VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes

Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I-transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I-transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I-transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a "winner-takes-all" mechanism of allelic exclusion

Antigenic variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression control.

African trypanosomes are lethal human and animal parasites that use antigenic variation for evasion of host adaptive immunity. To facilitate antigenic variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic variation requires transcription of a single VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organization all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context

Insights into antitrypanosomal drug mode-of-action from cytology-based profiling

Chemotherapy continues to have a major impact on reducing the burden of disease caused by trypanosomatids. Unfortunately though, the mode-of-action (MoA) of antitrypanosomal drugs typically remains unclear or only partially characterised. This is the case for four of five current drugs used to treat Human African Trypanosomiasis (HAT); eflornithine is a specific inhibitor of ornithine decarboxylase. Here, we used a panel of T. brucei cellular assays to probe the MoA of the current HAT drugs. The assays included DNA-staining followed by microscopy and quantitative image analysis, or flow cytometry; terminal dUTP nick end labelling to monitor mitochondrial (kinetoplast) DNA replication; antibody-based detection of sites of nuclear DNA damage; and fluorescent dye-staining of mitochondria or lysosomes. We found that melarsoprol inhibited mitosis; nifurtimox reduced mitochondrial protein abundance; pentamidine triggered progressive loss of kinetoplast DNA and disruption of mitochondrial membrane potential; and suramin inhibited cytokinesis. Thus, current antitrypanosomal drugs perturb distinct and specific cellular compartments, structures or cell cycle phases. Further exploiting the findings, we show that putative mitogen-activated protein-kinases contribute to the melarsoprol-induced mitotic defect, reminiscent of the mitotic arrest associated signalling cascade triggered by arsenicals in mammalian cells, used to treat leukaemia. Thus, cytology-based profiling can rapidly yield novel insight into antitrypanosomal drug MoA

Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite <i>Trypanosoma brucei</i>

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by emergent resistance and toxicity. Our understanding of drug mode-of-action and potential routes to resistance is also limited. Forward genetic approaches have revolutionised our understanding of drug mode-of-action in the related kinetoplastid parasite, Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNAi library for knockdowns that render cells resistant to the current anti-leishmanial drugs, sodium stibogluconate (antimonial), paromomycin, miltefosine and amphotericin-B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource

Role of Preoperative Nerve Conduction Studies for Penetrating Hand Injuries Involving the Median Palmar Cutaneous Nerve

Penetrating lacerations to the hand are a common cause of nerve injury and can lead to debilitating pain and numbness in the distribution of the nerve affected. Owing to an overlap in the cutaneous innervation from different sensory nerves, clinically identifying the injured nerve can be difficult. We present a novel case of isolated injury to the palmar cutaneous nerve from a penetrating knife injury which was detected using \u27comparison waveform\u27 nerve conduction studies. Using this technique, we can isolate injuries to the palmar cutaneous branch of the median nerve (PCBmdn) from the median nerve, dorsal radial sensory nerve, and lateral antebrachial cutaneous nerve. In addition, sensory nerve testing identified conduction block as the mechanism of injury, which resolved after surgery at 8 weeks postoperatively. Preoperative nerve conduction study can discern the level of nerve injury to PCBmdn only, thus eliminating the need for median and radial nerve exploration at the forearm, unnecessary incisions, pain, and scarring. The objective of this case report is to illustrate the value of preoperative comparison waveform nerve conduction study, particularly the PCBmdn, in patients presenting with neurologic deficits who have sustained penetrating lacerations to the hand

Health Diplomacy the Adaptation of Global Health Interventions to Local Needs in sub-Saharan Africa and Thailand: Evaluating Findings from Project Accept (HPTN 043).

