential varies, suggesting that their response to developmental signals is different. The R1 cell line is widely used for gene manipulation due to its good growth characteristics and highly efficient germline transmission. Hence, we analysed the expression of Notch, Wnt and Sonic Hedgehog (Shh) pathway genes during differentiation of R1 cells into early vascular lineages. Notch-, Wnt-and Shh-mediated signalling is important during embryonic development. Regulation of gene expression through these signalling molecules is a frequently used theme, resulting in context-dependent outcomes during development. Perturbing these pathways can result in severe and possibly lethal developmental phenotypes often due to primary cardiovascular defects. We report that during early spontaneous differentiation of R1 cells, Notch-1 and the Wnt target Brachyury are active whereas the Shh receptor is not detected. This expression pattern is similar to that seen in a mouse endothelial cell line. This temporal study of expression of genes representative of all three pathways in ES cell differentiation will aid in further analysis of cell signalling during vascular development.     

Sequencing of 15 622 gene‐bearing BACs clarifies the gene‐dense regions of the barley genome

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole‐genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene‐containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical‐mapped gene‐bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene‐enriched BACs and are characterized by high recombination rates, there are also gene‐dense regions with suppressed recombination. We made use of published map‐anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D‐genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley–Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map‐based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene‐dense but low recombination is particularly relevant.     

Differential scanning calorimetric and spectroscopic studies on the unfolding of Momordica charantia lectin. Similar modes of thermal and chemical denaturation

Thermal stability of Momordica charantia seed lectin (MCL) was investigated as a function of protein concentration, pH, scan rate, and at different ligand concentrations by using high-sensitivity differential scanning calorimetry (DSC). The DSC endotherm obtained at pH 7.4 consists of two entities with transition temperatures at ca. 333.7 K, and 338 K. The unfolding process is irreversible and could be described by a three-state model. For MCL tetramer ΔH_c/ΔH_v ratio is close to 4 for the first transition and ∼2 for the second transition, suggesting that four and two cooperative units are involved in the first and second transitions, respectively. In the presence of lactose both transitions shifted to higher temperatures, suggesting that ligand binds preferentially to the native conformation of MCL. Endotherms recorded as a function of pH indicate that MCL is more stable at lower pH. Chemical unfolding of MCL, induced by Gdn.HCl, was investigated by monitoring the intrinsic fluorescence properties of the protein. The results obtained indicate that chemical denaturation of MCL can also be described by a three-state process, involving an intermediate populated at ∼3–4 M Gdn.HCl. These observations suggest that the chemical and thermal unfolding processes are similar in that both of them proceed via an intermediate. The far UV and near UV CD spectra of MCL were nearly identical at different pH values and indicate that its secondary and tertiary structure do not change significantly with pH, suggesting that the structure of the protein is stable over a wide pH range.     

Host-age-dependent parasitism and reproductive success of Apanteles stantoni (Hymenoptera: Braconidae), a larval parasitoid of Diaphania indica (Lepidoptera: Pyralidae)

The relative suitability of five instars of Diaphania indica (Saunders) (Lepidoptera: Pyralidae) as a substrate for the development of a larval parasitoid, Apanteles stantoni Ashmead (Hymenoptera: Braconidae), was investigated. Maximum parasitism (22.25?±?1.21%) under laboratory conditions was observed in the early larval instars. The highest parasitoid emergence was recorded from the second (86.07?±?0.70%) and third (98.93?±?0.72%) instar larvae of D. indica, and that from the first larvae was 71.43?±?1.18%. The number of cocoons in each cluster, length and width of single cocoons, percentage emergence, sex ratio and adult longevity of A. stantoni collected from different instars of D. indica were also recorded. These results indicated that the life stage of the host when the parasitoid larvae complete their final instar is particularly important for their development. Therefore, considering the efficiency of parasitism and reproduction, the second-instar larvae of D. indica is the most suitable stage for mass rearing A. stantoni in the laboratory.     

Fluidization and Resolidification of the Human Bladder Smooth Muscle Cell in Response to Transient Stretch

Background

Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood.

Principal Findings

In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize.

Conclusions

Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin.     

Differential regulation of MRN (Mre11-Rad50-Nbs1) complex subunits and telomerase activity in cancer cells

Several lines of evidence suggest that cancer progression is associated with up-regulation or reactivation of telomerase and the underlying mechanism remains an active area of research. The heterotrimeric MRN complex, consisting of Mre11, Rad50 and Nbs1, which is required for the repair of double-strand breaks, plays a key role in telomere length maintenance. In this study, we show significant differences in the levels of expression of MRN complex subunits among various cancer cells and somatic cells. Notably, siRNA-mediated depletion of any of the subunits of MRN complex led to complete ablation of other subunits of the complex. Treatment of leukemia and prostate cancer cells with etoposide lead to increased expression of MRN complex subunits, with concomitant decrease in the levels of telomerase activity, compared to breast cancer cells. These studies raise the possibility of developing anti-cancer drugs targeting MRN complex subunits to sensitize a subset of cancer cells to radio- and/or chemotherapy.