High variability in the MHC class II DA beta chain of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.     

Ataxia telangiectasia mutated influences cytochrome c oxidase activity

Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1 h with the ATM inhibitor KU55933 caused a ∼50% reduction (P < 0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P < 0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity.     

Identification, replication, and fine-mapping of Loci associated with adult height in individuals of african ancestry

Adult height is a classic polygenic trait of high heritability (h ² ∼0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain ∼10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10⁻¹² and 2p14-rs4315565, P = 1.2×10⁻⁸). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10⁻⁴ for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits.     

Hands-Free Image Capture,Data Tagging and Transfer Using Google Glass: A Pilot Study for Improved Wound Care Management

Chronic wounds, including pressure ulcers, compromise the health of 6.5 million Americans and pose an annual estimated burden of $25 billion to the U.S. health care system. When treating chronic wounds, clinicians must use meticulous documentation to determine wound severity and to monitor healing progress over time. Yet, current wound documentation practices using digital photography are often cumbersome and labor intensive. The process of transferring photos into Electronic Medical Records (EMRs) requires many steps and can take several days. Newer smartphone and tablet-based solutions, such as Epic Haiku, have reduced EMR upload time. However, issues still exist involving patient positioning, image-capture technique, and patient identification. In this paper, we present the development and assessment of the SnapCap System for chronic wound photography. Through leveraging the sensor capabilities of Google Glass, SnapCap enables hands-free digital image capture, and the tagging and transfer of images to a patient’s EMR. In a pilot study with wound care nurses at Stanford Hospital (n=16), we (i) examined feature preferences for hands-free digital image capture and documentation, and (ii) compared SnapCap to the state of the art in digital wound care photography, the Epic Haiku application. We used the Wilcoxon Signed-ranks test to evaluate differences in mean ranks between preference options. Preferred hands-free navigation features include barcode scanning for patient identification, Z(15) = -3.873, p < 0.001, r = 0.71, and double-blinking to take photographs, Z(13) = -3.606, p < 0.001, r = 0.71. In the comparison between SnapCap and Epic Haiku, the SnapCap System was preferred for sterile image-capture technique, Z(16) = -3.873, p < 0.001, r = 0.68. Responses were divided with respect to image quality and overall ease of use. The study’s results have contributed to the future implementation of new features aimed at enhancing mobile hands-free digital photography for chronic wound care.     

In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy

Raman micro-spectroscopy combined with multivariate analysis was employed to monitor real-time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double-strand DNA breaks in human type II pneumocyte-like cells (A549 cell-line). Raman spectra of A549 cells exposed to 100 microM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over-expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro-spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Oxide-Based Solid-State Batteries: A Perspective on Composite Cathode Architecture

The garnet-type phase Li₇La₃Zr₂O₁₂ (LLZO) attracts significant attention as an oxide solid electrolyte to enable safe and robust solid-state batteries (SSBs) with potentially high energy density. However, while significant progress has been made in demonstrating compatibility with Li metal, integrating LLZO into composite cathodes remains a challenge. The current perspective focuses on the critical issues that need to be addressed to achieve the ultimate goal of an all-solid-state LLZO-based battery that delivers safety, durability, and pack-level performance characteristics that are unobtainable with state-of-the-art Li-ion batteries. This perspective complements existing reviews of solid/solid interfaces with more emphasis on understanding numerous homo- and heteroionic interfaces in a pure oxide-based SSB and the various phenomena that accompany the evolution of the chemical, electrochemical, structural, morphological, and mechanical properties of those interfaces during processing and operation. Finally, the insights gained from a comprehensive literature survey of LLZO–cathode interfaces are used to guide efforts for the development of LLZO-based SSBs.     

Characterization of a deep-sea microbial mat from an active cold seep at the Milano mud volcano in the Eastern Mediterranean Sea

A white, filamentous microbial mat at the Milano mud volcano in the Eastern Mediterranean Sea was sampled during the Medinaut cruise of the R/V Nadir in 1998. The composition of the mat community was characterized using a combination of phylogenetic and lipid biomarker methods. The mat sample was filtered through 0.2 and 5-microm filters to coarsely separate unicellular and filamentous bacteria. Analyses of 16S rRNA gene sequences amplified from the total community DNA from these fractions showed that similar archaeal populations were present in both fractions. However, the bacterial populations in the fractions differed from one another, and were more diverse than the archaeal ones. Lipid analysis showed that bacteria were the dominant members of the mat microbial community and the relatively low delta(13)C carbon isotope values of bulk bacterial lipids suggested the occurrence of methane- and sulfide-based chemo(auto)trophy. Consistent with this, the bacterial populations in the fractions were related to Alpha-, Gamma- and Epsilonproteobacteria, most of which were chemoautotrophic bacteria that utilize hydrogen sulfide (or reduced sulfur compounds) and/or methane. The most common archaeal 16S rRNA gene sequences were related to those of previously identified Archaea capable of anaerobic methane oxidation. Although the filamentous organisms observed in the mat were not conclusively identified, our results indicated that the Eastern Mediterranean deep-sea microbial mat community might be sustained on a combination of methane- and sulfide-driven chemotrophy.     

DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication

The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.