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1.  An update to the HIV-TRePS system: the development of new computational models that do not require a genotype to predict HIV treatment outcomes 
Objectives
The optimal individualized selection of antiretroviral drugs in resource-limited settings is challenging because of the limited availability of drugs and genotyping. Here we describe the development of the latest computational models to predict the response to combination antiretroviral therapy without a genotype, for potential use in such settings.
Methods
Random forest models were trained to predict the probability of a virological response to therapy (<50 copies HIV RNA/mL) following virological failure using the following data from 22 567 treatment-change episodes including 1090 from southern Africa: baseline viral load and CD4 cell count, treatment history, drugs in the new regimen, time to follow-up and follow-up viral load. The models were assessed during cross-validation and with an independent global test set of 1000 cases including 100 from southern Africa. The models' accuracy [area under the receiver-operating characteristic curve (AUC)] was evaluated and compared with genotyping using rules-based interpretation systems for those cases with genotypes available.
Results
The models achieved AUCs of 0.79–0.84 (mean 0.82) during cross-validation, 0.80 with the global test set and 0.78 with the southern African subset. The AUCs were significantly lower (0.56–0.57) for genotyping.
Conclusions
The models predicted virological response to HIV therapy without a genotype as accurately as previous models that included a genotype. They were accurate for cases from southern Africa and significantly more accurate than genotyping. These models will be accessible via the online treatment support tool HIV-TRePS and have the potential to help optimize antiretroviral therapy in resource-limited settings where genotyping is not generally available.
doi:10.1093/jac/dkt447
PMCID: PMC3956369  PMID: 24275116
antiretroviral therapy; resource-limited settings; genotyping
2.  Incomplete Reversibility of Estimated Glomerular Filtration Rate Decline Following Tenofovir Disoproxil Fumarate Exposure 
Jose, Sophie | Hamzah, Lisa | Campbell, Lucy J. | Hill, Teresa | Fisher, Martin | Leen, Clifford | Gilson, Richard | Walsh, John | Nelson, Mark | Hay, Phillip | Johnson, Margaret | Chadwick, David | Nitsch, Dorothea | Jones, Rachael | Sabin, Caroline A. | Post, Frank A. | Ainsworth, Jonathan | Anderson, Jane | Babiker, Abdel | Chadwick, David | Delpech, Valerie | Dunn, David | Fisher, Martin | Gazzard, Brian | Gilson, Richard | Gompels, Mark | Hay, Phillip | Hill, Teresa | Johnson, Margaret | Kegg, Stephen | Leen, Clifford | Nelson, Mark | Orkin, Chloe | Palfreeman, Adrian | Phillips, Andrew | Pillay, Deenan | Post, Frank | Sabin, Caroline | Sachikonye, Memory | Schwenk, Achim | Walsh, John | Hill, Teresa | Huntington, Susie | Josie, Sophie | Phillips, Andrew | Sabin, Caroline | Thornton, Alicia | Dunn, David | Glabay, Adam | Orkin, C. | Garrett, N. | Lynch, J. | Hand, J. | de Souza, C. | Fisher, M. | Perry, N. | Tilbury, S. | Churchill, D. | Gazzard, B. | Nelson, M. | Waxman, M. | Asboe, D. | Mandalia, S. | Delpech, V. | Anderson, J. | Munshi, S. | Korat, H. | Poulton, M. | Taylor, C. | Gleisner, Z. | Campbell, L. | Babiker, Abdel | Dunn, David | Glabay, Adam | Gilson, R. | Brima, N. | Williams, I. | Schwenk, A. | Ainsworth, J. | Wood, C. | Miller, S. | Johnson, M. | Youle, M. | Lampe, F. | Smith, C. | Grabowska, H. | Chaloner, C. | Puradiredja, D. | Walsh, J. | Weber, J. | Ramzan, F. | Mackie, N. | Winston, A. | Leen, C. | Wilson, A. | Gompels, M. | Allan, S. | Palfreeman, A. | Moore, A. | Chadwick, D. | Wakeman, K. | Kegg, Stephen | Main, Paul | Mitchell,  | Hunter,  | Sachikonye, Memory | Hay, Phillip | Dhillon, Mandip
The Journal of Infectious Diseases  2014;210(3):363-373.
Background. Tenofovir disoproxil fumarate (TDF) has been linked to renal impairment, but the extent to which this impairment is reversible is unclear. We aimed to investigate the reversibility of renal decline during TDF therapy.
Methods. Cox proportional hazards models assessed factors associated with discontinuing TDF in those with an exposure duration of >6 months. In those who discontinued TDF therapy, linear piecewise regression models estimated glomerular filtration rate (eGFR) slopes before initiation of, during, and after discontinuation of TDF therapy. Factors associated with not achieving eGFR recovery 6 months after discontinuing TDF were assessed using multivariable logistic regression.
Results. We observed declines in the eGFR during TDF exposure (mean slopes, −15.7 mL/minute/1.73 m2/year [95% confidence interval {CI}, −20.5 to −10.9] during the first 3 months and −3.1 mL/minute/1.73 m2/year [95% CI, −4.6 to −1.7] thereafter) and evidence of eGFR increases following discontinuation of TDF therapy (mean slopes, 12.5 mL/minute/1.73 m2/year [95% CI, 8.9–16.1] during the first 3 months and 0.8 mL/minute/1.73 m2/year [95% CI, .1–1.5] thereafter). Following TDF discontinuation, 38.6% of patients with a decline in the eGFR did not experience recovery. A higher eGFR at baseline, a lower eGFR after discontinuation of TDF therapy, and more-prolonged exposure to TDF were associated with an increased risk of incomplete recovery 6 months after discontinuation of TDF therapy.
