Targeted activation of human ether-à-go-go-related gene channels rescues electrical instability induced by the R56Q+/- long QT syndrome variant.

AIMS: Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. METHODS AND RESULTS: The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. CONCLUSION: Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs.

Combined GM-CSF and G-CSF administration mobilizes CD4+ CD25hi Foxp3hi Treg in leukapheresis products of rhesus monkeys.

Early phase clinical trials are evaluating the feasibility, safety, and therapeutic potential of ex vivo expanded regulatory T cells (Treg) in transplantation. A limitation is the paucity of naturally occurring Treg numbers in peripheral blood. Hence, protracted ex vivo expansion is required to obtain sufficient Treg in order to meet target cell doses. Because cytokine administration has been used successfully to mobilize immune cells to the peripheral blood in experimental and clinical studies, we hypothesized that granulocyte macrophage-colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) administration would enhance Treg percentages in leukapheresis products of rhesus monkeys. Following combined GM-CSF and G-CSF administration, the incidence of Treg in peripheral blood and leukapheresis products was elevated significantly, where approximately 3.7 × 106 /kg CD4+ CD25hi Foxp3hi or 6.8 × 106 /kg CD4+ CD25hi CD127lo Treg can be collected from individual products. Mobilized Treg expressed a comparable repertoire of surface markers, chemokine receptors, and transcription factors to naïve monkey peripheral blood Treg. Furthermore, when expanded ex vivo, mobilized leukapheresis product and peripheral blood Treg exhibited similar ability to suppress autologous CD4+ and CD8+ T cell proliferation. These observations indicate that leukapheresis products from combined GM-CSF- and G-CSF-mobilized individuals are a comparatively rich source of Treg and may circumvent long-term ex vivo expansion required for therapeutic application.

CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus.

BACKGROUND: Hydractinia symbiolongicarpus, a colonial cnidarian, is a tractable model system for many cnidarian-specific and general biological questions. Until recently, tests of gene function in Hydractinia have relied on laborious forward genetic approaches, randomly integrated transgenes, or transient knockdown of mRNAs. RESULTS: Here, we report the use of CRISPR/Cas9 genome editing to generate targeted genomic insertions in H. symbiolonigcarpus. We used CRISPR/Cas9 to promote homologous recombination of two fluorescent reporters, eGFP and tdTomato, into the Eukaryotic elongation factor 1 alpha (Eef1a) locus. We demonstrate that the transgenes are expressed ubiquitously and are stable over two generations of breeding. We further demonstrate that CRISPR/Cas9 genome editing can be used to mark endogenous proteins with FLAG or StrepII-FLAG affinity tags to enable in vivo and ex vivo protein studies. CONCLUSIONS: This is the first account of CRISPR/Cas9 mediated knockins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9 inserted transgene in a cnidarian. The ability to precisely insert exogenous DNA into the Hydractinia genome will enable sophisticated genetic studies and further development of functional genomics tools in this understudied cnidarian model.

Prolonged California aridity linked to climate warming and Pacific sea surface temperature.

California has experienced a dry 21(st) century capped by severe drought from 2012 through 2015 prompting questions about hydroclimatic sensitivity to anthropogenic climate change and implications for the future. We address these questions using a Holocene lake sediment record of hydrologic change from the Sierra Nevada Mountains coupled with marine sediment records from the Pacific. These data provide evidence of a persistent relationship between past climate warming, Pacific sea surface temperature (SST) shifts and centennial to millennial episodes of California aridity. The link is most evident during the thermal-maximum of the mid-Holocene (~8 to 3 ka; ka = 1,000 calendar years before present) and during the Medieval Climate Anomaly (MCA) (~1 ka to 0.7 ka). In both cases, climate warming corresponded with cooling of the eastern tropical Pacific despite differences in the factors producing increased radiative forcing. The magnitude of prolonged eastern Pacific cooling was modest, similar to observed La Niña excursions of 1(o) to 2 °C. Given differences with current radiative forcing it remains uncertain if the Pacific will react in a similar manner in the 21st century, but should it follow apparent past behavior more intense and prolonged aridity in California would result.

Predictors of response to steroid therapy for eosinophilic esophagitis and treatment of steroid-refractory patients.

BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) is commonly treated with swallowed (topical) corticosteroids (tCS). However, few factors have been described that predict outcomes of steroid therapy. We aimed to identify factors associated with nonresponse to tCS and report outcomes of second-line treatment for patients with steroid-refractory EoE. METHODS: We performed a retrospective cohort study by using the University of North Carolina EoE Clinicopathologic Database to identify patients who received tCS for EoE from 2006 through 2013. Demographic, symptom, endoscopic, and histologic data were extracted from medical records. Immunohistochemistry was performed on archived biopsies. Responders and nonresponders to tCS were compared. RESULTS: Of 221 patients with EoE who received tCS, 71% had endoscopic improvement, 79% had symptomatic improvement, and 57% had histologic response (<15 eosinophils/high-power field). After multivariate logistic regression, esophageal dilation at the baseline examination predicted nonresponse (odds ratio, 2.9; 95% confidence interval, 1.4-6.3), and abdominal pain predicted response (odds ratio for nonresponse, 0.31; 95% confidence interval, 0.12-0.83); no other clinical features were predictive. On the basis of immunohistochemical analysis, higher baseline levels of tryptase (244 vs 157 mast cells/mm(2), P = .04) and eotaxin-3 (2425 vs 239 cells/mm(2), P = .02) were associated with steroid response, but levels of major basic protein were not. Among 27 steroid-refractory patients, a mean of 2 additional therapies were tried; only 48% of the patients eventually responded to any second-line therapy. CONCLUSIONS: On the basis of a retrospective analysis of a large group of patients with EoE, only 57% have a histologic response to steroid therapy. Baseline esophageal dilation and decreased levels of mast cells and eotaxin-3 predicted which patients would not respond to therapy. Combining clinical factors and immunohistochemistry might therefore be used to direct therapy.

Evaluation of Histologic Cutpoints for Treatment Response in Eosinophilic Esophagitis.

AIM: No consensus exists on the definition of successful treatment in eosinophilic esophagitis (EoE). The aim of this study was to identify the optimal histologic cutpoint to define successful treatment of EoE by assessing rates of symptomatic and endoscopic improvement. MATERIALS AND METHODS: We performed a retrospective cohort study utilizing the University of North Carolina EoE Clinicopathologic Database between 2006 and 2013. Rates of symptomatic and endoscopic improvement were determined, as were post-treatment eosinophil counts. The area under the receiver operator characteristic curve (AUC) was calculated for symptomatic and endoscopic response at several possible eosinophil count cutpoints (eos/hpf). Predictors of response were also assessed. RESULTS: Of 224 treatments in 199 patients, 76% were associated with symptomatic improvement, 68% with endoscopic improvement, and 60% with both. Of treatments that resulted in a post-treatment count of <15 eos/hpf, 90% were associated with an endoscopic response, 88% with a symptomatic response, and 81% with both symptomatic and endoscopic responses. Using a <15 eos/hpf threshold, the area under the curves (AUCs) were 0.70, 0.78, and 0.75 for symptomatic, endoscopic, and symptomatic/endoscopic responses, respectively. Lower histologic cut-points did not result in a substantial gain in response, but decreased the AUC. CONCLUSION: In this large cohort of EoE patients, rates of symptomatic and endoscopic improvement were generally associated with histologic improvement. A histologic cutoff for treatment response of <15 eos/hpf may balance clinical outcomes and test performance.

Elongated TCR alpha chain CDR3 favors an altered CD4 cytokine profile.

