Peptide-Folding Triggered Phase Separation and Lipid Membrane Destabilization in Cholesterol-Rich Lipid Vesicles.

Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems.

A Robust Liposomal Platform for Direct Colorimetric Detection of Sphingomyelinase Enzyme and Inhibitors.

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick's, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

A key to the backdoor into the castle: The clinical ramifications of immunoediting driven by antigenic competition.

Over the last decade the field of cancer biology has gained considerable data on genomic heterogeneity. This situation creates challenges and possibly opportunities for cancer treatment. The evolution of the tumor at all stages also requires the growing malignancy to confront and avoid the immune system. What we describe here is the interaction of two immune phenomena that work together to change the characteristics of the tumor, i.e., antigenic competition and immune editing. These two systems are mutually functional and their interaction is capable of altering the characteristics of the tumor for protection and survival in an immune competent host as well as restricting the diversity of the tumor clones. Therefore, the final outcome of these interactions can also become the key to the backdoor into the castle. Through an additional immune manipulation, autologous tumor cell immunization, we can achieve prevention of disease recurrence after surgical resection and by analyzing induced human monoclonal antibodies to the neoantigens, gain in site into the restriction of diversity of the mutant clones. These findings may also open the door for a pathway to immune prevention of cancer.

Cytosolic 5'-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis.

OBJECTIVES: Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. MATERIALS AND METHODS: Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. RESULTS: Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. INTERPRETATION: Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.

No effect of AR polyG polymorphism on spinal and bulbar muscular atrophy phenotype.

BACKGROUND AND PURPOSE: Disease severity varies considerably among patients with Spinal and Bulbar Muscular Atrophy (SBMA). Our aim was to investigate the role of androgen receptor (AR) polymorphic repeats in SBMA phenotype. METHODS: We analyzed the length of AR polyQ and polyG tracts in 159 SBMA patients. RESULTS: No relationship between polyG size or polyG/polyQ haplotypes and clinical phenotype was found. An independent negative correlation between polyQ-length and onset of weakness was confirmed (P < 0.001). CONCLUSIONS: The negative results of our study prompt to continue the search for potential disease modifiers in SBMA outside the AR gene.

Zebra body myopathy is caused by a mutation in the skeletal muscle actin gene (ACTA1).

We present follow up data on the original case of 'zebra body myopathy' published by Lake and Wilson in 1975. Pathological features in a second biopsy performed at the age of 29 years included a wide variation in fibre size, multiple split fibres, excess internal nuclei and endomysial connective tissue, rimmed vacuoles, accumulation of myofibrillar material and large 'wiped out' areas lacking stain for oxidative enzymes. The presence of nemaline rods and actin-like filaments in addition to small zebra bodies suggested ACTA1 as a candidate gene. This has been confirmed by the identification of a novel c.1043T.p.Leu348Gln mutation, which probably occurred de novo. This case illustrates that the myopathy associated with zebra bodies is part of the spectrum of myopathies associated with the ACTA1 gene. It also highlights that accumulation of actin filaments is not confined to severe neonatal ACTA1 cases and that progression of weakness can occur in congenital myopathies, as the patient is now wheelchair bound and can only stand with the aid of a walking frame.

Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli.

Droplet interface bilayers (DIBs) provide an exciting new platform for the study of membrane proteins in stable bilayers of controlled composition. To date, the successful reconstitution and activity measurement of membrane proteins in DIBs has relied on the use of the synthetic lipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). We report the functional reconstitution of the mechanosensitive channel of large conductance (MscL) into DIBs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a lipid of significantly greater biological relevance than DPhPC. MscL functionality has been demonstrated using a fluorescence-based assay, showing that dye flow occurs across the DIB when MscL is gated by the cysteine reactive chemical 2-(trimethylammonium)ethyl methane thiosulfonate bromide (MTSET). MscL has already been the subject of a number of studies investigating its interaction with the membrane. We propose that this method will pave the way for future MscL studies looking in detail at the effects of controlled composition or membrane asymmetry on MscL activity using biologically relevant lipids and will also be applicable to other lipid-protein systems, paving the way for the study of membrane proteins in DIBs with biologically relevant lipids.

SEPN1-related myopathies: clinical course in a large cohort of patients.