Conclusions. This study shows that a decline in the eGFR during TDF therapy was not fully reversible in one third of patients and suggests that prolonged TDF exposure at a low eGFR should be avoided.
doi:10.1093/infdis/jiu107
PMCID: PMC4091582  PMID: 24585896
tenofovir; highly active antiretroviral therapy; eGFR; eGFR slopes; renal function; kidney
3.  Nurse-Led Intervention to Improve Knowledge of Medications in Survivors of Stroke or Transient Ischemic Attack: A Cluster Randomized Controlled Trial 
Introduction
Limited evidence exists on effective interventions to improve knowledge of preventive medications in patients with chronic diseases, such as stroke. We investigated the effectiveness of a nurse-led intervention, where a component was to improve knowledge of prevention medications, in patients with stroke or transient ischemic attack (TIA).
Methods
Prospective sub-study of the Shared Team Approach between Nurses and Doctors for Improved Risk Factor Management, a randomized controlled trial of risk factor management. We recruited patients aged ≥18 years and hospitalized for stroke/TIA. The intervention comprised an individualized management program, involving nurse-led education, and management plan with medical specialist oversight. The outcome, participants’ knowledge of secondary prevention medications at 12 months, was assessed using questionnaires. A score of ≥5 was considered as good knowledge. Effectiveness of the intervention on knowledge of medications was determined using logistic regression.
Results
Between May 2014 and January 2015, 142 consecutive participants from the main trial were included in this sub-study, 64 to usual care and 78 to the intervention (median age 68.9 years, 68% males, and 79% ischemic stroke). In multivariable analyses, we found no significant difference between intervention groups in knowledge of medications. Factors independently associated with good knowledge (score ≥5) at 12 months included higher socioeconomic position (OR 4.79, 95% CI 1.76, 13.07), greater functional ability (OR 1.69, 95% CI 1.17, 2.45), being married/living with a partner (OR 3.12, 95% CI 1.10, 8.87), and using instructions on pill bottle/package as an administration aid (OR 4.82, 95% CI 1.76, 13.22). Being aged ≥65 years was associated with poorer knowledge of medications (OR 0.24, 95% CI 0.08, 0.71), while knowledge was worse among those taking three medications (OR 0.15, 95% CI 0.03, 0.66) or ≥4 medications (OR 0.09, 95% CI 0.02, 0.44), when compared to participants taking fewer (≤2) prevention medications.
Conclusion
There was no evidence that the nurse-led intervention was effective for improving knowledge of secondary prevention medications in patients with stroke/TIA at 12 months. However, older patients and those taking more medications should be particularly targeted for more intensive education.
Trial registration
Australian New Zealand Clinical Trials Registry (ACTRN12688000166370).
doi:10.3389/fneur.2016.00205
PMCID: PMC5114293  PMID: 27917150
randomized controlled trial; stroke; nursing intervention; patient medication knowledge; secondary prevention
4.  Gain-of-function mutation in TRPV4 identified in patients with osteonecrosis of the femoral head 
Journal of Medical Genetics  2016;53(10):705-709.
Background
Osteonecrosis of the femoral head is a debilitating disease that involves impaired blood supply to the femoral head and leads to femoral head collapse.
Methods
We use whole-exome sequencing and Sanger sequencing to analyse a family with inherited osteonecrosis of the femoral head and fluorescent Ca2+ imaging to functionally characterise the variant protein.
Results
We report a family with four siblings affected with inherited osteonecrosis of the femoral head and the identification of a c.2480_2483delCCCG frameshift deletion followed by a c.2486T>A substitution in one allele of the transient receptor potential vanilloid 4 (TRPV4) gene. TRPV4 encodes a Ca2+-permeable cation channel known to play a role in vasoregulation and osteoclast differentiation. While pathogenic TRPV4 mutations affect the skeletal or nervous systems, association with osteonecrosis of the femoral head is novel. Functional measurements of Ca2+ influx through mutant TRPV4 channels in HEK293 cells and patient-derived dermal fibroblasts identified a TRPV4 gain of function. Analysis of channel open times, determined indirectly from measurement of TRPV4 activity within a cluster of TRPV4 channels, revealed that the TRPV4 gain of function was caused by longer channel openings.
Conclusions
These findings identify a novel TRPV4 mutation implicating TRPV4 and altered calcium homeostasis in the pathogenesis of osteonecrosis while reinforcing the importance of TRPV4 in bone diseases and vascular endothelium.
doi:10.1136/jmedgenet-2016-103829
PMCID: PMC5035228  PMID: 27330106
TRPV4; osteonecrosis of the femoral head; novel mutation; calcium channel
5.  Transient contractions of urinary bladder smooth muscle are drivers of afferent nerve activity during filling 
The Journal of General Physiology  2016;147(4):323-335.
As the urinary bladder fills, the rate of rise (but not the frequency) of spontaneous contractions of smooth muscle in the bladder wall increases, thereby eliciting increased afferent nerve activity that signals the fullness of the bladder.
Activation of afferent nerves during urinary bladder (UB) filling conveys the sensation of UB fullness to the central nervous system (CNS). Although this sensory outflow is presumed to reflect graded increases in pressure associated with filling, UBs also exhibit nonvoiding, transient contractions (TCs) that cause small, rapid increases in intravesical pressure. Here, using an ex vivo mouse bladder preparation, we explored the relative contributions of filling pressure and TC-induced pressure transients to sensory nerve stimulation. Continuous UB filling caused an increase in afferent nerve activity composed of a graded increase in baseline activity and activity associated with increases in intravesical pressure produced by TCs. For each ∼4-mmHg pressure increase, filling pressure increased baseline afferent activity by ∼60 action potentials per second. In contrast, a similar pressure elevation induced by a TC evoked an ∼10-fold greater increase in afferent activity. Filling pressure did not affect TC frequency but did increase the TC rate of rise, reflecting a change in the length-tension relationship of detrusor smooth muscle. The frequency of afferent bursts depended on the TC rate of rise and peaked before maximum pressure. Inhibition of small- and large-conductance Ca2+-activated K+ (SK and BK) channels increased TC amplitude and afferent nerve activity. After inhibiting detrusor muscle contractility, simulating the waveform of a TC by gently compressing the bladder evoked similar increases in afferent activity. Notably, afferent activity elicited by simulated TCs was augmented by SK channel inhibition. Our results show that afferent nerve activity evoked by TCs represents the majority of afferent outflow conveyed to the CNS during UB filling and suggest that the maximum TC rate of rise corresponds to an optimal length-tension relationship for efficient UB contraction. Furthermore, our findings implicate SK channels in controlling the gain of sensory outflow independent of UB contractility.