BACKGROUND: CD4 T lymphocyte activation requires T cell receptor (TCR) engagement by peptide/MHC (major histocompatibility complex) (pMHC). The TCR complementarity-determining region 3 (CDR3) contains variable α and ß loops critical for pMHC recognition. During any immune response, tuning of TCR usage through progressive clonal selection occurs. Th1 and Th2 cells operate at different avidities for activation and display distinct transcriptional programs, although polarization may be plastic, influenced by pathogens and cytokines. We therefore hypothesized that CDR3αß sequence features may intrinsically influence CD4 phenotype during progression of a response. RESULTS: We show that CD4 polarization involves distinct CDR3α usage: Th1 and Th17 cells favored short TCR CDR3α sequences of 12 and 11 amino acids, respectively, while Th2 cells favored elongated CDR3α loops of 14 amino acids, with lower predicted affinity. The dominant Th2- and Th1-derived TCRα sequences with 14 amino acid CDR3 loops and 12 amino acid CDR3 loops, respectively, were expressed in TCR transgenics. The functional impact of these TCRα transgenes was assessed after in vivo priming with a peptide/adjuvant. The short, Th1-derived receptor transgenic T cell lines made IFNγ, but not IL-4, 5 or 13, while the elongated, Th2-derived receptor transgenic T cell lines made little or no IFNγ, but increased IL-4, 5 and 13 with progressive re-stimulations, mirrored by GATA-3 up-regulation. T cells from primed Th2 TCRα transgenics selected dominant TCR Vß expansions, allowing us to generate TCRαß transgenics carrying the favored, Th2-derived receptor heterodimer. Primed T cells from TCRαß transgenics made little or no IL-17 or IFNγ, but favored IL-9 after priming with Complete Freund's adjuvant and IL-4, 5, 9, 10 and 13 after priming with incomplete Freund's. In tetramer-binding studies, this transgenic receptor showed low binding avidity for pMHC and polarized T cell lines show TCR avidity for Th17 > Th1 > Th2. While transgenic expression of a Th2-derived, 'elongated' TCR-CDR3α and the TCRαß pair, clearly generated a program shifted away from Th1 immunity and with low binding avidity, cytokine-skewing could be over-ridden by altering peptide challenge dose. CONCLUSION: We propose that selection from responding clones with distinctive TCRs on the basis of functional avidity can direct a preference away from Th1 effector responses, favoring Th2 cytokines.

Evolution of the bomolochiform superfamily complex (Copepoda: Cyclopoida): new insights from ssrDNA and morphology, and origin of Umazuracolids from polychaete-infesting ancestors rejected.

Poecilostome cyclopoids are among the most morphologically diverse copepods, having established symbiotic relationships with teleosts, elasmobranchs and invertebrate hosts belonging to no fewer than 14 marine phyla. Many parasitic lineages display radically divergent body plans and on that basis have traditionally been placed at higher taxonomic rank than they deserve. The most recent example is the monotypic family Umazuracolidae, established for a derived fish parasite with bomolochiform affinities. Phylogenetic analysis of complete ssrDNA (18S) sequences of 44 species belonging to 21 families of cyclopoid copepods shows that there is no support for the familial distinctiveness of the Umazuracolidae. Both maximum parsimony tree reconstruction and Bayesian inference, operating under the GTR+I+Γ model of nucleotide substitution, unambiguously placed Umazuracola elongatus in the Taeniacanthidae within the predominantly fish parasitic bomolochiform complex, refuting the original suggestion of a shared most recent common ancestry with polychaete symbionts. The phylogenies also revealed that the bomolochiform families and the Clausidiidae (and allies) form a monophyletic group, the clausidiiform complex, with high nodal support under both methods. Bayesian inference suggested a diphyletic origin of the "Poecilostomatoida" with the clausidiiform family-group holding a basal position while the traditional cyclopoid families form a monophyletic group in apposition to a second poecilostomatoid clade; however, maximum parsimony results were equivocal, depending on outgroup selection. Scrutiny of the morphological characters diagnosing the monotypic families Tegobomolochidae and Tuccidae demonstrated that they merely represent derived lineages within more inclusive taxa, the former being related to a group of nostril-inhabiting genera within the Bomolochidae, the latter forming the sistergroup of Taeniacanthodes within the Taeniacanthidae. The taeniacanthid genus Makrostrotos occupies a position at the base of the bomolochiform complex and is fixed as the type of a new family, Makrostrotidae. Although both morphological and molecular evidence hint that the Bomolochidae is nested within a paraphyletic Taeniacanthidae, the status quo of maintaining both families is favoured here pending additional molecular data. The bomolochiform complex, comprising the Bomolochidae, Taeniacanthidae, Telsidae and Makrostrotidae, is attributed superfamilial rank as the Bomolochoidea. A recent controversial phylogenetic analysis of the poecilostomatoid families is shown to be flawed, being based on a dataset containing imperfect or misleading information, and characters whose states were wrongly assessed.

Genetic diversity of Ascaris in southwestern Uganda.

