CACNA1H missense mutations associated with amyotrophic lateral sclerosis alter Cav3.2 T-type calcium channel activity and reticular thalamic neuron firing

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. In a recent study by Steinberg and colleagues, 2 recessive missense mutations were identified in the Ca_v3.2 T-type calcium channel gene (CACNA1H), in a family with an affected proband (early onset, long duration ALS) and 2 unaffected parents. We have introduced and functionally characterized these mutations using transiently expressed human Ca_v3.2 channels in tsA-201 cells. Both of these mutations produced mild but significant changes on T-type channel activity that are consistent with a loss of channel function. Computer modeling in thalamic reticular neurons suggested that these mutations result in decreased neuronal excitability of thalamic structures. Taken together, these findings implicate CACNA1H as a susceptibility gene in amyotrophic lateral sclerosis.     

The L33F darunavir resistance mutation acts as a molecular anchor reducing the flexibility of the HIV-1 protease 30s and 80s loops

HIV-1 protease (PR) is a 99 amino acid protein responsible for proteolytic processing of the viral polyprotein – an essential step in the HIV-1 life cycle. Drug resistance mutations in PR that are selected during antiretroviral therapy lead to reduced efficacy of protease inhibitors (PI) including darunavir (DRV). To identify the structural mechanisms associated with the DRV resistance mutation L33F, we performed X-ray crystallographic studies with a multi-drug resistant HIV-1 protease isolate that contains the L33F mutation (MDR769 L33F). In contrast to other PR L33F DRV complexes, the structure of MDR769 L33F complexed with DRV reported here displays the protease flaps in an open conformation. The L33F mutation increases noncovalent interactions in the hydrophobic pocket of the PR compared to the wild-type (WT) structure. As a result, L33F appears to act as a molecular anchor, reducing the flexibility of the 30s loop (residues 29–35) and the 80s loop (residues 79–84). Molecular anchoring of the 30s and 80s loops leaves an open S1/S1′ subsite and distorts the conserved hydrogen-bonding network of DRV. These findings are consistent with previous reports despite structural differences with regards to flap conformation.     

Lineage specific evolution of the VNTR composite retrotransposon central domain and its role in retrotransposition of gibbon LAVA elements

Background

VNTR (Variable Number of Tandem Repeats) composite retrotransposons - SVA (SINE-R-VNTR-Alu), LAVA (LINE-1-Alu-VNTR-Alu), PVA (PTGR2-VNTR-Alu) and FVA (FRAM-VNTR-Alu) - are specific to hominoid primates. Their assembly, the evolution of their 5’ and 3’ domains, and the functional significance of the shared 5’ Alu-like region are well understood. The central VNTR domain, by contrast, has long been assumed to represent a more or less random collection of 30-50 bp GC-rich repeats. It is only recently that it attracted attention in the context of regulation of SVA expression.

Results

Here we provide evidence that the organization of the VNTR is non-random, with conserved repeat unit (RU) arrays at both the 5’ and 3’ ends of the VNTRs of human, chimpanzee and orangutan SVA and gibbon LAVA. The younger SVA subfamilies harbour highly organized internal RU arrays. The composition of these arrays is specific to the human/chimpanzee and orangutan lineages, respectively. Tracing the development of the VNTR through evolution we show for the first time how tandem repeats evolve within the constraints set by a functional, non-autonomous non-LTR retrotransposon in two different families - LAVA and SVA - in different hominoid lineages. Our analysis revealed that a microhomology-driven mechanism mediates expansion/contraction of the VNTR domain at the DNA level.Elements of all four VNTR composite families have been shown to be mobilized by the autonomous LINE1 retrotransposon in trans. In case of SVA, key determinants of mobilization are found in the 5’ hexameric repeat/Alu-like region. We now demonstrate that in LAVA, by contrast, the VNTR domain determines mobilization efficiency in the context of domain swaps between active and inactive elements.

Conclusions

The central domain of VNTR composites evolves in a lineage-specific manner which gives rise to distinct structures in gibbon LAVA, orangutan SVA, and human/chimpanzee SVA. The differences observed between the families and lineages are likely to have an influence on the expression and mobilization of the elements.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1543-z) contains supplementary material, which is available to authorized users.     

Antiangiogenic Arming of an Oncolytic Vaccinia Virus Enhances Antitumor Efficacy in Renal Cell Cancer Models