doi:10.1085/jgp.201511550
PMCID: PMC4810069  PMID: 26976828
6.  Gain-of-function mutation in TRPV4 identified in patients with osteonecrosis of the femoral head 
Journal of medical genetics  2016;53(10):705-709.
Background
Osteonecrosis of the femoral head is a debilitating disease that involves impaired blood supply to the femoral head and leads to femoral head collapse.
Methods
We use whole-exome sequencing and Sanger sequencing to analyse a family with inherited osteonecrosis of the femoral head and fluorescent Ca2+ imaging to functionally characterise the variant protein.
Results
We report a family with four siblings affected with inherited osteonecrosis of the femoral head and the identification of a c.2480_2483delCCCG frameshift deletion followed by a c.2486T>A substitution in one allele of the transient receptor potential vanilloid 4 (TRPV4) gene. TRPV4 encodes a Ca2+-permeable cation channel known to play a role in vasoregulation and osteoclast differentiation. While pathogenic TRPV4 mutations affect the skeletal or nervous systems, association with osteonecrosis of the femoral head is novel. Functional measurements of Ca2+ influx through mutant TRPV4 channels in HEK293 cells and patient-derived dermal fibroblasts identified a TRPV4 gain of function. Analysis of channel open times, determined indirectly from measurement of TRPV4 activity within a cluster of TRPV4 channels, revealed that the TRPV4 gain of function was caused by longer channel openings.
Conclusions
These findings identify a novel TRPV4 mutation implicating TRPV4 and altered calcium homeostasis in the pathogenesis of osteonecrosis while reinforcing the importance of TRPV4 in bone diseases and vascular endothelium.
doi:10.1136/jmedgenet-2016-103829
PMCID: PMC5035228  PMID: 27330106
7.  Pharmacological inhibitors of TRPV4 channels reduce cytokine production, restore endothelial function and increase survival in septic mice 
Scientific Reports  2016;6:33841.
Sepsis is characterized by systemic inflammation, edema formation and hypo-perfusion leading to organ dysfunction and ultimately death. Activation of the transient receptor potential vanilloid type 4 (TRPV4) channel is associated with edema formation and circulatory collapse. Here, we show that TRPV4 channels are involved in the hyper-inflammatory response and mortality associated with sepsis. Pharmacological inhibition of TRPV4 channels in mice reduced mortality in lipopolysaccharide and cecal-ligation-and-puncture models of sepsis, but not in a tumor necrosis factor-α (TNFα)-induced sepsis model. These protective effects of TRPV4 channel inhibition were attributable to prevention of the sepsis-induced surge of a broad spectrum of pro-inflammatory cytokines, including TNFα, interleukin (IL)-1 and IL-6, and subsequent preservation of endothelial cell function, including Ca2+ signaling, integrity and endothelium-dependent vasodilation. These results suggest that TRPV4 antagonists may be of therapeutic utility in the management of sepsis.
doi:10.1038/srep33841
PMCID: PMC5031985  PMID: 27653046
8.  Dysfunction of mouse cerebral arteries during early aging 
Aging leads to a gradual decline in the fidelity of cerebral blood flow (CBF) responses to neuronal activation, resulting in an increased risk for stroke and dementia. However, it is currently unknown when age-related cerebrovascular dysfunction starts or which vascular components and functions are first affected. The aim of this study was to examine the function of microcirculation throughout aging in mice. Microcirculation was challenged by inhalation of 5% and 10% CO2 or by forepaw stimulation in 6-week, 8-month, and 12-month-old FVB/N mice. The resulting dilation of pial vessels and increase in CBF was measured by intravital fluorescence microscopy and laser Doppler fluxmetry, respectively. Neurovascular coupling and astrocytic endfoot Ca2+ were measured in acute brain slices from 18-month-old mice. We did not reveal any changes in CBF after CO2 reactivity up to an age of 12 months. However, direct visualization of pial vessels by in vivo microscopy showed a significant, age-dependent loss of CO2 reactivity starting at 8 months of age. At the same age neurovascular coupling was also significantly affected. These results suggest that aging does not affect cerebral vessel function simultaneously, but starts in pial microvessels months before global changes in CBF are detectable.
doi:10.1038/jcbfm.2015.107
PMCID: PMC4640303  PMID: 26058694
aging; cerebral blood flow; CO2 reactivity; in vivo microscopy; mice; neurovascular coupling
9.  Efficacy and Safety of Elvitegravir/Cobicistat/Emtricitabine/Tenofovir Disoproxil Fumarate in Asian Subjects with Human Immunodeficiency Virus 1 Infection: A Sub-Analysis of Phase 3 Clinical Trials 
Infection & Chemotherapy  2016;48(3):219-224.
