Hepatitis C Virus Phylogenetic Clustering Is Associated with the Social-Injecting Network in a Cohort of People Who Inject Drugs

It is hypothesized that social networks facilitate transmission of the hepatitis C virus (HCV). We tested for association between HCV phylogeny and reported injecting relationships using longitudinal data from a social network design study. People who inject drugs were recruited from street drug markets in Melbourne, Australia. Interviews and blood tests took place three monthly (during 2005–2008), with participants asked to nominate up to five injecting partners at each interview. The HCV core region of individual isolates was then sequenced and phylogenetic trees were constructed. Genetic clusters were identified using bootstrapping (cut-off: 70%). An adjusted Jaccard similarity coefficient was used to measure the association between the reported injecting relationships and relationships defined by clustering in the phylogenetic analysis (statistical significance assessed using the quadratic assignment procedure). 402 participants consented to participate; 244 HCV infections were observed in 238 individuals. 26 genetic clusters were identified, with 2–7 infections per cluster. Newly acquired infection (AOR = 2.03, 95% CI: 1.04–3.96, p = 0.037, and HCV genotype 3 (vs. genotype 1, AOR = 2.72, 95% CI: 1.48–4.99) were independent predictors of being in a cluster. 54% of participants whose infections were part of a cluster in the phylogenetic analysis reported injecting with at least one other participant in that cluster during the study. Overall, 16% of participants who were infected at study entry and 40% of participants with newly acquired infections had molecular evidence of related infections with at least one injecting partner. Likely transmission clusters identified in phylogenetic analysis correlated with reported injecting relationships (adjusted Jaccard coefficient: 0.300; p<0.001). This is the first study to show that HCV phylogeny is associated with the injecting network, highlighting the importance of the injecting network in HCV transmission.     

Advanced Liposomal Vectors as Cancer Vaccines in Melanoma Immunotherapy

Malignant tumors represent a major source of disability and account for more than one of five deaths in Western countries. Among the different cancers, melanoma harbors two distinctive features. First, its has long been recognized as an immunogenic tumor, and second, an unprecedented rise in incidence is currently observed, in face of few therapeutic options. Thus, melanoma represent an ideal target for a cancer immunotherapy program. To date, a number of immunodominant epitopes from tumor associated antigens (TAA) are used as cancer vaccines in clinical trials, in spite of an acknowledged rapid degradation in vivo and low immunogenicity. However, most of the immunotherapy trials reported so far do not achieve consistent clinical results. Hence, there is an urgent need for the development of a carrier system and strong adjuvants suitable for a TAA-based cancer immunotherapy. Liposomes and their further development as virosomes with added adjuvancy may address both these issues. We report here our experience in the tailoring of dedicated advanced liposomal vectors that were developed in the context of an upcoming immunotherapy clinical trial for melanoma.     

The Role of Hypoxia Inducible Factor-1 Alpha in Bypassing Oncogene-Induced Senescence

Oncogene induced senescence (OIS) is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR), senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on Ras^V12-induced senescence in human diploid fibroblasts (HDFs). We showed here that hypoxia prevents execution of oncogene induced senescence (OIS), through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21^CIP1 and p16^INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α). In addition, hypoxia also decreased marks of H-Ras^V12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21^CIP1 but not p16^INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21^CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.     

Variable Myopathic Presentation in a Single Family with Novel Skeletal RYR1 Mutation

