Haplotyping cryptic Adélie penguin taxa using low-cost,high-resolution melt curves

Distinguishing morphologically cryptic taxa, by definition, requires genetic data such as DNA sequences. However, DNA sequences may not be obtained easily for taxa from remote sites. Here we provide the details of a high-resolution melt-curve-based method using taxon-specific primers that can distinguish two taxa of Adélie penguins, and that will be usable in Antarctica when combined with some of the newly developed field-deployable thermal cyclers. We suggest that the wider adoption of field-deployable polymerase-chain-reaction-based techniques will enable faster assignation of haplotype to individuals in situ, and so allow the targeting of observations and sample collection to specimens relevant to the research question. Targeting individuals will also reduce the need to repeatedly handle animals and reduce the time and travel required to complete field work.     

A 2.6 A structure of a serpin polymer and implications for conformational disease.

The function of the serpins as proteinase inhibitors depends on their ability to insert the cleaved reactive centre loop as the fourth strand in the main A beta-sheet of the molecule upon proteolytic attack at the reactive centre, P1-P1'. This mechanism is vulnerable to mutations which result in inappropriate intra- or intermolecular loop insertion in the absence of cleavage. Intermolecular loop insertion is known as serpin polymerisation and results in a variety of diseases, most notably liver cirrhosis resulting from mutations of the prototypical serpin alpha1-antitrypsin. We present here the 2.6 A structure of a polymer of alpha1-antitrypsin cleaved six residues N-terminal to the reactive centre, P7-P6 (Phe352-Leu353). After self insertion of P14 to P7, intermolecular linkage is affected by insertion of the P6-P3 residues of one molecule into the partially occupied beta-sheet A of another. This results in an infinite, linear polymer which propagates in the crystal along a 2-fold screw axis. These findings provide a framework for understanding the uncleaved alpha1-antitrypsin polymer and fibrillar and amyloid deposition of proteins seen in other conformational diseases, with the ordered array of polymers in the crystal resulting from slow accretion of the cleaved serpin over the period of a year.     

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.     

The Epidemiology of Helicobacter pylori and Public Health Implications

This article presents a review of the literature on the epidemiology and public health implications of Helicobacter pylori infection published from April 2008 through to March 2009. The authors used MeSH terms "Helicobacter infections epidemiology,""Helicobacter infections prevention and control" to search multiple databases (PubMed, Embase, Cochrane, Cochrane Library, EBMR, BIOSIS), and independently searched PubMed using the term "Helicobacter" with "Epidemiology,""Transmission,""Prevalence" or "Environment." Articles without topical relevance were excluded. Two additional papers known to the authors were added. The identified literature is summarized by subtopic: reviews; prevalence; incidence; transmission; risk factors; and public health policy.     

Implementation of an insecticide-treated net subsidy scheme under a public-private partnership for malaria control in Tanzania – challenges in implementation

Background

In the past decade there has been increasing visibility of malaria control efforts at the national and international levels. The factors that have enhanced this scenario are the availability of proven interventions such as artemisinin-based combination therapy, the wide scale use of insecticide-treated nets (ITNs) and a renewed emphasis in indoor residual house-spraying. Concurrently, there has been a window of opportunity of financial commitments from organizations such as the Global Fund for HIV/AIDS, Tuberculosis and Malaria (GFATM), the President's Malaria Initiative and the World Bank Booster programme.

Methods

The case study uses the health policy analysis framework to analyse the implementation of a public-private partnership approach embarked upon by the government of Tanzania in malaria control – 'The Tanzania National Voucher Scheme'- and in this synthesis, emphasis is on the challenges faced by the scheme during the pre-implementation (2001 – 2004) and implementation phases (2004 – 2005). Qualitative research tools used include: document review, interview with key informants, stakeholder's analysis, force-field analysis, time line of events, policy characteristic analysis and focus group discussions. The study is also complemented by a cross-sectional survey, which was conducted at the Rufiji Health Demographic Surveillance Site, where a cohort of women of child-bearing age were followed up regarding access and use of ITNs.