The efficacy and safety of a single tablet regimen (STR) of elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (E/C/F/TDF) were analyzed in Phase 3 clinical trials in antiretroviral therapy (ART)-naïve and ART-experienced Asian subjects infected with human immunodeficiency virus (HIV)-1. Studies GS-US-236-102 and GS-US-236-103 were randomized, double-blind, placebo-controlled, 144-week studies conducted in ART-naïve subjects, comparing E/C/F/TDF versus efavirenz (EFV)/F/TDF or ritonavir-boosted atazanavir (ATV+RTV) plus emtricitabine/tenofovir DF (F/TDF), respectively. Studies GS-US-236-115 and GS-US-236-121 were randomized, open-label, 96-week long conducted in ART-experienced subjects, who switched to E/C/F/TDF from ritonavir-boosted protease inhibitors (PI+RTV)+F/TDF, or non-nucleoside reverse transcriptase inhibitors (NNRTI)+F/TDF regimens. The E/C/F/TDF appeared to have sustained efficacy and safety and was well tolerated in the small number of ART-naïve and ART-experienced Asian subjects.
doi:10.3947/ic.2016.48.3.219
PMCID: PMC5048004  PMID: 27704731
Human immunodeficiency virus; Antiretroviral therapy; Asian Elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate
10.  The S100P/RAGE signaling pathway regulates expression of microRNA-21 in colon cancer cells 
FEBS letters  2015;589(18):2388-2393.
S100P signaling through the receptor for advanced glycation end-products (RAGE) contributes to colon cancer invasion and metastasis, but the mechanistic features of this process are obscure. Here, we investigate whether activation of S100P/RAGE signaling regulates oncogenic microRNA-21 (miR-21). We show that exogenous S100P up-regulates miR-21 levels in human colon cancer cells, whereas knockdown of S100P results in a decrease of miR-21. Furthermore, blockage of RAGE with anti-RAGE antibody suppresses S100P induction of miR-21. In addition, we found that S100P induction of miR-21 expression involves ERK and is suppressed by the MEK inhibitor U0126. Also, S100P treatment stimulates the enrichment of c-Fos, and AP-1 family members, at the miR-21 gene promoter.
doi:10.1016/j.febslet.2015.07.010
PMCID: PMC4582666  PMID: 26193421
11.  Mechanistic insights into a TIMP3-sensitive pathway constitutively engaged in the regulation of cerebral hemodynamics 
eLife  null;5:e17536.
Cerebral small vessel disease (SVD) is a leading cause of stroke and dementia. CADASIL, an inherited SVD, alters cerebral artery function, compromising blood flow to the working brain. TIMP3 (tissue inhibitor of metalloproteinase 3) accumulation in the vascular extracellular matrix in CADASIL is a key contributor to cerebrovascular dysfunction. However, the linkage between elevated TIMP3 and compromised cerebral blood flow (CBF) remains unknown. Here, we show that TIMP3 acts through inhibition of the metalloprotease ADAM17 and HB-EGF to regulate cerebral arterial tone and blood flow responses. In a clinically relevant CADASIL mouse model, we show that exogenous ADAM17 or HB-EGF restores cerebral arterial tone and blood flow responses, and identify upregulated voltage-dependent potassium channel (KV) number in cerebral arterial myocytes as a heretofore-unrecognized downstream effector of TIMP3-induced deficits. These results support the concept that the balance of TIMP3 and ADAM17 activity modulates CBF through regulation of myocyte KV channel number.
DOI: http://dx.doi.org/10.7554/eLife.17536.001
eLife digest
There are currently no effective treatments or cures for small blood vessel diseases of the brain, which lead to strokes and subsequent decreases in mental abilities. Normally, smooth muscle cells that surround the vessels relax or contract to regulate blood flow and ensure the right amount of oxygen and nutrients reaches the different regions of the brain. In a syndrome called CADASIL, which is the most common form of inherited small vessel disease, a genetic mutation causes the smooth muscle cells to weaken over time.
The accumulation of several proteins – including one called TIMP3 – around the smooth muscle cells plays a key role in the smooth muscle cell weakening seen in CADASIL. Capone et al. have now studied mice that display the symptoms of CADASIL to investigate how TIMP3 decreases blood flow through blood vessels in the brain. This revealed that TIMP3 inactivates another protein called ADAM17. The latter protein is normally responsible for starting a signaling pathway that helps smooth muscle cells to regulate blood flow according to the needs of the brain cells. Artificially adding more ADAM17 to the brains of the CADASIL mice reduced their symptoms of small vessel disease.
Using smooth muscle cells freshly isolated from the brains of CADASIL mice, Capone et al. also demonstrated that abnormal TIMP3-ADAM17 signaling increases the number of voltage-dependent potassium channels in the membrane of the muscle cells. Having too many of these channels impairs the flow of blood through vessels in the brain.
Further experiments are needed to investigate whether correcting TIMP3-ADAM17 signaling could prevent strokes in people with inherited CADASIL. It also remains to be seen whether similar signaling mechanisms are at play in other small vessel diseases.
DOI: http://dx.doi.org/10.7554/eLife.17536.002
doi:10.7554/eLife.17536
PMCID: PMC4993587  PMID: 27476853
cerebral blood flow; myogenic tone; voltage-gated potassium channel; cerebral small vessel disease; CADASIL; ADAM17; Mouse
12.  Effects of changing sea ice on marine mammals and subsistence hunters in northern Alaska from traditional knowledge interviews 
Biology Letters  2016;12(8):20160198.
