Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Effects of Pilates,McKenzie and Heckscher training on disease activity,spinal motility and pulmonary function in patients with ankylosing spondylitis: a randomized controlled trial

The optimal management of ankylosis spondylitis (AS) involves a combination of nonpharmacologic and pharmacologic treatment aiming to maximize health-related quality of life. The primary objective of our study was to demonstrate the benefits of an original multimodal exercise program combining Pilates, McKenzie and Heckscher techniques on pulmonary function in patients with AS, while secondary objectives were to demonstrate the benefits of the same program on function and disease activity. This is a randomized controlled study on ninety-six consecutive patients with AS (axial disease subset), assigned on a 1:1 rationale into two groups based on their participation in the Pilates, McKenzie and Heckscher (group I) or in the classical kinetic program (group II). The exercise program consisted of 50-min sessions performed 3 times weekly for 48 weeks. Standard assessments were done at week 0 and 48 and included pain, modified Schober test (mST) and finger–floor distance (FFD), chest expansion (CE) and vital capacity (VC), as well as disease activity Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), functional Bath Ankylosing Spondylitis Functional Index (BASFI) and metrology index Bath Ankylosing Spondylitis Metrology Index (BASMI). Groups were comparable at baseline; we demonstrated significant improvement between baseline and after 48 weeks of regular kinetic training for all AS-related parameters in both groups. However, significant improvement was found in pain, lumbar spine motility (mST, FFD), BASFI, BASDAI and BASMI in AS performing the specific multimodal exercise program at the end of study (p = 0.001). Although there were significant improvements in CE in both groups as compared to baseline (group I, p = 0.001; group II, p = 0.002), this parameter increased significantly only in group I (p = 0.001). VC measurements were not significantly changed at the end of the study (group I, p = 0.127; group II, p = 0.997), but we found significant differences within groups (p = 0.011). A multimodal training combining Pilates, McKenzie and Heckscher exercises performed regularly should be included in the routine management of patients with AS for better control of function, disease activity and pulmonary function.     

Evidence for altered inflammatory and repair responses in symptomatic carotid plaques from elderly patients

Objective

Most acute cardiovascular events are caused by rupture of an atherosclerotic plaque. The incidence of cardiovascular events increases with age and inflammation is generally considered to be the main cause of increased plaque vulnerability. However, the relationship between age and plaque inflammation has not yet been fully clarified. The aim of our study was to determine if age-dependent plaque vulnerability is associated with increased plaque inflammation.

Methods

We collected 200 endarterectomy specimens, 103 of which were from patients 70 years or older. One-hundred and five patients had a recent cerebrovascular event, whereas the rest were asymptomatic despite significant carotid stenosis. Smooth muscle cell, lipid and macrophage content were analyzed by histology. Cytokines, growth factors and extracellular matrix proteins were analyzed in whole plaque homogenates by immunoassays and biochemical methods.

Results

Plaques from elderly patients contained less IFN-γ, TNF-α, fractalkine, sCD40L, and elastin. Lipid and macrophage content was higher in plaques from symptomatic compared to asymptomatic patients in the elderly group, but not in younger patients. The elastin and collagen content was lower in plaques from symptomatic patients in both age groups. Plaques associated with symptoms also contained more TNF-α, IL-1β, IL-6, sCD40L, MIP-1β, MCP-1, RANTES and VEGF, regardless of age.

Conclusions

Our data imply that increased plaque vulnerability in the symptomatic elderly patients is associated with increased lipid accumulation and impaired tissue repair, rather than with increased plaque inflammation, compared to younger individuals.     

Factors Associated with Systemic Hypertension in Asthma

Purpose

Asthmatics have unique characteristics that may influence cardiovascular morbidity. We tested the association of lower airway caliber, obstructive sleep apnea (OSA), and other asthma-related factors, with systemic hypertension (HTN).

Methods

Asthma individuals at specialty clinics completed the Sleep Apnea scale of the Sleep Disorders Questionnaire (SA-SDQ). Medical records were reviewed for diagnosed HTN, OSA and comorbidities, spirometry, and current medications. FEV₁% predicted was categorized as ≥80 (reference), 70–79, 60–69, and <60. SA-SDQ ≥36 for men and ≥32 for women defined high OSA risk.