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.In 2002 renal cell cancer accounted for more than 200,000 cases and 100,000 deaths worldwide (33). Unfortunately, chemotherapy, radiotherapy, and immunotherapy yield low response rates (9, 17) in this cancer type. Thus, prognosis for patients is poor, especially when the disease is metastatic, as median survival is only 8 months (19). Although recently approved drugs, such as sorafenib, sunitinib, temsirolimus, and bevacizumab, have provided additional tools for treatment of renal cell cancer (7), they are usually not curative, and thus new treatment approaches are needed.Oncolytic vaccinia viruses are promising agents for cancer treatment and have shown compelling results in preclinical tumor models (40, 42, 45). Moreover, good safety and preliminary evidence of antitumor efficacy were seen in phase 1 clinical trials (22, 26, 32). Vaccinia virus has a strong oncolytic effect due to its fast replication cycle (45) and a high innate tropism to cancer tissue (34). Tumor targeting can be further improved by deleting vaccinia virus genes that are necessary for replication in normal cells but not in cancer cells. For example, deletions of either thymidine kinase (TK) or vaccinia virus growth factor (VGF) or both have been shown to reduce pathogenicity compared to wild-type virus (3, 5, 27). To enhance antitumor potency, oncolytic vaccinia viruses can be armed with therapeutic transgenes, such as immunostimulatory factors (26) or suicide genes (14, 16, 35). With regard to kidney cancer, an arming approach with antiangiogenenic molecules seems logical, considering the high vascularization characteristic of renal tumors (20).Vascular endothelial growth factor (VEGF) is a major player in tumor angiogenesis and is highly expressed in renal cell cancers (29). VEGF binds to the fms-like-tyrosine kinase receptor (flt-1 or VEGFR-1) and kinase domain region receptor (KDR or VEGFR-2) with high affinity (13). The soluble vascular endothelial growth factor receptor 1-Ig fusion protein (VEGFR-1-Ig) used in this study is derived from the membrane-bound VEGFR-1 and binds human and murine VEGF without inducing vascular endothelial cell mitogenesis (31). Blocking VEGF with this or closely related molecules has been shown to inhibit tumor growth in several cancer models (18, 21, 25, 39).Although tumor cell selective replication can be enhanced by deletion of TK and/or VGF to reduce pathogenicity (3, 5, 27), high doses of attenuated vaccinia virus may increase serum cytokine concentrations which parallel the onset of toxic events, as seen with other viral vectors (2, 38). The potential “early” toxicity associated with oncolytic vaccinia viruses has not been completely elucidated heretofore (36, 46).Given the high vascularization of renal cell cancers and the pressing need to generate new antitumor agents with increased safety and efficacy, we hypothesized that an oncolytic vaccinia virus targeted by TK and VGF deletions and armed with VEGFR-1-Ig would exhibit enhanced antitumor efficacy due to its antiangiogenic properties in renal cell cancer models compared to a nonarmed control virus, allowing reduction of the treatment dose.     

A Simple Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.     

Assaying the proton transport and regulation of UCP1 using solid supported membranes

The uncoupling protein 1 (UCP1) is a mitochondrial protein that carries protons across the inner mitochondrial membrane. It has an important role in non-shivering thermogenesis, and recent evidence suggests its role in human adult metabolism. Using rapid solution exchange on solid supported membranes, we succeeded in measuring electrical currents generated by the transport activity of UCP1. The protein was purified from mouse brown adipose tissue, reconstituted in liposomes and absorbed on solid supported membranes. A fast pH jump activated the ion transport, and electrical signals could be recorded. The currents were characterized by a fast rise and a slow decay, were stable over time, inhibited by purine nucleotides and activated by fatty acids. This new assay permits direct observation of UCP1 activity in controlled cell-free conditions, and opens up new possibilities for UCP1 functional characterization and drug screening because of its robustness and its potential for automation.     

Plants,microorganisms, and soil temperatures contribute to a decrease in methane fluxes on a drained Arctic floodplain

As surface temperatures are expected to rise in the future, ice‐rich permafrost may thaw, altering soil topography and hydrology and creating a mosaic of wet and dry soil surfaces in the Arctic. Arctic wetlands are large sources of CH₄, and investigating effects of soil hydrology on CH₄ fluxes is of great importance for predicting ecosystem feedback in response to climate change. In this study, we investigate how a decade‐long drying manipulation on an Arctic floodplain influences CH₄‐associated microorganisms, soil thermal regimes, and plant communities. Moreover, we examine how these drainage‐induced changes may then modify CH₄ fluxes in the growing and nongrowing seasons. This study shows that drainage substantially lowered the abundance of methanogens along with methanotrophic bacteria, which may have reduced CH₄ cycling. Soil temperatures of the drained areas were lower in deep, anoxic soil layers (below 30 cm), but higher in oxic topsoil layers (0–15 cm) compared to the control wet areas. This pattern of soil temperatures may have reduced the rates of methanogenesis while elevating those of CH₄ oxidation, thereby decreasing net CH₄ fluxes. The abundance of Eriophorum angustifolium, an aerenchymatous plant species, diminished significantly in the drained areas. Due to this decrease, a higher fraction of CH₄ was alternatively emitted to the atmosphere by diffusion, possibly increasing the potential for CH₄ oxidation and leading to a decrease in net CH₄ fluxes compared to a control site. Drainage lowered CH₄ fluxes by a factor of 20 during the growing season, with postdrainage changes in microbial communities, soil temperatures, and plant communities also contributing to this reduction. In contrast, we observed CH₄ emissions increased by 10% in the drained areas during the nongrowing season, although this difference was insignificant given the small magnitudes of fluxes. This study showed that long‐term drainage considerably reduced CH₄ fluxes through modified ecosystem properties.