Results

The major challenges observed include: the re-introduction of taxes on mosquito nets and related products, procurement and tendering procedures in the implementation of the GFATM, and organizational arrangements and free delivery of mosquito nets through a Presidential initiative.

Conclusion

The lessons gleaned from this synthesis include: (a) the consistency of the stakeholders with a common vision, was an important strength in overcoming obstacles, (b) senior politicians often steered the policy agenda when the policy in question was a 'crisis event', the stakes and the visibility were high, (c) national stakeholders in policy making have an advantage in strengthening alliances with international organizations, where the latter can become extremely influential in solving bottlenecks as the need arises, and (d) conflict can be turned into an opportunity, for example the Presidential initiative has inadvertently provided Tanzania with important lessons in the organization of 'catch-up' campaigns.     

New insight into serpin polymerization and aggregation

We recently solved the crystallographic structure of a dimeric form of the serpin antithrombin which has fundamentally changed the way we think about serpin polymerization. Like for other diseases that have protein deposition as a hallmark, the serpinopathies are associated with discrete inter-protomer linkage followed by subsequent association into larger fibrils and aggregates. Polymerization of the serpins is an off-pathway event that occurs during folding in the endoplasmic reticulum. Our structure reveals the nature of the polymerogenic folding intermediate, the reason that the inter-protomer linkage is hyperstable, and suggests a mechanism of lateral association of polymers into soluble fibrils and insoluble aggregates. While the basis of cellular toxicity is still unclear, novel therapeutic approaches targeting the folding intermediate or the lateral association event are now conceivable.Key words: aggregation, conformation, folding, polymerization, serpin, structure, toxicityThe serpins are serine protease inhibitors that utilize a unique and well characterized β-sheet expansion event as a necessary part of their mechanism.^1–3 This conformational/topological change from a five-stranded (N in Fig. 1) to a six-stranded β-sheet A (L in Fig. 1) results in a doubling of the protein''s stability, and is driven by a free-energy term of around −32 kcal/mol.⁴ Thus, the native form of serpins is metastable in defiance of the Anfinsen principle,⁵ requiring a folding pathway that kinetically traps the five-stranded state. This unusual protein folding requirement allows for an off-pathway event known as polymerization, where one protomer completes the A sheet of another.⁶ For secreted serpins, this occurs in the endoplasmic reticulum (ER) where polymers are seen to accumulate as insoluble proteinaceous inclusions. Polymerization has been described for several serpins and is always associated with a loss of functional levels due to accumulation within the cell, and occasionally it is associated with the death of the secretory cells through a poorly understood mechanism.^7–9 The best examples of the latter are provided by the Z variant of α₁-antitrypsin (α₁AT) leading to liver disease and the Syracuse variant of neuroserpin that causes early onset dementia.Open in a separate windowFigure 1Serpin folding and polymerization. The pathway of serpin folding proceeds from the unfolded state (U) to the native state (N) via a stable intermediate (M*). The native conformation is the only active state, and is composed of a five-stranded A sheet (red) and a 20 residue reactive centre loop (RCL, yellow). Serpin inhibitory function requires the native conformation to be a kinetically trapped metastable state. Completion of sheet A by incorporation of the RCL as strand 4, to form the latent (L) state, results in the doubling of the serpin''s thermodynamic stability (the six strands are labelled on L). Folding and unfolding of native serpins is known to proceed via a stable intermediate denoted M*, which also corresponds to the polymerogenic form.^24–26 The key feature of the M* state is that strand 5 is not yet incorporated into sheet A, and can thus insert in an intermolecular fashion to form off-pathway polymers (P, each protomer of the pentamer is in a different colour). The polymers have complete A sheets and are thus hyperstable. As a consequence of polymerization, the linker region (cyan), containing helix I, remains unfolded. We hypothesize that the hydrophobic linker (indicated by the oval) is responsible for the lateral association of polymers into insoluble aggregates.We recently solved a crystal structure of a self-terminating (closed) serpin dimer that revealed a large domain swap including the fourth and fifth strands of β-sheet A.