OBJECTIVE: To assess the clinical course and genotype-phenotype correlations in patients with selenoprotein-related myopathy (SEPN1-RM) due to selenoprotein N1 gene (SEPN1) mutations for a retrospective cross-sectional study. METHODS: Forty-one patients aged 1-60 years were included. Clinical data including scoliosis, respiratory function, and growth measurements were collected by case note review. RESULTS: Mean age at onset was 2.7 years, ranging from birth to the second decade of life. All but 2 remained independently ambulant: one lost ambulation at age 5 years and another in his late 50s. The mean age of starting nocturnal noninvasive ventilation (NIV) was 13.9 years. One child required full-time NIV at the age of 1 year while in 2 cases NIV was started at 33 years. Two patients died from respiratory failure at the age of 10 and 22 years, respectively. The mean age at scoliosis onset was 10 years, in most cases preceded by rigidity of the spine. Fourteen patients had successful spinal surgery (mean age 13.9 years). Twenty-one were underweight; however, overt feeding difficulties were not a feature. CONCLUSIONS: This study describes the largest population affected by SEPN1-RM reported so far. Our findings show that the spectrum of severity is wider than previously reported. Respiratory insufficiency generally develops by 14 years but may occur as early as in infancy or not until the fourth decade. Motor abilities remain essentially static over time even in patients with early presentation. Most adult patients remain ambulant and fully employed.

The clinical, histochemical, and molecular spectrum of PEO1 (Twinkle)-linked adPEO.

BACKGROUND: Mutations in the Twinkle (PEO1) gene are a recognized cause of autosomal dominant progressive external ophthalmoplegia (adPEO), resulting in the accumulation of multiple mitochondrial DNA (mtDNA) deletions and cytochrome c oxidase (COX)-deficient fibers in skeletal muscle secondary to a disorder of mtDNA maintenance. Patients typically present with isolated extraocular muscle involvement, with little apparent evidence of the clinical heterogeneity documented in other mtDNA maintenance disorders, in particular POLG-related disease. METHODS: We reviewed the clinical, histochemical, and molecular genetics analysis of 33 unreported patients from 26 families together with all previous cases described in the literature to define the clinical phenotype associated with PEO1 mutations. RESULTS: Ptosis and ophthalmoparesis were almost universal clinical features among this cohort, with 52% (17/33) reporting fatigue and 33% (11/33) having mild proximal myopathy. Features consistent with CNS involvement were rarely described; however, in 24% (8/33) of the patients, cardiac abnormalities were reported. Mitochondrial histochemical changes observed in muscle showed remarkable variability, as did the secondary mtDNA deletions, which in some patients were only detected by PCR-based assays and not Southern blotting. Moreover, we report 7 novel PEO1 variants. CONCLUSIONS: Our data suggest a shared clinical phenotype with variable mild multiorgan involvement, and that the contribution of PEO1 mutations as a cause of adPEO may well be underestimated. Direct sequencing of the PEO1 gene should be considered in adPEO patients prior to muscle biopsy.

Problems with botulinum toxin treatment in mitochondrial cytopathy: case report and review of the literature.

Botulinum toxin type A (BTXA) is widely used in neurological therapeutics for a variety of indications such as dystonia, spasticity, hyperhidrosis, and hypersalivation. It is relatively contraindicated in disorders of neuromuscular transmission, in individuals with known hypersensitivity or bleeding disorders, and during pregnancy. Two patients are presented with initially undetermined multisystem neurological disorders and excessive sialorrhoea, later diagnosed as mitochondrial cytopathy, who had side effects after treatment with ultrasound guided BTXA injections. Published reports on the use of BTXA injections in hypersalivation of various causes are reviewed, along with the proposed mechanisms of hypersensitivity to BTXA in patients with mitochondrial cytopathies. Clinicians should be cautious when using BTXA injections in such patients because of the significant risk of side effects.

Andersen-Tawil syndrome: new potassium channel mutations and possible phenotypic variation.

