Mitochondrial diseases and genetic defects of ATP synthase

ATP synthase is a key enzyme of mitochondrial energy conversion. In mammals, it produces most of cellular ATP. Alteration of ATP synthase biogenesis may cause two types of isolated defects: qualitative when the enzyme is structurally modified and does not function properly, and quantitative when it is present in insufficient amounts. In both cases the cellular energy provision is impaired, and diminished use of mitochondrial DeltamuH+ promotes ROS production by the mitochondrial respiratory chain. The primary genetic defects have so far been localized in mtDNA ATP6 gene and nuclear ATP12 gene, however, involvement of other nuclear genes is highly probable.     

Rapid protocol for electroporation of Clostridium perfringens

A rapid and simple method has been developed for the electroporation of Clostridium perfringens with plasmid DNA. The new improvements, harvesting cells early in the logarithmic stage of growth, keeping the cells at room temperature and the absence of post-shock incubation on ice increased transformation efficiency by one order of magnitude.     

Review of impact categories and environmental indicators for life cycle assessment of geotechnical systems

Life cycle assessment (LCA) has only had limited application in the geotechnical engineering discipline, though it has been widely applied to civil engineering systems such as pavements and roadways. A review of previous geotechnical LCAs showed that most studies have tracked a small set of impact categories, such as energy and global warming potential. Accordingly, currently reported environmental indicators may not effectively or fully capture important environmental impacts and tradeoffs associated with geotechnical systems, including those associated with land and soil resources. This research reviewed previous studies, methods, and models for assessment of land use and soil‐related impacts to understand their applicability to geotechnical LCA. The results of this review show that critical gaps remain in current knowledge and practice. In particular, further development or refinement of environmental indicators, impact categories, and cause–effect pathways is needed as they pertain to geotechnical applications—specifically those related to soil quality, soil functions, and the ecosystem services soils provide. In addition, many existing methods emerge from research on land use and land use change related to other disciplines (e.g., agriculture). For applicability to geotechnical projects, the resolution of many of these methods and resulting indicators need to be downscaled from the landscape/macro scale to the project scale. In the near term, practitioners of geotechnical LCA should begin tracking changes to soil properties and report impacts to land and soil resources qualitatively.     

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.     

Syntaxin 11 marks a distinct intracellular compartment recruited to the immunological synapse of NK cells to colocalize with cytotoxic granules

The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11-deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell-type-specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-containing compartment, which was clearly distinct from cytotoxic granules or Rab27a-expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK-sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11-deficent allo-specific cytotoxic T-lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11-expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a-expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule-mediated cytotoxicity.     

Comprehensive evaluation of one-carbon metabolism pathway gene variants and renal cell cancer risk

Introduction

Folate and one-carbon metabolism are linked to cancer risk through their integral role in DNA synthesis and methylation. Variation in one-carbon metabolism genes, particularly MTHFR, has been associated with risk of a number of cancers in epidemiologic studies, but little is known regarding renal cancer.

Methods

Tag single nucleotide polymorphisms (SNPs) selected to produce high genomic coverage of 13 gene regions of one-carbon metabolism (ALDH1L1, BHMT, CBS, FOLR1, MTHFR, MTR, MTRR, SHMT1, SLC19A1, TYMS) and the closely associated glutathione synthesis pathway (CTH, GGH, GSS) were genotyped for 777 renal cell carcinoma (RCC) cases and 1,035 controls in the Central and Eastern European Renal Cancer case-control study. Associations of individual SNPs (n = 163) with RCC risk were calculated using unconditional logistic regression adjusted for age, sex and study center. Minimum p-value permutation (Min-P) tests were used to identify gene regions associated with risk, and haplotypes were evaluated within these genes.

Results

The strongest associations with RCC risk were observed for SLC19A1 (P_min-P = 0.03) and MTHFR (P_min-P = 0.13). A haplotype consisting of four SNPs in SLC19A1 (rs12483553, rs2838950, rs2838951, and rs17004785) was associated with a 37% increased risk (p = 0.02), and exploratory stratified analysis suggested the association was only significant among those in the lowest tertile of vegetable intake.

Conclusions

To our knowledge, this is the first study to comprehensively examine variation in one-carbon metabolism genes in relation to RCC risk. We identified a novel association with SLC19A1, which is important for transport of folate into cells. Replication in other populations is required to confirm these findings.     

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short‐lifetime near‐infrared dyes and time‐gated imaging

We introduce a simple new approach for time‐resolved multiplexed analysis of complex systems using near‐infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user‐friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time‐gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely‐available software, has the advantage of time‐resolved NIR imaging, including better tissue penetration and background‐free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image‐guided surgery or optical tomography.      

Acute Treatment with Docosahexaenoic Acid Complexed to Albumin Reduces Injury after a Permanent Focal Cerebral Ischemia in Rats

Docosahexaenoic acid complexed to albumin (DHA-Alb) is highly neuroprotective after temporary middle cerebral artery occlusion (MCAo), but whether a similar effect occurs in permanent MCAo is unknown. Male Sprague-Dawley rats (270–330 g) underwent permanent MCAo. Neurological function was evaluated on days 1, 2 and 3 after MCAo. We studied six groups: DHA (5 mg/kg), Alb (0.63 or 1.25 g/kg), DHA-Alb (5 mg/kg+0.63 g/kg or 5 mg/kg+1.25 g/kg) or saline. Treatment was administered i.v. at 3 h after onset of stroke (n = 7–10 per group). Ex vivo imaging of brains and histopathology were conducted on day 3. Saline- and Alb-treated rats developed severe neurological deficits but were not significantly different from one another. In contrast, rats treated with low and moderate doses of DHA-Alb showed improved neurological score compared to corresponding Alb groups on days 2 and 3. Total, cortical and subcortical lesion volumes computed from T2 weighted images were reduced following a moderate dose of DHA-Alb (1.25 g/kg) by 25%, 22%, 34%, respectively, compared to the Alb group. The total corrected, cortical and subcortical infarct volumes were reduced by low (by 36–40%) and moderate doses (by 34–42%) of DHA-Alb treatment compared to the Alb groups. In conclusion, DHA-Alb therapy is highly neuroprotective in permanent MCAo in rats. This treatment can provide the basis for future therapeutics for patients suffering from ischemic stroke.     

Isolation and characterization of high affinity aptamers against DNA polymerase iota

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.