Marine mammals are important sources of food for indigenous residents of northern Alaska. Changing sea ice patterns affect the animals themselves as well as access to them by hunters. Documenting the traditional knowledge of Iñupiaq and Yupik hunters concerning marine mammals and sea ice makes accessible a wide range of information relevant to understanding the ecosystem to which humans belong. We interviewed hunters in 11 coastal villages from the northern Bering Sea to the Beaufort Sea. Hunters reported extensive changes in sea ice and weather that have affected the timing of marine mammal migrations, their distribution and behaviour and the efficacy of certain hunting methods. Amidst these changes, however, hunters cited offsetting technological benefits, such as more powerful and fuel-efficient outboard engines. Other concerns included potential impacts to subsistence hunting from industrial activity such as shipping and oil and gas development. While hunters have been able to adjust to some changes, continued environmental changes and increased disturbance from human activity may further challenge their ability to acquire food in the future. There are indications, however, that innovation and flexibility provide sources of resilience.
doi:10.1098/rsbl.2016.0198
PMCID: PMC5014017  PMID: 27555644
Arctic marine mammals; sea ice; Alaska; indigenous communities; traditional knowledge
13.  Direct-acting antivirals for acute hepatitis C in HIV-infected MSM 
AIDS (London, England)  2016;30(13):2137-2139.
doi:10.1097/QAD.0000000000001157
PMCID: PMC4966917  PMID: 27465281
14.  Efavirenz and Metabolites in Cerebrospinal Fluid: Relationship with CYP2B6 c.516G→T Genotype and Perturbed Blood-Brain Barrier Due to Tuberculous Meningitis 
Efavirenz (EFZ) has been associated with neuropsychiatric side effects. Recently, the 8-hydroxy-EFZ (8OH-EFZ) metabolite has been shown to be a potent neurotoxin in vitro, inducing neuronal damage at concentrations of 3.3 ng/ml. EFZ induced similar neuronal damage at concentrations of 31.6 ng/ml. We investigated the effect of genotype and blood-brain barrier integrity on EFZ metabolite concentrations in cerebrospinal fluid (CSF). We measured CSF drug concentrations in subjects from two separate study populations: 47 subjects with tuberculous meningitis (TBM) coinfection in Vietnam receiving 800 mg EFZ with standard antituberculous treatment and 25 subjects from the PARTITION study in the United Kingdom without central nervous system infection receiving 600 mg EFZ. EFZ and metabolite concentrations in CSF and plasma were measured and compared with estimates of effectiveness and neurotoxicity from available published in vitro and in vivo data. The effect of the CYP2B6 c.516G→T genotype (GG genotype, fast EFV metabolizer status; GT genotype, intermediate EFV metabolizer status; TT genotype, slow EFV metabolizer status) was examined. The mean CSF concentrations of EFZ and 8OH-EFZ in the TBM group were 60.3 and 39.3 ng/ml, respectively, and those in the no-TBM group were 15.0 and 5.9 ng/ml, respectively. Plasma EFZ and 8OH-EFZ concentrations were similar between the two groups. CSF EFZ concentrations were above the in vitro toxic concentration in 76% of samples (GG genotype, 61%; GT genotype, 90%; TT genotype, 100%) in the TBM group and 13% of samples (GG genotype, 0%; GT genotype, 18%; TT genotype, 50%) in the no-TBM group. CSF 8OH-EFZ concentrations were above the in vitro toxic concentration in 98% of the TBM group and 87% of the no-TBM group; levels were independent of genotype but correlated with the CSF/plasma albumin ratio. Potentially neurotoxic concentrations of 8OH-EFZ are frequently observed in CSF independently of the CYP2B6 genotype, particularly in those with impaired blood-brain barrier integrity.
doi:10.1128/AAC.00280-16
PMCID: PMC4958147  PMID: 27161633
15.  Virological failure and development of new resistance mutations according to CD4 count at combination antiretroviral therapy initiation 
Jose, S | Quinn, K | Dunn, D | Cox, A | Sabin, C | Fidler, S | Ainsworth, Jonathan | Allan, Sris | Anderson, Jane | Babiker, Abdel | Chadwick, David | Delpech, Valerie | Fisher, Martin | Gazzard, Brian | Gilson, Richard | Gompels, Mark | Hay, Phillip | Hill, Teresa | Johnson, Margaret | Kegg, Stephen | Leen, Clifford | Martin, Fabiola | Nelson, Mark | Orkin, Chloe | Palfreeman, Adrian | Phillips, Andrew | Pillay, Deenan | Post, Frank | Pritchard, Jillian | Sachikonye, Memory | Schwenk, Achim | Tariq, Anjum | Walsh, John | Thornton, Alicia | Glabay, Adam | Perry, N | Tilbury, S | Youssef, E | Churchill, D | Everett, R | Asboe, D | Mandalia, S | Korat, H | Taylor, C | Gleisner, Z | Ibrahim, F | Campbell, L | Brima, N | Williams, I | Youle, M | Lampe, F | Smith, C | Tsintas, R | Chaloner, C | Hutchinson, S | Huntington, S | Mackie, N | Winston, A | Weber, J | Ramzan, F | Carder, M | Lynch, J | Hand, J | Souza, C | Munshi, S | Miller, S | Wood, C | Wilson, A | Morris, S | Allan, S | Palfreeman, A | Memon, K | ewszuk, A | Chadwick, D | Cope, E | Gibson, J | Kegg, S | Main, P | Mitchell, Dr | Hunter, Dr | Hay, P | Dhillon, M | Martin, F | Russell‐Sharpe, S | Harte, A | Clay, S | Tariq, A | Spencer, H | Jones, R | Atkinson, C | Delpech, V | Sachikonye, M | Aitken, Celia | Pozniak, Anton | Cane, Patricia | Clark, Duncan | Collins, Simon | Douthwaite, Samuel | White, Ellen | Fraser, Christophe | Geretti, Anna Maria | Hale, Antony | Hué, Stéphane | Kaye, Steve | Kellam, Paul | Lazarus, Linda | Leigh‐Brown, Andrew | Mbisa, Tamyo | Moses, Samuel | Nastouli, Eleni | Smit, Erasmus | Templeton, Kate | Tilston, Peter | Webster, Daniel | Zhang, Hongyi | Greatorex, Jane | Mullen, Jane | Tandy, Richard | Fawcett, Tracy | Hopkins, Mark | Ashton, Lynn | Booth, Claire | Garcia‐Diaz, Ana | Shepherd, Jill | Schmid, Matthias L | Payne, Brendan | Pere, Spiro | Hubb, Jonathan | Kirk, Stuart | Gunson, Rory | Bradley‐Stewart, Amanda
HIV Medicine  2015;17(5):368-372.
