DNase 2 Is the Main DNA-Degrading Enzyme of the Stratum Corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA

Examine and Evaluate a Process to Use Salt Caverns to Receive Ship Borne Liquefied Natural Gas: Final Report

The goal of the U.S. Department of Energy cooperative research project is to define, describe, and validate, a process to utilize salt caverns to receive and store the cargoes of LNG ships. The project defines the process as receiving LNG from a ship, pumping the LNG up to cavern injection pressures, warming it to cavern compatible temperatures, injecting the warmed vapor directly into salt caverns for storage, and distribution to the pipeline network. The performance of work under this agreement is based on U.S. Patent 5,511,905, and other U.S. and Foreign pending patent applications. The cost sharing participants in the research are The National Energy Technology Laboratory (U.S. Department of Energy), BP America Production Company, Bluewater Offshore Production Systems (U.S.A.), Inc., and HNG Storage, L.P. Initial results indicate that a salt cavern based receiving terminal could be built at about half the capital cost, less than half the operating costs and would have significantly higher delivery capacity, shorter construction time, and be much more secure than a conventional liquid tank based terminal. There is a significant body of knowledge and practice concerning natural gas storage in salt caverns, and there is a considerable body of knowledge and practice in handling LNG, but there has never been any attempt to develop a process whereby the two technologies can be combined. Salt cavern storage is infinitely more secure than surface storage tanks, far less susceptible to accidents or terrorist acts, and much more acceptable to the community. The project team developed conceptual designs of two salt cavern based LNG terminals, one with caverns located in Calcasieu Parish Louisiana, and the second in Vermilion block 179 about 50 miles offshore Louisiana. These conceptual designs were compared to conventional tank based LNG terminals and demonstrate superior security, economy and capacity. The potential for the development of LNG receiving terminals, utilizing salt caverns for storage and the existing comprehensive pipeline system has profound implications for the next generation of LNG terminals. LNG imports are expected to become an increasingly more important part of the U.S. energy supply and the capacities to receive LNG securely, safely, and economically must be expanded. Salt cavern LNG receiving terminals both in onshore and offshore locations can be quickly built and provide additional import capacity into the U.S. exceeding 6-10 Bcf/day in the aggregate

Genetic basis for <i>Saccharomyces cerevisiae</i> biofilm in liquid medium

Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the Σ1278b background and found 71 genes that were essential for biofilm development. Quantitative northern blots further revealed that AIM1, ASG1, AVT1, DRN1, ELP4, FLO8, FMP10, HMT1, KAR5, MIT1, MRPL32, MSS11, NCP1, NPR1, PEP5, PEX25, RIM8, RIM101, RGT1, SNF8, SPC2, STB6, STP22, TEC1, VID24, VPS20, VTC3, YBL029W, YBL029C-A, YFL054C, YGR161W-C, YIL014C-A, YIR024C, YKL151C, YNL200C, YOR034C-A, and YOR223W controlled biofilm through FLO11 induction. Almost all deletion mutants that were unable to form biofilms in liquid medium also lost the ability to form surface-spreading biofilm colonies (mats) on agar and 69% also lost the ability to grow invasively. The protein kinase A isoform Tpk3p functioned specifically in biofilm and mat formation. In a tpk3 mutant, transcription of FLO11 was induced three-fold compared with wild-type, but biofilm development and cell–cell adhesion was absent, suggesting that Tpk3p regulates FLO11 positive posttranscriptionally and negative transcriptionally. The study provides a resource of biofilm-influencing genes for additional research on biofilm development and suggests that the regulation of FLO11 is more complex than previously anticipated

Genetic Basis for Saccharomyces cerevisiae Biofilm in Liquid Medium

Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the Σ1278b background and found 71 genes that were essential for biofilm development. Quantitative northern blots further revealed that AIM1, ASG1, AVT1, DRN1, ELP4, FLO8, FMP10, HMT1, KAR5, MIT1, MRPL32, MSS11, NCP1, NPR1, PEP5, PEX25, RIM8, RIM101, RGT1, SNF8, SPC2, STB6, STP22, TEC1, VID24, VPS20, VTC3, YBL029W, YBL029C-A, YFL054C, YGR161W-C, YIL014C-A, YIR024C, YKL151C, YNL200C, YOR034C-A, and YOR223W controlled biofilm through FLO11 induction. Almost all deletion mutants that were unable to form biofilms in liquid medium also lost the ability to form surface-spreading biofilm colonies (mats) on agar and 69% also lost the ability to grow invasively. The protein kinase A isoform Tpk3p functioned specifically in biofilm and mat formation. In a tpk3 mutant, transcription of FLO11 was induced three-fold compared with wild-type, but biofilm development and cell–cell adhesion was absent, suggesting that Tpk3p regulates FLO11 positive posttranscriptionally and negative transcriptionally. The study provides a resource of biofilm-influencing genes for additional research on biofilm development and suggests that the regulation of FLO11 is more complex than previously anticipated

Irreversible transformation of ferromagnetic ordered stripe domains in single-shot IR pump - resonant X-ray scattering probe experiments