Despite the common occurrence of ascariasis in southwestern Uganda, helminth control in the region has been limited. To gain further insights into the genetic diversity of Ascaris in this area, a parasitological survey in mothers (n=41) and children (n=74) living in two villages, Habutobere and Musezero, was carried out. Adult Ascaris worms were collected from infected individuals by chemo-expulsion using pyrantel pamoate treatment. Genetic diversity within these worms was assessed by inspection of DNA sequence variation in a mitochondrial marker and length polymorphism at microsatellite loci. Overall prevalence of ascariasis was 42.5% in mothers and 30.4% in their children and a total of 98 worms was examined from 18 hosts. Sequence analysis of a portion of the mitochondrial cytochrome c oxidase subunit 1 gene revealed 19 different haplotypes, 13 of which had not been previously encountered. Microsatellite analysis using eight loci provided evidence for high gene flow between worm populations from the two villages but comparing these worms with others obtained in a prior study on Unguja, Zanzibar, confirmed little genetic exchange and mixing of worm populations between the two areas. By adding to our understanding of the genetic diversity of Ascaris in Africa, this study provides useful information for monitoring changes in parasite population structure in the face of ongoing and future control.

Cancrincolidae (Copepoda, Harpacticoida) associated with land crabs: a semiterrestrial leaf of the ameirid tree.

Morphological evidence suggests harpacticoid copepods have recurrently entered into symbiosis with other crustaceans but only members of the family Cancrincolidae have successfully made the transition from marine habitats to semiterrestrial hosts. Cancrincolids are primarily amphi-Atlantic in distribution (with one outlier in the western Pacific) and typically inhabit the gill chambers of grapsoidean land crabs belonging to the families Grapsidae, Sesarmidae, Varunidae and Gecarcinidae. Morphologically, they are difficult to place because they exhibit unusual autapomorphies and the shared derived characters claimed to unite them with the primitively marine Ameiridae are equivocal. Both maximum parsimony and Bayesian inference solutions based on SSU rDNA gene sequences show topological congruence in placing the Cancrincolidae within the Ameiridae and in firmly resolving it as the sistergroup of taxa that have been reported as obligate or commensal associates of crayfish. This relationship is further supported by swimming leg sexual dimorphism and mandibular palp morphology. Morphological comparison with ameirid copepods revealed the majority of synapomorphies previously proposed in support of cancrincolid monophyly and familial distinctiveness can be attributed to heterochrony. The progressive evolution of cancrincolid associates appears to be largely concordant with the sequential adaptation to terrestriality by their grapsoidean hosts. The current amphi-Atlantic distribution of Cancrincola may suggest its possibly free-living ancestor had already assumed a virtually continuous distribution along the northern seaboard of Gondwana prior to the opening of the South Atlantic during the early Cretaceous, implying symbiotic relationships were established only much later when grapsoidean crabs started to emerge, radiate and diversify in the mid-Tertiary (15-35 mya). The adoption of semi-terrestriality in cancrincolid copepods can be viewed as yet another independent attempt (incursion) to colonize low-salinity environments by members of the family Ameiridae. The possible sistergroup relationships of the Ameiridae and the position of the genus Argestigens Willey are discussed.

Extraordinary host switching in siphonostomatoid copepods and the demise of the Monstrilloida: integrating molecular data, ontogeny and antennulary morphology.

Copepods exhibit an astounding variety of lifestyles, host associations and morphology, to the extent that their crustacean affinities may be obscured. Relationships among the ten copepod orders based on morphological characters remain equivocal. Here we test the ordinal status of the enigmatic Monstrilloida using SSU rDNA gene sequences, comparative morphological data (antennulary sensory interface) and ontogenetic data (caudal ramus setation patterns). Bayesian analysis unexpectedly revealed the Monstrilloida are nested within a fish-parasitic clade of the Siphonostomatoida and share a common ancestor with the stem species of the caligiform families (sea-lice). This unforeseen relationship is congruent with both antennulary and caudal ramus morphology. The divergence of the monstrilloids from an ectoparasitic, vertebrate-associated ancestor involved radical changes in host utilization, body plan and life cycle strategy, a combination rarely observed and probably unique in metazoan parasites. Adult monstrilloids secondarily returned to a free-living, predator-exposed mode of life and we postulate the pressure on maintaining a functional approaching-predator detection system has progenetically delayed the suppression (as in post-copepodid caligiform instars) of the 5-point antennulary sensory array. The homoplastic evolution of the frontal filament in Siphonostomatoida is discussed.