Objectives
No randomized controlled trials have yet reported an individual patient benefit of initiating combination antiretroviral therapy (cART) at CD4 counts > 350 cells/μL. It is hypothesized that earlier initiation of cART in asymptomatic and otherwise healthy individuals may lead to poorer adherence and subsequently higher rates of resistance development.
Methods
In a large cohort of HIV‐positive individuals, we investigated the emergence of new resistance mutations upon virological treatment failure according to the CD4 count at the initiation of cART.
Results
Of 7918 included individuals, 6514 (82.3%), 996 (12.6%) and 408 (5.2%) started cART with a CD4 count ≤ 350, 351–499 and ≥ 500 cells/μL, respectively. Virological rebound occurred while on cART in 488 (7.5%), 46 (4.6%) and 30 (7.4%) with a baseline CD4 count ≤ 350, 351–499 and ≥ 500 cells/μL, respectively. Only four (13.0%) individuals with a baseline CD4 count > 350 cells/μL in receipt of a resistance test at viral load rebound were found to have developed new resistance mutations. This compared to 107 (41.2%) of those with virological failure who had initiated cART with a CD4 count < 350 cells/μL.
Conclusions
We found no evidence of increased rates of resistance development when cART was initiated at CD4 counts above 350 cells/μL.
doi:10.1111/hiv.12302
PMCID: PMC4949527  PMID: 26306942
antiretroviral therapy; CD4 count; HIV resistance; virological failure
16.  CSF/plasma HIV-1 RNA discordance even at low levels is associated with up-regulation of host inflammatory mediators in CSF☆ 
Cytokine  2016;83:139-146.
Highlights
•Discordant HIV in CSF is associated with raised inflammatory mediators in CSF.•CSF mediators are raised with discordance both at high and low levels.•Discordance on ultrasensitive testing can also be also associated with raised mediators.
Introduction
HIV-1 RNA can be found at higher levels in cerebrospinal fluid (CSF) than in plasma, termed CSF/plasma discordance. The clinical significance of CSF/plasma discordance is not known and the degree of discordance considered important varies. We aimed to determine whether a panel of CSF cytokines, chemokines and associated mediators were raised in patients with CSF/plasma discordance at different levels.
Methods
A nested case-control study of 40 CSF samples from the PARTITION study. We used a cytometric bead array to measure CSF mediator concentrations in 19 discordant and 21 non-discordant samples matched for plasma HIV-1 RNA. Discordant samples were subdivided into ‘high discordance’ (>1log10) and ‘low discordance’ (0.5–1log10, or ultrasensitive discordance). CSF mediators significant in univariate analysis went forward to two-way unsupervised hierarchical clustering based on the patterns of relative mediator concentrations.
Results
In univariate analysis 19 of 21 CSF mediators were significantly higher in discordant than non-discordant samples. There were no significant differences between samples with high versus low discordance. The samples grouped into two clusters which corresponded to CSF/plasma discordance (p < 0.0001). In cluster one all mediators had relatively high abundance; this included 18 discordant samples and three non-discordant samples. In cluster two all mediators had relatively low abundance; this included 18 non-discordant samples and one non-discordant sample with ultrasensitive discordance only.
Conclusions
CSF/plasma discordance is associated with potentially damaging neuroinflammatory process. Patients with discordance at lower levels (ie. 0.5–1log10) should also be investigated as mediator profiles were similar to those with discordance >1log10. Sensitive testing may have a role to determine whether ultrasensitive discordance is present in those with low level CSF escape.
doi:10.1016/j.cyto.2016.04.004
PMCID: PMC4889775  PMID: 27131579
HIV; Cerebrospinal fluid; CSF escape; Sanctuary site; Inflammation
17.  Sequencing, de novo assembly and annotation of a pink bollworm larval midgut transcriptome 
GigaScience  2016;5:28.
Background
The pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) is one of the world’s most important pests of cotton. Insecticide sprays and transgenic cotton producing toxins of the bacterium Bacillus thuringiensis (Bt) are currently used to manage this pest. Bt toxins kill susceptible insects by specifically binding to and destroying midgut cells, but they are not toxic to most other organisms. Pink bollworm is useful as a model for understanding insect responses to Bt toxins, yet advances in understanding at the molecular level have been limited because basic genomic information is lacking for this cosmopolitan pest. Here, we have sequenced, de novo assembled and annotated a comprehensive larval midgut transcriptome from a susceptible strain of pink bollworm.
Findings
A de novo transcriptome assembly for the midgut of P. gossypiella was generated containing 46,458 transcripts (average length of 770 bp) derived from 39,874 unigenes. The size of the transcriptome is similar to published midgut transcriptomes of other Lepidoptera and includes up to 91 % annotated contigs. The dataset is publicly available in NCBI and GigaDB as a resource for researchers.
Conclusions
Foundational knowledge of protein-coding genes from the pink bollworm midgut is critical for understanding how this important insect pest functions. The transcriptome data presented here represent the first large-scale molecular resource for this species, and may be used for deciphering relevant midgut proteins critical for xenobiotic detoxification, nutrient digestion and allocation, as well as for the discovery of protein receptors important for Bt intoxication.
Electronic supplementary material
The online version of this article (doi:10.1186/s13742-016-0130-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13742-016-0130-9
PMCID: PMC4916539  PMID: 27333791
Pectinophora gossypiella; Pink bollworm; RNA-Seq; Transcriptome; Midgut; Bacillus thuringiensis
18.  National survey of risk factors for non-communicable disease in Vietnam: prevalence estimates and an assessment of their validity 
BMC Public Health  2016;16:498.