We describe an autosomal recessive heterogeneous congenital myopathy in a large consanguineous family. The disease is characterized by variable severity, progressive course in 3 of 4 patients, myopathic face without ophthalmoplegia and proximal muscle weakness. Absence of cores was noted in all patients. Genome wide linkage analysis revealed a single locus on chromosome 19q13 with Zmax = 3.86 at θ = 0.0 and homozygosity of the polymorphic markers at this locus in patients. Direct sequencing of the main candidate gene within the candidate region, RYR1, was performed. A novel homozygous A to G nucleotide substitution (p.Y3016C) within exon 60 of the RYR1 gene was found in patients. ARMS PCR was used to screen for the mutation in all available family members and in an additional 150 healthy individuals. This procedure confirmed sequence analysis and did not reveal the A to G mutation (p.Y3016C) in 300 chromosomes from healthy individuals. Functional analysis on EBV immortalized cell lines showed no effect of the mutation on RyR1 pharmacological activation or the content of intracellular Ca²⁺ stores. Western blot analysis demonstrated a significant reduction of the RyR1 protein in the patient’s muscle concomitant with a reduction of the DHPRα1.1 protein. This novel mutation resulting in RyR1 protein decrease causes heterogeneous clinical presentation, including slow progression course and absence of centrally localized cores on muscle biopsy. We suggest that RYR1 related myopathy should be considered in a wide variety of clinical and pathological presentation in childhood myopathies.     

The Influence of Electric Field and Confinement on Cell Motility

The ability of cells to sense and respond to endogenous electric fields is important in processes such as wound healing, development, and nerve regeneration. In cell culture, many epithelial and endothelial cell types respond to an electric field of magnitude similar to endogenous electric fields by moving preferentially either parallel or antiparallel to the field vector, a process known as galvanotaxis. Here we report on the influence of dc electric field and confinement on the motility of fibroblast cells using a chip-based platform. From analysis of cell paths we show that the influence of electric field on motility is much more complex than simply imposing a directional bias towards the cathode or anode. The cell velocity, directedness, as well as the parallel and perpendicular components of the segments along the cell path are dependent on the magnitude of the electric field. Forces in the directions perpendicular and parallel to the electric field are in competition with one another in a voltage-dependent manner, which ultimately govern the trajectories of the cells in the presence of an electric field. To further investigate the effects of cell reorientation in the presence of a field, cells are confined within microchannels to physically prohibit the alignment seen in 2D environment. Interestingly, we found that confinement results in an increase in cell velocity both in the absence and presence of an electric field compared to migration in 2D.     

Comparative morphology of porpoise (Cetacea: Phocoenidae) pterygoid sinuses: Phylogenetic and functional implications

High‐resolution X‐ray computed tomographic scans were used to examine pterygoid sinus morphology within extant porpoise species and one delphinid (Tursiops truncatus), in order to consider: 1) intraspecific and interspecific variation among the studied species; 2) the most parsimonious sequence of character acquisition; and 3) the potential functional roles of the preorbital lobes of the sinuses in sound reflection. Scans revealed that the pterygoid/palatine regions are mediolaterally broader in the earliest diverging phocoenid (Neophocaena phocaenoides) and Tursiops truncatus than the dorsoventrally elongated sinuses observed in other species. Rostrocaudal lengths of the sphenoidal regions of the sinuses in all individuals studied are proportionally similar, indicating conservatism in this region across species. The neonate Phocoena phocoena has shorter preorbital lobes than adults, but they are still proportionally longer than Neophocaena phocaenoides and Phocoena spinipinnis. The preorbital lobes broaden mediolaterally to varying degrees across species; in particular, Phocoenoides dalli has the largest dorsal and lateral expansion of this region. Assuming the highest pulse frequency produced by porpoises is 150 kHz, all regions of the preorbital lobes are thick enough to reflect the wavelengths produced. In addition, the neonate preorbital lobes are not as elongated as they are in adults, and the dorsal third of this region may not reflect sound to the same extent. This study reinforces the importance of using nondestructive methods to quantify variation in endocranial anatomy and the value of CT data for recovering phylogenetically useful information, as well as functional roles sinuses play in concert with the soft tissue head anatomy for biosonar. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Disassembly of Escherichia coli RecA E38K/��C17 Nucleoprotein Filaments Is Required to Complete DNA Strand Exchange

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/ΔC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/ΔC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/ΔC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.