Effect of pravastatin on body composition and markers of cardiovascular disease in HIV-infected men--a randomized, placebo-controlled study.

OBJECTIVES: To determine the effect of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, on markers of cardiovascular risk and lipodystrophy in HIV-infected, protease inhibitor (PI)-treated men with hypercholesterolaemia. METHODS: A randomized, placebo-controlled, 16-week study was carried out on 33 HIV-infected, hypercholesterolaemic men (fasting total cholesterol > 6.5 mmol/L) on PI-containing therapy. Patients commenced dietary assessment and advice at week 0 and were randomized to 12 weeks pravastatin (40 mg each night) or placebo from week 4. The primary endpoint was the time-weighted change (TWAUC) in total cholesterol from week 0. Secondary endpoints included TWAUC cholesterol from week 4 (start of pravastatin), total and regional body fat, fasting lipids, glucose, insulin, and markers of cardiovascular risk. RESULTS: Of 33 men randomized (pravastatin n = 16, mean age 48 years), 31 completed the study. Groups were matched for baseline cholesterol and body composition. Although there was no significant between-group difference in TWAUC cholesterol from week 0 (pravastatin -0.6 +/- 1.0 versus placebo -0.4 +/- 1.0 mmol/L/week; P = 0.8), TWAUC cholesterol from week 4 decreased more in the pravastatin group (-0.8 +/- 1.0 versus -0.3 +/- 0.9 mmol/L/week; P = 0.04). Neither triglycerides nor dietary intake changed. Subcutaneous fat increased significantly with pravastatin (+0.72 +/- 1.55 versus +0.19 +/- 0.48 kg change in limb fat, P < 0.04; +5.2 +/- 8.7 versus -1.3 +/- 13.7 cm change in abdominal subcutaneous fat, P = 0.02). Apart from homocystine, which decreased in the pravastatin group, there were no significant differences in other cardiovascular, lipid or glucose parameters. CONCLUSIONS: Despite limited effects on cholesterol, 12 weeks use of pravastatin 40 mg each night in HIV-infected men with hypercholesterolaemia resulted in significant increases in subcutaneous fat.

A randomized, double-blind study of gemfibrozil for the treatment of protease inhibitor-associated hypertriglyceridaemia.

BACKGROUND: Hypertriglyceridaemia is common in patients with HIV, especially those taking protease inhibitors or with lipodystrophy, frequently observed at levels associated with accelerated cardiac disease. This study aimed to explore the efficacy and safety of gemfibrozil for hypertriglyceridemia in patients with HIV infection. METHODS: A 16-week, randomized, double-blind, comparative study of low saturated fat diet versus low saturated fat diet with gemfibrozil 600 mg twice daily in patients with triglycerides > or = 3mmol/l receiving protease inhibitor therapy. Following a 4-week period of dietary intervention alone, patients were randomized to gemfibrozil or matching placebo. The primary outcome was the difference in mean change in fasting triglycerides at week 16 between the two groups. RESULTS: 37 men were randomized (17 gemfibrozil, 20 placebo) with median fasting triglycerides 5.6 mmol/l. Mean changes in triglycerides from week 4 to week 16 were -1.22 mmol/l and +0.35 mmol/l for the gemfibrozil and placebo groups respectively (between-group mean difference of 1.57 mmol/l; 95% confidence interval, -6.7 to 3.5; = 0.08). Only one patient treated had triglycerides return to a desirable range (< or = 2.00 mmol/l). No significant changes in the other metabolic parameters were observed. Gemfibrozil was well tolerated, did not appear to induce additional protease inhibitor toxicity, and did not induce changes in CD4 lymphocyte counts or HIV RNA load. CONCLUSIONS: Gemfibrozil is safe and demonstrated at most, modest efficacy for hypertriglyceridemia in HIV-infected patients receiving protease inhibitors. Given the level of response, however, it is unclear whether these reductions will confer clinical benefit, at least in the presence of continued protease inhibitor use.