Background
To estimate the prevalence of non-communicable disease (NCD) risk factors at a provincial level in Vietnam, and to assess whether the summary estimates allow reliable inferences to be drawn regarding regional differences in risk factors and associations between them.
Methods
Participants (n = 14706, 53.5 % females) aged 25–64 years were selected by multi-stage stratified cluster sampling from eight provinces each representing one of the eight geographical regions of Vietnam. Measurements were made using the World Health Organization STEPS protocols. Data were analysed using complex survey methods.
Results
Differences by sex in mean years of schooling (males 8.26 ± 0.20, females 7.00 ± 0.18), proportions of current smokers (males 57.70 %, females 1.73 %), and binge-drinkers (males 25.11 %, females 0.63 %), and regional differences in diet, reflected the geographical and socio-cultural characteristics of the country. Provinces with a higher proportion of urban population had greater mean levels of BMI (r = 0.82), and lesser proportions of active people (r = −0.89). The associations between the summary estimates were generally plausible (e.g. physical activity and BMI, r = −0.80) but overstated, and with some anomalous findings due to characterisation of smoking and hypertension by STEPS protocols.
Conclusions
This report provides an extensive description of the sex-specific and regional distribution of NCD risk factors in Vietnam and an account of some health-related consequences of industrialisation in its early stages. The STEPS protocols can be utilized to provide aggregate data for valid between-population comparisons, but with important caveats identified.
doi:10.1186/s12889-016-3160-4
PMCID: PMC4902939  PMID: 27286818
Non-communicable disease; Risk factors; Prevalence; Ecological inference
19.  Discordant CSF/plasma HIV-1 RNA in patients with unexplained low-level viraemia 
Journal of Neurovirology  2016;22(6):852-860.
The central nervous system has been proposed as a sanctuary site where HIV can escape antiretroviral control and develop drug resistance. HIV-1 RNA can be at higher levels in CSF than plasma, termed CSF/plasma discordance. We aimed to examine whether discordance in CSF is associated with low level viraemia (LLV) in blood. In this MRC-funded multicentre study, we prospectively recruited patients with LLV, defined as one or more episode of unexplained plasma HIV-1 RNA within 12 months, and undertook CSF examination. Separately, we prospectively collected CSF from patients undergoing lumbar puncture for a clinical indication. Patients with durable suppression of viraemia and no evidence of CNS infection were identified as controls from this group. Factors associated with CSF/plasma HIV-1 discordance overall were examined. One hundred fifty-three patients were recruited across 13 sites; 40 with LLV and 113 undergoing clinical lumbar puncture. Seven of the 40 (18 %) patients with LLV had CSF/plasma discordance, which was significantly more than 0/43 (0 %) with durable suppression in blood from the clinical group (p = 0.005). Resistance associated mutations were shown in six CSF samples from discordant patients with LLV (one had insufficient sample for testing), which affected antiretroviral therapy at sampling in five. Overall discordance was present in 20/153 (13 %) and was associated with nadir CD4 but not antiretroviral concentrations in plasma or CSF. CSF/plasma discordance is observed in patients with LLV and is associated with antiretroviral resistance associated mutations in CSF. The implications for clinical practice require further investigation.
doi:10.1007/s13365-016-0448-1
PMCID: PMC5127885  PMID: 27194435
HIV; Cerebrospinal fluid; Antiretroviral agents; Drug resistance; Viral; Central nervous system
20.  Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent 
Introduction
Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands.
Methods
The CB2-selective agonist JWH-015 was used to investigate mechanisms downstream of CB2 activation in mouse and human breast cancer cell lines in vitro and in a murine mammary tumor model.
Results
JWH-015 treatment significantly reduced primary tumor burden and metastasis of luciferase-tagged murine mammary carcinoma 4T1 cells in immunocompetent mice in vivo. Furthermore, JWH-015 reduced the viability of murine 4T1 and human MCF7 mammary carcinoma cells in vitro by inducing apoptosis. JWH-015-mediated reduction of breast cancer cell viability was not dependent on Gαi signaling in vitro or modified by classical pharmacological blockade of CB1, GPR55, TRPV1, or TRPA1 receptors. JWH-015 effects were calcium dependent and induced changes in MAPK/ERK signaling.
Conclusion
The results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor.
doi:10.2147/BCTT.S100393
PMCID: PMC4847606  PMID: 27186076
cannabinoid receptor-2; CB2; breast cancer; JWH-015; MAPK/ERK; apoptosis; calcium
21.  Vascular Inward Rectifier K+ Channels as External K+ Sensors in the Control of Cerebral Blood Flow 
For decades it has been known that external potassium (K+) ions are rapid and potent vasodilators that increase cerebral blood flow (CBF). Recent studies have implicated the local release of K+ from astrocytic endfeet—which encase the entirety of the parenchymal vasculature—in the dynamic regulation of local CBF during neurovascular coupling (NVC). It has been proposed that the activation of strong inward rectifier K+ (KIR) channels in the vascular wall by external K+ is a central component of these hyperemic responses; however, a number of significant gaps in our knowledge remain. Here, we explore the concept that vascular KIR channels are the major extracellular K+ sensors in the control of CBF. We propose that K+ is an ideal mediator of NVC, and discuss KIR channels as effectors that produce rapid hyperpolarization and robust vasodilation of cerebral arterioles. We provide evidence that KIR channels, of the KIR2 subtype in particular, are present in both the endothelial and smooth muscle cells of parenchymal arterioles and propose that this dual positioning of KIR2 channels increases the robustness of the vasodilation to external K+, enables the endothelium to be actively engaged in neurovascular coupling, and permits electrical signaling through the endothelial syncytium to promote upstream vasodilation to modulate CBF.
doi:10.1111/micc.12190
PMCID: PMC4404517  PMID: 25641345
KIR channels; neurovascular coupling; cerebral blood flow; functional hyperemia; parenchymal arteriole; capillary; smooth muscle; endothelium; astrocytic endfoot
22.  Can Hepatitis C Virus (HCV) Direct-Acting Antiviral Treatment as Prevention Reverse the HCV Epidemic Among Men Who Have Sex With Men in the United Kingdom? Epidemiological and Modeling Insights 
Epidemiological data and modeling suggest a continuing hepatitis C virus (HCV) epidemic among human immunodeficiency virus-diagnosed men who have sex with men in the United Kingdom. Substantial reductions in HCV transmission could be achieved through scale-up of HCV treatment and behavioral intervention.
