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Background Several small molecule corrector and potentiator drugs have recently been licensed for Cystic Fibrosis (CF) therapy. However, other aspects of the disease, especially inflammation, are less effectively treated by these drugs. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy. Methods IB3–1 CF lung epithelial cells were treated with different Vertex (VX) drugs, digitoxin, and various drug mixtures, and ELISA assays were used to assess suppression of baseline and TNFα-activated secretion of cytokines and chemokines. Transcriptional responses to these drugs were assessed by RNA-seq and compared with gene expression in AAV-[ wildtype ]CFTR-treated IB3–1 (S9) cells. We also compared in vitro gene expression signatures with in vivo data from biopsied nasal epithelial cells from digitoxin-treated CF patients. Results CF cells exposed to digitoxin exhibited significant suppression of both TNFα/NFκB signaling and downstream secretion of IL-8, IL-6 and GM-CSF, with or without co-treatment with VX drugs. No evidence of drug-drug interference was observed. RNA-seq analysis showed that gene therapy-treated CF lung cells induced changes in 3134 genes. Among these, 32.6% were altered by digitoxin treatment in the same direction. Shared functional gene ontology themes for genes suppressed by both digitoxin and gene therapy included inflammation (84 gene signature), and cell-cell interactions and fibrosis (49 gene signature), while genes elevated by both were enriched for epithelial differentiation (82 gene signature). A new analysis of mRNA data from digitoxin-treated CF patients showed consistent trends in expression for genes in these signatures. Conclusions Adjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients.

Increased recognition of the business case for managing corporate impacts on the environment has helped drive increasingly detailed and quantified corporate environmental goals. Foremost among these are goals of no net loss (NNL) and net positive impact (NPI). We assess the scale and growth of such corporate goals. Since the first public, company-wide NNL/NPI goal in 2001, 32 companies have set similar goals, of which 18 specifically include biodiversity. Mining companies have set the most NNL/NPI goals, and the majority of those that include biodiversity, despite the generally lower total global impact of the mining industry on biodiversity compared to the agriculture or forestry industries. This could be linked to the mining industry's greater participation in best practice bodies, high-profile impacts, and higher profit margins per area of impact. The detail and quality of present goals vary widely. We examined specific NNL/NPI goals and assessed the extent to which their key components were likely to increase the effectiveness of these goals in benefiting biodiversity and managing business risk. Nonetheless, outcomes are more important than goals, and we urge conservationists to work with companies to both support and monitor their efforts to achieve increasingly ambitious environmental goals.

Conservation investment, particularly for charismatic and wide-ranging large mammal species, needs to be evidence-based. Despite the prevalence of this theme within the literature, examples of robust data being generated to guide conservation policy and funding decisions are rare. We present the first published case-study of tiger conservation in Indochina, from a site where an evidence-based approach has been implemented for this iconic predator and its prey. Despite the persistence of extensive areas of habitat, Indochina's tiger and ungulate prey populations are widely supposed to have precipitously declined in recent decades. The Seima Protection Forest (SPF), and broader Eastern Plains Landscape, was identified in 2000 as representing Cambodia's best hope for tiger recovery; reflected in its designation as a Global Priority Tiger Conservation Landscape. Since 2005 distance sampling, camera-trapping and detection-dog surveys have been employed to assess the recovery potential of ungulate and tiger populations in SPF. Our results show that while conservation efforts have ensured that small but regionally significant populations of larger ungulates persist, and density trends in smaller ungulates are stable, overall ungulate populations remain well below theoretical carrying capacity. Extensive field surveys failed to yield any evidence of tiger, and we contend that there is no longer a resident population within the SPF. This local extirpation is believed to be primarily attributable to two decades of intensive hunting; but importantly, prey densities are also currently below the level necessary to support a viable tiger population. Based on these results and similar findings from neighbouring sites, Eastern Cambodia does not currently constitute a Tiger Source Site nor meet the criteria of a Global Priority Tiger Landscape. However, SPF retains global importance for many other elements of biodiversity. It retains high regional importance for ungulate populations and potentially in the future for Indochinese tigers, given adequate prey and protection.

Post-traumatic stress disorder (PTSD) is psychiatric disease, which can occur following exposure to traumatic events. PTSD may be acute or chronic, and can have a waxing and waning course of symptoms. It has been hypothesized that proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) or plasma might be mediators of the psychophysiological mechanisms relating a history of trauma exposure to changes in behavior and mental health disorders, and medical morbidity. Here we test the cytokine/chemokine hypothesis for PTSD by examining levels of 17 classical cytokines and chemokines in CSF, sampled at 0900 hours, and in plasma sampled hourly for 24 h. The PTSD and healthy control patients are from the NIMH Chronic PTSD and healthy control cohort, initially described by Bonne et al. (2011), in which the PTSD patients have relatively low comorbidity for major depressive disorder (MDD), drug or alcohol use. We find that in plasma, but not CSF, the bivariate MCP4 (CCL13)/ MCP1(CCL2) ratio is ca. twofold elevated in PTSD patients compared with healthy controls. The MCP-4/MCP-1 ratio is invariant over circadian time, and is independent of gender, body mass index or the age at which the trauma was suffered. By contrast, MIP-1β is a candidate biomarker for PTSD only in females, whereas TARC is a candidate biomarker for PTSD only in males. It remains to be discovered whether these disease-specific differences in circadian expression for these specific immune signaling molecules are biomarkers, surrogates, or drivers for PTSD, or whether any of these analytes could contribute to therapy.

Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy.

Amyloid β protein (AβP) is the 40- to 42-residue polypeptide implicated in the pathogenesis of Alzheimer disease. We have incorporated this peptide into phosphatidylserine liposomes and then fused the liposomes with a planar bilayer. When incorporated into bilayers the AβP forms channels, which generate linear current-voltage relationships in symmetrical solutions. A permeability ratio, PK/PCI, of 11 for the open AβP channel was estimated from the reversal potential of the channel current in asymmetrical KCI solutions. The permeability sequence for different cations, estimated from the reversal potential of the AβP-channel current for each system of asymmetrical solutions, is PCs> PLi> PCa≥ PK> PNa. AβP-channel current (either Cs+or Ca2+as charge carriers) is blocked reversibly by tromethamine (millimolar range) and irreversibly by Al3+(micromolar range). The inhibition of the AβP-channel current by these two substances depends on transmembrane potential, suggesting that the mechanism of blockade involves direct interaction between tromethamine (or Al3+) and sites within the AβP channel. Hitherto, AβP has been presumed to be neurotoxic. On the basis of the present data we suggest that the channel activity of the polypeptide may be responsible for some or all of its neurotoxic effects. We further propose that a useful strategy for drug discovery for treatment of Alzheimer disease may include screening compounds for their ability to block or otherwise modify AβP channels.

Annexin-A7 (ANXA7) tumor suppressor role has been shown in various tumors, and ANXA7 expression has been particularly lost in androgen-resistant prostate cancers. In this study, we studied ANXA7 regulation in normal prostate versus androgen-sensitive and -resistant prostate cancer cells. Deletion mapping analysis showed lowest ANXA7-promoter activities in androgen-sensitive LNCaP prostate cancer cells. Genomatix analysis of ANXA7 promoter identified a cluster of steroid nuclear hormone receptor elements, including V$GREF (V$GRE.02/ARE.02). Gelshift analysis clearly indicated distinct nuclear protein occupancy at this ANXA7-promoter site (−1086/−890) in prostate cancer (LNCaP, DU145, and PC3) versus normal prostate (PrEC) cells. In matrix-assisted laser desorption time-of-flight mass spectrometry-based search for ANXA7 nuclear regulators, we identified several heterogeneous nuclear ribonucleoproteins (hnRNPs) (A1, A2/B1 and K) attached to the steroid-associated ANXA7-promoter site in the androgen-resistant PC3 prostate cancer cells with high ANXA7 gene copy number, but not in PrEC. The hnPNP role in ANXA7 regulation (that was validated by hnRNPA2/B1 antibody interference) resulted in multiple ANXA7 cDNA and protein products in PC3, but not in PrEC. Ingenuity pathways analysis showed plausible molecular paths between ANXA7 and the hnRNP-associated network in prostate cancer progression. Thus, a multi-hnRNP complex can be responsible for aberrant ANXA7 transcription and splicing, thereby affecting ANXA7 expression pattern and tumor suppressor function in prostate cancer.

We have recently shown that the Alzheimer disease 40-residue amyloid β-protein [Aβ P-(1-40)] can form cation-selective channels when incorporated into planar lipid bilayers by fusion of liposomes containing the peptide. Since Aβ P-(1-40) comprises portions of the putative extracellular and membrane-spanning domains of the amyloid precursor protein (APP751), we suggested that the channel-forming property could be the underlying cause of amyloid neurotoxicity. The peptide has been proposed to occur in vivo in both membrane-bound and soluble forms, and we now report that soluble Aβ P-(1-40) can also form similar channels in solvent-free lipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposomemediated incorporation, the amyloid channel activity in the patch pipet exhibits multiple conductance levels between 40 and 400 pS, cation selectivity, and sensitivity to tromethamine (Tris). Further studies with Aβ P channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective. Amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would be homeostatically dissipative. We therefore interpret these data as providing further support for the concept that cell death in Alzheimer disease may be due to amyloid ion-channel activity.

The Alzheimer disease 40-residue amyloid β protein (Aβ P[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+has been reported to bind to Aβ P[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+binding for the Aβ P[1-40] channel. Provided the Aβ P[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+blocks the open channel in a dosedependent manner. For Aβ P[1-40] channels in the giant conductance mode (>400 pS), Zn2+doses in the millimolar range were required to exert substantial blockade. The Zn2+chelator o-phenanthroline reverses the blockade. We also found that Zn2+modulates Aβ P[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on Aβ P[1-40] channels indicating asymmetric Zn2+-Aβ P[1-40] interactions at the entrance to the pore.

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2+, although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin O-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 microM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.