Study-based global health interventions, especially those that are conducted on an international or multi-site basis, frequently require site-specific adaptations in order to (1) respond to socio-cultural differences in risk determinants, (2) to make interventions more relevant to target population needs, and (3) in recognition of 'global health diplomacy' issues. We report on the adaptations development, approval and implementation process from the Project Accept voluntary counseling and testing, community mobilization and post-test support services intervention. We reviewed all relevant documentation collected during the study intervention period (e.g. monthly progress reports; bi-annual steering committee presentations) and conducted a series of semi-structured interviews with project directors and between 12 and 23 field staff at each study site in South Africa, Zimbabwe, Thailand and Tanzania during 2009. Respondents were asked to describe (1) the adaptations development and approval process and (2) the most successful site-specific adaptations from the perspective of facilitating intervention implementation. Across sites, proposed adaptations were identified by field staff and submitted to project directors for review on a formally planned basis. The cross-site intervention sub-committee then ensured fidelity to the study protocol before approval. Successfully-implemented adaptations included: intervention delivery adaptations (e.g. development of tailored counseling messages for immigrant labour groups in South Africa) political, environmental and infrastructural adaptations (e.g. use of local community centers as VCT venues in Zimbabwe); religious adaptations (e.g. dividing clients by gender in Muslim areas of Tanzania); economic adaptations (e.g. co-provision of income generating skills classes in Zimbabwe); epidemiological adaptations (e.g. provision of 'youth-friendly' services in South Africa, Zimbabwe and Tanzania), and social adaptations (e.g. modification of terminology to local dialects in Thailand: and adjustment of service delivery schedules to suit seasonal and daily work schedules across sites). Adaptation selection, development and approval during multi-site global health research studies should be a planned process that maintains fidelity to the study protocol. The successful implementation of appropriate site-specific adaptations may have important implications for intervention implementation, from both a service uptake and a global health diplomacy perspective

Global mean surface temperature and climate sensitivity of the early Eocene Climatic Optimum (EECO), Paleocene–Eocene Thermal Maximum (PETM), and latest Paleocene

Accurate estimates of past global mean surface temperature (GMST) help to contextualise future climate change and are required to estimate the sensitivity of the climate system to CO2 forcing through Earth's history. Previous GMST estimates for the latest Paleocene and early Eocene (∼57 to 48 million years ago) span a wide range (∼9 to 23 ∘C higher than pre-industrial) and prevent an accurate assessment of climate sensitivity during this extreme greenhouse climate interval. Using the most recent data compilations, we employ a multi-method experimental framework to calculate GMST during the three DeepMIP target intervals: (1) the latest Paleocene (∼57 Ma), (2) the Paleocene–Eocene Thermal Maximum (PETM; 56 Ma), and (3) the early Eocene Climatic Optimum (EECO; 53.3 to 49.1 Ma). Using six different methodologies, we find that the average GMST estimate (66 % confidence) during the latest Paleocene, PETM, and EECO was 26.3 ∘C (22.3 to 28.3 ∘C), 31.6 ∘C (27.2 to 34.5 ∘C), and 27.0 ∘C (23.2 to 29.7 ∘C), respectively. GMST estimates from the EECO are ∼10 to 16 ∘C warmer than pre-industrial, higher than the estimate given by the Intergovernmental Panel on Climate Change (IPCC) 5th Assessment Report (9 to 14 ∘C higher than pre-industrial). Leveraging the large “signal” associated with these extreme warm climates, we combine estimates of GMST and CO2 from the latest Paleocene, PETM, and EECO to calculate gross estimates of the average climate sensitivity between the early Paleogene and today. We demonstrate that “bulk” equilibrium climate sensitivity (ECS; 66 % confidence) during the latest Paleocene, PETM, and EECO is 4.5 ∘C (2.4 to 6.8 ∘C), 3.6 ∘C (2.3 to 4.7 ∘C), and 3.1 ∘C (1.8 to 4.4 ∘C) per doubling of CO2. These values are generally similar to those assessed by the IPCC (1.5 to 4.5 ∘C per doubling CO2) but appear incompatible with low ECS values (<1.5 per doubling CO2)

Role of G protein‐coupled receptor kinases (GRKs) in β2‐adrenoceptor‐mediated glucose uptake

Truncation of the C‐terminal tail of the β2‐AR, transfection of βARKct or over‐expression of a kinase‐dead GRK mutant reduces isoprenaline‐stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the β2‐AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO‐GLUT4myc cells expressing wild‐type and mutant β2‐ARs were generated and receptor affinity for [3H]‐CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by β2‐AR agonists, cAMP accumulation, GLUT4 translocation, [3H]‐2‐deoxyglucose uptake, and β2‐AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between β2‐AR and β‐arrestin2 or between β2‐AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to β2‐AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to β2‐AR agonists occurred in CHO‐GLUT4myc cells expressing β2‐ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild‐type β2‐AR. However, β2‐ARs lacking phosphorylation sites failed to recruit β‐arrestin2 and did not internalize. GRK2 knock‐down or GRK2 inhibitors decreased isoprenaline‐stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the β2‐AR is not associated with isoprenaline‐ or BRL37344‐stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock‐down or GRK2 inhibition reduces isoprenaline‐stimulated glucose uptake