Background. We report on the hepatitis C virus (HCV) epidemic among human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) in the United Kingdom and model its trajectory with or without scaled-up HCV direct-acting antivirals (DAAs).
Methods. A dynamic HCV transmission model among HIV–diagnosed MSM in the United Kingdom was calibrated to HCV prevalence (antibody [Ab] or RNA positive), incidence, and treatment from 2004 to 2011 among HIV-diagnosed MSM in the UK Collaborative HIV Cohort (UK CHIC). The epidemic was projected with current or scaled-up HCV treatment, with or without a 20% behavioral risk reduction.
Results. HCV prevalence among HIV-positive MSM in UK CHIC increased from 7.3% in 2004 to 9.9% in 2011, whereas primary incidence was flat (1.02–1.38 per 100 person-years). Over the next decade, modeling suggests 94% of infections are attributable to high-risk individuals, comprising 7% of the population. Without treatment, HCV chronic prevalence could have been 38% higher in 2015 (11.9% vs 8.6%). With current treatment and sustained virological response rates (status quo), chronic prevalence is likely to increase to 11% by 2025, but stabilize with DAA introduction in 2015. With DAA scale-up to 80% within 1 year of diagnosis (regardless of disease stage), and 20% per year thereafter, chronic prevalence could decline by 71% (to 3.2%) compared to status quo in 2025. With additional behavioral interventions, chronic prevalence could decline further to <2.5% by 2025.
Conclusions. Epidemiological data and modeling suggest a continuing HCV epidemic among HIV-diagnosed MSM in the United Kingdom driven by high-risk individuals, despite high treatment rates. Substantial reductions in HCV transmission could be achieved through scale-up of DAAs and moderately effective behavioral interventions.
doi:10.1093/cid/ciw075
PMCID: PMC4826456  PMID: 26908813
hepatitis C virus; HIV; men who have sex with men; antiviral treatment; prevention
23.  Disease Modification of Breast Cancer–Induced Bone Remodeling by Cannabinoid 2 Receptor Agonists 
Most commonly originating from breast malignancies, metastatic bone cancer causes bone destruction and severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain, and drug-induced side effects; furthermore, recent findings suggest that patients are severely undertreated for their cancer pain. Strong analgesics, namely opiates, are first-line therapy in alleviating cancer-related pain despite the severe side effects, including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which are associated with highly undesirable side effects, including nephrotoxicity and osteonecrosis of the jaw. In contrast, cannabinoid receptor 2 (CB2) receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB2 agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB1/CB2 agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Here we demonstrate for the first time that CB2 agonists reduce breast cancer–induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression. Studies used the spontaneously-occurring murine mammary cell line (66.1) implanted into the femur intramedullary space; measurements of spontaneous pain, bone loss, and cancer proliferation were made. The systemic administration of a CB2 agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB2-mediated effects in vivo were reversed by concurrent treatment with a CB2 antagonist/inverse agonist but not with a CB1 antagonist/inverse agonist. In vitro, JWH015 reduced cancer cell proliferation and inflammatory mediators that have been shown to promote pain, bone loss, and proliferation. Taken together, these results suggest CB2 agonists as a novel treatment for breast cancer–induced bone pain, in which disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone pain, and increased survival without the major side effects of current therapeutic options.
doi:10.1002/jbmr.1732
PMCID: PMC4745976  PMID: 22903605
CANNABINOID 2 RECEPTOR; BONE; CANCER; PAIN; CYTOKINE; CHEMOKINE
24.  A Vulnerability Assessment of Fish and Invertebrates to Climate Change on the Northeast U.S. Continental Shelf 
PLoS ONE  2016;11(2):e0146756.
Climate change and decadal variability are impacting marine fish and invertebrate species worldwide and these impacts will continue for the foreseeable future. Quantitative approaches have been developed to examine climate impacts on productivity, abundance, and distribution of various marine fish and invertebrate species. However, it is difficult to apply these approaches to large numbers of species owing to the lack of mechanistic understanding sufficient for quantitative analyses, as well as the lack of scientific infrastructure to support these more detailed studies. Vulnerability assessments provide a framework for evaluating climate impacts over a broad range of species with existing information. These methods combine the exposure of a species to a stressor (climate change and decadal variability) and the sensitivity of species to the stressor. These two components are then combined to estimate an overall vulnerability. Quantitative data are used when available, but qualitative information and expert opinion are used when quantitative data is lacking. Here we conduct a climate vulnerability assessment on 82 fish and invertebrate species in the Northeast U.S. Shelf including exploited, forage, and protected species. We define climate vulnerability as the extent to which abundance or productivity of a species in the region could be impacted by climate change and decadal variability. We find that the overall climate vulnerability is high to very high for approximately half the species assessed; diadromous and benthic invertebrate species exhibit the greatest vulnerability. In addition, the majority of species included in the assessment have a high potential for a change in distribution in response to projected changes in climate. Negative effects of climate change are expected for approximately half of the species assessed, but some species are expected to be positively affected (e.g., increase in productivity or move into the region). These results will inform research and management activities related to understanding and adapting marine fisheries management and conservation to climate change and decadal variability.
doi:10.1371/journal.pone.0146756
PMCID: PMC4739546  PMID: 26839967

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