The evolution of a magnetic domain structure upon excitation by an intense, femtosecond Infra-Red (IR) laser pulse has been investigated using single-shot based time-resolved resonant X-ray scattering at the X-ray Free Electron laser LCLS. A well-ordered stripe domain pattern as present in a thin CoPd alloy film has been used as prototype magnetic domain structure for this study. The fluence of the IR laser pump pulse was sufficient to lead to an almost complete quenching of the magnetization within the ultrafast demagnetization process taking place within the first few hundreds of femtoseconds following the IR laser pump pulse excitation. On longer time scales this excitation gave rise to subsequent irreversible transformations of the magnetic domain structure. Under our specific experimental conditions, it took about 2 nanoseconds before the magnetization started to recover. After about 5 nanoseconds the previously ordered stripe domain structure had evolved into a disordered labyrinth domain structure. Surprisingly, we observe after about 7 nanoseconds the occurrence of a partially ordered stripe domain structure reoriented into a novel direction. It is this domain structure in which the sample's magnetization stabilizes as revealed by scattering patterns recorded long after the initial pump-probe cycle. Using micro-magnetic simulations we can explain this observation based on changes of the magnetic anisotropy going along with heat dissipation in the film.Comment: 16 pages, 6 figure

An integrative taxonomic revision and redefinition of Gephyromantis (Laurentomantis) malagasius based on archival DNA analysis reveals four new mantellid frog species from Madagascar

The subgenus Laurentomantis in the genus Gephyromantis contains some of the least known amphibian species of Madagascar. The six currently valid nominal species are rainforest frogs known from few individuals, hampering a full understanding of the species diversity of the clade. We assembled data on specimens collected during field surveys over the past 30 years and integrated analysis of mitochondrial and nuclear-encoded genes of 88 individuals, a comprehensive bioacoustic analysis, and morphological comparisons to delimit a minimum of nine species-level lineages in the subgenus. To clarify the identity of the species Gephyromantis malagasius, we applied a target-enrichment approach to a sample of the 110 year-old holotype of Microphryne malagasia Methuen and Hewitt, 1913 to assign this specimen to a lineage based on a mitochondrial DNA barcode. The holotype clustered unambiguously with specimens previously named G. ventrimaculatus. Consequently we propose to consider Trachymantis malagasia ventrimaculatus Angel, 1935 as a junior synonym of Gephyromantis malagasius. Due to this redefinition of G. malagasius, no scientific name is available for any of the four deep lineages of frogs previously subsumed under this name, all characterized by red color ventrally on the hindlimbs. These are here formally named as Gephyromantis fiharimpe sp. nov., G. matsilo sp. nov., G. oelkrugi sp. nov., and G. portonae sp. nov. The new species are distinguishable from each other by genetic divergences of >4% uncorrected pairwise distance in a fragment of the 16S rRNA marker and a combination of morphological and bioacoustic characters. Gephyromantis fiharimpe and G. matsilo occur, respectively, at mid-elevations and lower elevations along a wide stretch of Madagascar’s eastern rainforest band, while G. oelkrugi and G. portonae appear to be more range-restricted in parts of Madagascar’s North East and Northern Central East regions. Open taxonomic questions surround G. horridus, to which we here assign specimens from Montagne d’Ambre and the type locality Nosy Be; and G. ranjomavo, which contains genetically divergent populations from Marojejy, Tsaratanana, and Ampotsidy.info:eu-repo/semantics/publishedVersio

Endogenous HMGB1 regulates autophagy

HMGB1 displaces Bcl-2 from Beclin1 to induce and sustain autophagy in response to cell stress

TOM40 Mediates Mitochondrial Dysfunction Induced by α-Synuclein Accumulation in Parkinson's Disease.

Alpha-synuclein (α-Syn) accumulation/aggregation and mitochondrial dysfunction play prominent roles in the pathology of Parkinson's disease. We have previously shown that postmortem human dopaminergic neurons from PD brains accumulate high levels of mitochondrial DNA (mtDNA) deletions. We now addressed the question, whether alterations in a component of the mitochondrial import machinery -TOM40- might contribute to the mitochondrial dysfunction and damage in PD. For this purpose, we studied levels of TOM40, mtDNA deletions, oxidative damage, energy production, and complexes of the respiratory chain in brain homogenates as well as in single neurons, using laser-capture-microdissection in transgenic mice overexpressing human wildtype α-Syn. Additionally, we used lentivirus-mediated stereotactic delivery of a component of this import machinery into mouse brain as a novel therapeutic strategy. We report here that TOM40 is significantly reduced in the brain of PD patients and in α-Syn transgenic mice. TOM40 deficits were associated with increased mtDNA deletions and oxidative DNA damage, and with decreased energy production and altered levels of complex I proteins in α-Syn transgenic mice. Lentiviral-mediated overexpression of Tom40 in α-Syn-transgenic mice brains ameliorated energy deficits as well as oxidative burden. Our results suggest that alterations in the mitochondrial protein transport machinery might contribute to mitochondrial impairment in α-Synucleinopathies

Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis