Designed Azolopyridinium Salts Block Protective Antigen Pores In Vitro and Protect Cells from Anthrax Toxin

Background:Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric PA63 binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA63-channel in a dose dependent way.Methodology/Principal Findings:Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the PA63-channel in the μM range, when both, inhibitor and PA63 are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of PA63-channel function also efficiently block intoxication of the cells by the combination lethal factor and PA63 in the same concentration range as they block the channels in vitro.Conclusions/Significance:These results strongly argue in favor of a transport of lethal factor through the PA63-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax. © 2013 Beitzinger et al

Potential plasma markers of type 1 and type 2 leprosy reactions: a preliminary report

<p>Abstract</p> <p>Background</p> <p>The clinical management of leprosy Type 1 (T1R) and Type 2 (T2R) reactions pose challenges mainly because they can cause severe nerve injury and disability. No laboratory test or marker is available for the diagnosis or prognosis of leprosy reactions. This study simultaneously screened plasma factors to identify circulating biomarkers associated with leprosy T1R and T2R among patients recruited in Goiania, Central Brazil.</p> <p>Methods</p> <p>A nested case-control study evaluated T1R (n = 10) and TR2 (n = 10) compared to leprosy patients without reactions (n = 29), matched by sex and age-group (+/- 5 years) and histopathological classification. Multiplex bead based technique provided profiles of 27 plasma factors including 16 pro inflammatory cytokines: tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), interleukin (IL)- IL12p70, IL2, IL17, IL1 β, IL6, IL15, IL5, IL8, macrophage inflammatory protein (MIP)-1 alpha (MIP1α), 1 beta (MIP1β), regulated upon activation normal T-cell expressed and secreted (RANTES), monocyte chemoattractrant protein 1 (MCP1), CC-chemokine 11 (CCL11/Eotaxin), CXC-chemokine 10 (CXCL10/IP10); 4 anti inflammatory interleukins: IL4, IL10, IL13, IL1Rα and 7 growth factors: IL7, IL9, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor BB (PDGF BB), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF).</p> <p>Results</p> <p>Elevations of plasma CXCL10 (P = 0.004) and IL6 (p = 0.013) were observed in T1R patients compared to controls without reaction. IL6 (p = 0.05), IL7 (p = 0.039), and PDGF-BB (p = 0.041) were elevated in T2R. RANTES and GMCSF were excluded due to values above and below detection limit respectively in all samples.</p> <p>Conclusion</p> <p>Potential biomarkers of T1R identified were CXCL10 and IL6 whereas IL7, PDGF-BB and IL6, may be laboratory markers of TR2. Additional studies on these biomarkers may help understand the immunopathologic mechanisms of leprosy reactions and indicate their usefulness for the diagnosis and for the clinical management of these events.</p

Staff perspectives of barriers to women accessing birthing services in Nepal: A qualitative study

Background: Nepal has made significant progress with regard to reducing the maternal mortality ratio but a major challenge remains the under-utilisation of skilled birth attendants who are predominantly facility based. Studies have explored women's views of the barriers to facility birth; however the voices of staff who offer services have not been studied in detail. This research explores the views of staff as to the key reasons why pregnant women do not give birth in a maternity-care facility. Methods: This mixed methods study comprised qualitative interviews and non-participant observation. The study was conducted in two small non-governmental hospitals, one semi-rural and one urban, in Kathmandu Valley. Twenty interviews were conducted with health care providers and other staff in these hospitals. The interviews were undertaken with the aid of a Nepali translator, with some interviews being held in English. Twenty-five hours of non-participant observation was conducted in both maternity hospitals . Both observation and interview data were analysed thematically. Ethical approval was granted by the Nepal Research Health Council and Bournemouth University's Ethics Committee. Results: Key themes that emerged from the analysis reflected barriers that women experience in accessing services at different conceptual levels and resembled the three phases of delay model by Thaddeus and Maine. This framework is used to present the barriers. First Phase Delays are: 1) lack of awareness that the facility/services exist; 2) women being too busy to attend; 3) poor services; 4) embarrassment; and 5) financial issues. Themes for the second Phase of Delay are: 1) birthing on the way; and 2) by-passing the facility in favour of one further away. The final Phase involved: 1) absence of an enabling environment; and 2) disrespectful care. Conclusion: This study highlights a multitude of barriers, not all of the same importance or occuring at the same time in the pregnancy journey. It is clear that staff are aware of many of the barriers for women in reaching the facility to give birth, and these fit with previous literature of women's views. However, staff had limited insight into barriers occuring within the facility itself and were more likely to suggest that this was a problem for other institutions and not theirs