¹⁰ We then modelled an open polymerization competent polymer based on the structure (P in Fig. 1) that explained their facile propagation, the hyperstability and flexibility of the inter-protomer linkage, and also suggested a structure for the polymerogenic folding intermediate (M* in Fig. 1). We proposed that the final step in folding to the native state is the insertion of strand 5 into β-sheet A and the association of the coiled linker domain to the ‘bottom’ of the molecule. This event would leave the fourth strand (the reactive centre loop) accessible to serve as bait for proteolytic attack, necessary for the functioning of the serpin mechanism. While many details are yet to be confirmed, the position of certain polymerogenic mutations on and underlying strand 5A supports the proposal.¹⁰One unexpected implication of our model is the requisite unfolding and exposure of helix I and the following coiled region in linear serpin polymers. Exposure of this ‘linker region’ was verified in linear polymers of serpins antithrombin and α₁AT through limited proteolysis and fluorescence studies, and explains the observation that polymers are hydrophobic and exhibit an increased propensity towards aggregation. Unglycosylated serpins typically aggregate when polymerized in vitro (with heat or low concentrations of chaotrophes), even at vanishingly low concentrations, whereas high concentrations are required to observe aggregation of glycosylated serpin polymers. We hypothesized that aggregation/precipitation occurs via lateral association of linear polymers, either through specific β-strand linkage or non-specific hydrophobic interactions involving the linker region. Sequence analysis of helix I suggest that it is a ‘frustrated’ β-strand¹¹ for several serpins including α₁AT, supporting the idea that aggregates of serpin polymers form through an extended β-sheet mechanism akin to other ‘conformational diseases.’¹²While there are clear parallels between the ‘serpinopathies’ and conformational diseases such as Alzheimer, Huntington and the prion encephalopathies (e.g., ordered intermolecular linkage, β-sheet expansion, cell death, dementia, accumulation of insoluble aggregates, domain-swapping),^12,13 the detailed molecular mechanism revealed by our crystal structure is unique to the serpins. Domain swaps in other proteins are generally characterized by normal activity and stability, and may not play a role in the secondary association event that leads to the toxic species.^14,15 For serpins the domain swap leads to hyperstability and the exposure of hydrophobic regions not seen in the monomeric state. Another key difference is the manner of cellular toxicity and the nature of the toxic species. It is becoming clear that for Alzheimer, Huntington and other conformational diseases the toxic fragments are likely to be the soluble (proto)-fibrils, not the insoluble aggregates (inclusions).^16,17 Serpin polymerization generally leads to disease through loss of secretion of the active species, and only in two special cases is it through gain-of-function cellular toxicity, and although the toxic mechanisms are incompletely resolved, they appear to involve the insoluble aggregates.The most common cause of cirrhosis among children is the homozygous Z mutation in α₁AT.¹⁸ Antitrypsin is expressed at high levels by hepatocytes (1.3–3.5 g/l in blood plasma) and its expression can increase in response to infection and other stimuli.¹⁹ However, only about one-third of the homozygous carriers ever manifest liver disease¹⁸ and it never occurs in carriers of a single Z allele, indicating that hepatocytes are generally well equipped to deal with the mutant protein. Soluble Z α₁AT in the ER binds to chaperones and is subsequently targeted to the ERAD pathway for clearance by the proteosome, and insoluble polymers and aggregates are thought to activate autophagy for degradation in the lysosomes.^20,21 In such a model, accumulation of polymers and cellular toxicity only occur when the proteosomal and autophagic pathways have been saturated by the high level of expression of mutant α₁AT in the liver.²¹ In contrast, neuroserpin is expressed at low levels in neurons and mutation leads to polymerization and dementia in an autosomal dominant fashion.^22,23 However, cell death and disease are still associated with accumulation of inclusion bodies within the ER, and the toxic mechanisms are likely to be similar.⁹In summary, we have elucidated a novel mechanism of serpin polymerization that involves a hyperstable domain swap of a folding intermediate. Formation of linear polymers exposes hydrophobic regions that mediate lateral polymer association and eventually leads to intra ER accretion and cellular toxicity. Our proposal suggests new avenues for the rational design of compounds to combat the diseases caused by serpin polymerization, either through targeting the folding intermediate or the lateral association of soluble polymers.     

Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted approximately 20 A, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.