Results

Among 812 asthmatics (mean age ± standard deviation: 46 ± 14 years), HTN was diagnosed in 191 (24 %), OSA in 65 (8 %), and OSA or high OSA risk (combined OSA variable) in 239 (29 %). HTN was more prevalent in lower FEV₁% categories (p < 0.0001), in subjects with OSA, and those with combined OSA variable (55 vs. 21 % and 46 vs. 14 %, respectively, both p < 0.0001). With adjustment for covariates, associations with HTN remained significant for some FEV₁% categories (70–79 % odds ratio = 1.60 [95 % CI 0.90–2.87]; 60–69 % 2.73 [1.28–5.79]; <60 % 0.96 [0.43–2.14]), and for OSA (2.20 [1.16–4.19]). The combined OSA variable in comparison with OSA alone demonstrated a stronger association with HTN (3.17 [1.99–5.04]) in a reiteration of this model. Inhaled corticosteroids (ICS) at lowest doses, in comparison to no ICS use had an independent “protective” association with HTN (0.44 [0.22–0.90]).

Conclusions

In this young population, worse lower airways obstruction and OSA were associated with HTN. In contrast, lower ICS doses attenuated likelihood for HTN. Adequate control of airway inflammation at appropriate ICS doses, and screening for OSA may reduce the burden of HTN in asthma.     

Non–Small Cell Lung Cancer: Epidemiology,Screening, Diagnosis,and Treatment

Lung cancer remains the leading cause of cancer deaths in the United States. In the past decade, significant advances have been made in the science of non–small cell lung cancer (NSCLC). Screening has been introduced with the goal of early detection. The National Lung Screening Trial found a lung cancer mortality benefit of 20% and a 6.7% decrease in all-cause mortality with the use of low-dose chest computed tomography in high-risk individuals. The treatment of lung cancer has also evolved with the introduction of several lines of tyrosine kinase inhibitors in patients with EGFR, ALK, ROS1, and NTRK mutations. Similarly, immune checkpoint inhibitors (ICIs) have dramatically changed the landscape of NSCLC treatment. Furthermore, the results of new trials continue to help us understand the role of these novel agents and which patients are more likely to benefit; ICIs are now part of the first-line NSCLC treatment armamentarium as monotherapy, combined with chemotherapy, or after definite chemoradiotherapy in patients with stage III unresectable NSCLC. Expression of programmed cell death protein-ligand 1 in malignant cells has been studied as a potential biomarker for response to ICIs. However, important drawbacks exist that limit its discriminatory potential. Identification of accurate predictive biomarkers beyond programmed cell death protein-ligand 1 expression remains essential to select the most appropriate candidates for ICI therapy. Many questions remain unanswered regarding the proper sequence and combinations of these new agents; however, the field is moving rapidly, and the overall direction is optimistic.     

Therapy of cryptococcosis with a combination of flucytosine and amphotericin B.

In a prospective study from May 1971 to November 1973, 20 consecutive patients with a diagnosis of disseminated cryptococcosis were treated for six weeks with a combination of amphotericin B (20 mg daily) intravenously and flucytosine (150 mg/kg daily) orally. Fifteen patients has culturally docummented Cryptococcus neoformans meningitis, and three died of infection early in therapy. Of the remaining 12 patients, eight were alive and well eight to 34 months after therapy, and four died of other causes. None of the surviving patients has relapsed. Hematologic complications developed in nine patients, three of whom had no underlying lymphoreticular disorder or therapy with known cytotoxic agents. Renal insufficiency of mild degree occurred in only six patients. A shorter period of hospitalization and reduction in toxicity of amphotericin B suggest that combined therapy is a safe and efficacious alternative to other regimens.     

Pathogenesis of hyperuricemia: recent advances

The literature on the pathogenesis of hyperuricemia have been limited to the discussion of metabolic syndromes associated with risk factors for atherosclerosis and hyperuricemia and the genetics of the juvenile form of hyperuricemic nephropathies. A few new mutations in the hypoxanthine-guanine phosphoribosyltransferase gene, which result in Lesch-Nyhan syndrome, have been described. In addition, some new insight has been gained in the renal handling of uric acid by the human kidney.     

Application of survival classification and regression tree analysis for identification of subgroups of risk in patients with heart failure and reduced left ventricular ejection fraction

The International Journal of Cardiovascular Imaging - The aim of this study was to identify by classification and regression tree (CART) analysis groups of patients with different survival patterns...