A Synthetic Uric Acid Analog Accelerates Cutaneous Wound Healing in Mice

Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice. Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions

Associations of familial risk factors with social fears and social phobia: evidence for the continuum hypothesis in social anxiety disorder?

We examined parental psychopathology and family environment in subthreshold and DSM-IV threshold conditions of social anxiety disorder (SAD) in a representative cohort sample of 1,395 adolescents. Offspring and parental psychopathology was assessed using the DIA-X/M-CIDI; recalled parental rearing and family functioning via questionnaire. Diagnostic interviews in parents were supplemented by family history reports from offspring. The cumulative lifetime incidence was 23.07% for symptomatic SAD, and 18.38 and 7.41% for subthreshold and threshold SAD, respectively. The specific parent-to-offspring association for SAD occurred for threshold SAD only. For subthreshold and threshold SAD similar associations were found with other parental anxiety disorders, depression and substance use disorders. Parental rearing behaviour, but not family functioning, was associated with offspring threshold SAD, and although less strong and less consistent, also with subthreshold SAD. Results suggest a continued graded relationship between familial risk factors and offspring SAD. Parental psychopathology and negative parental styles may be used defining high-risk groups to assign individuals with already subthreshold conditions of SAD to early intervention programs

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

Insulin Degrading Enzyme Induces a Conformational Change in Varicella-Zoster Virus gE, and Enhances Virus Infectivity and Stability

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines

Alternative splicing of barley clock genes in response to low temperature:evidence for alternative splicing conservation

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley clock genes to assess whether temperature-dependent AS responses also occur in a monocotyledonous crop species. We identify changes in AS of various barley core clock genes including the barley orthologues of Arabidopsis AtLHY and AtPRR7 which showed the most pronounced AS changes in response to low temperature. The AS events modulate the levels of functional and translatable mRNAs, and potentially protein levels, upon transition to cold. There is some conservation of AS events and/or splicing behaviour of clock genes between Arabidopsis and barley. In addition, novel temperature-dependent AS of the core clock gene HvPPD-H1 (a major determinant of photoperiod response and AtPRR7 orthologue) is conserved in monocots. HvPPD-H1 showed a rapid, temperature-sensitive isoform switch which resulted in changes in abundance of AS variants encoding different protein isoforms. This novel layer of low temperature control of clock gene expression, observed in two very different species, will help our understanding of plant adaptation to different environments and ultimately offer a new range of targets for plant improvement

Indigenous biosecurity: Māori responses to kauri dieback and myrtle rust in Aotearoa New Zealand

It is widely acknowledged that Indigenous peoples have traditional knowledge relevant to modern environmental management. By asserting roles within associated science and policy networks, such Indigenous Knowledge (IK) can be seen as part of the resistance to colonisation that includes protest, treaty making, political and economic empowerment, legislation, cultural renaissance and regulatory influence. In New Zealand, these achievements inform attempts by Māori (the Indigenous people of New Zealand) to manage forest ecosystems and cultural keystone species. This chapter presents two case studies of how indigenous participation in modern biosecurity through the example of Māori asserting and contributing to forest management. While progress is often frustratingly slow for indigenous participants, significant gains in acceptance of Māori cultural frameworks have been achieved