OBJECTIVE: To evaluate clinical, genetic, and electrophysiologic features of patients with Andersen-Tawil syndrome (ATS) in the United Kingdom. METHODS: Clinical and neurophysiologic evaluation was conducted of 11 families suspected to have ATS. Molecular genetic analysis of each proband was performed by direct DNA sequencing of the entire coding region of KCNJ2. Control samples were screened by direct DNA sequencing. The electrophysiologic consequences of several new mutations were studied in an oocyte expression system. RESULTS: All 11 ATS families harbored pathogenic mutations in KCNJ2 with six mutations not previously reported. Some unusual clinical features including renal tubular defect, CNS involvement, and dental and phonation abnormalities were observed. Five mutations (T75M, D78G, R82Q, L217P, and G300D) were expressed, all of which resulted in nonfunctional channels when expressed alone, and co-expression with wild-type (WT) KCNJ2 demonstrated a dominant negative effect. CONCLUSION: Six new disease-causing mutations in KCNJ2 were identified, one of which was in a PIP2 binding site. Molecular expression studies indicated that five of the mutations exerted a dominant negative effect on the wild-type allele. KCNJ2 mutations are an important cause of ATS in the UK.

Immunotherapy with autologous tumor cell-BCG vaccine in patients with colon cancer: a prospective study of medical and economic benefits.

We have completed a multicenter, randomized controlled phase III clinical trial in Stages II and III colon cancer patients with active specific immunotherapy (ASI) using autologous tumor cells with an immunomodulating adjuvant bacillus Callmette-Guerin (BCG) vaccine (OncoVAX) in an adjuvant setting. In this study, patients were randomized to receive either OncoVAX therapy or no therapy after surgical resection of the primary tumor and stratified by stage of disease. Since the biologic essence of the effective tumor immunotherapy is the presence in the vaccine of a minimum number of viable, metabolically active, autologous tumor cells, the processing of the vaccine product, occurred within 48 h after surgery. Analysis of prognostic benefit in the pivotal phase III trial, with a 5.8 year median follow-up, showed that a beneficial effect of OncoVAX is statistically significant for all endpoints including recurrence-free interval, overall survival, and recurrence-free survival in Stage II colon cancer patients. Surgery alone cures approximately 65% of Stage II (Dukes B2, B3) colon cancer patients. In the remaining patients, OncoVAX in an adjuvant setting, significantly prolongs recurrence-free interval (57.1% relative risk reduction) and significantly improves 5-year overall survival and recurrence-free survival. No statistically significant prognostic benefits were achieved in Stage III (Duke's C1-C3) patients. A health economics assessment was performed on these results in Stage II colon cancer patients using disease-free survival and overall survival (for the entire intent-to-treat population). Cost-effectiveness, cost-utility and sensitivity analysis were applied with, cost of life years, recurrence-free life years and quality adjusted life years (QALYs) as the primary endpoints to this analysis. The perspective of the economic analysis was the current direct medical cost established by the health care providers. The introduction of new technologies often leads to additional costs. This report verified that the use of OncoVAX for patients with Stage II colon cancer not only has significant prognostic benefit and positive clinical outcomes, but also showed that OncoVAX therapy yields impressive health economics benefits.

Unusual muscle disease in HIV infected patients.

Two patients presented with proximal muscle weakness, a normal or minor elevation of creatine phosphokinase (CPK) and normal findings on electromyography. Muscle biopsy in one patient revealed CD8+ polymyositis, and in the other showed ddI induced myopathy. These cases illustrate the importance of muscle biopsy in identifying the underlying pathology in HIV infected patients with muscle weakness and little or no abnormality in laboratory investigations.

Primary pyruvate dehydrogenase E3 binding protein deficiency with mild hyperlactataemia and hyperalaninaemia.

A case of pyruvate dehydrogenase E3 binding protein deficiency is reported in a 24-year-old male with encephalomyopathy. Blood lactate was only minimally elevated, as was alanine.

Human T cell leukaemia virus type I associated neuromuscular disease causing respiratory failure.

Polymyositis and inclusion body myositis have rarely been described in association with human T cell leukaemia virus type I (HTLV-I) infection. Most of such patients have coexisting HTLV-I associated myelopathy (HAM). Two patients with HTLV-I infection, myopathy, and respiratory failure are described. The muscle biopsy specimen of the first patient bore the histological features of inclusion body myositis and there was no evidence of concurrent myelopathy. The second patient had HAM, and her muscle biopsy showed non-specific myopathic and neuropathic changes. Both patients developed respiratory muscle weakness over eight years after diagnosis of myopathy, leading to hypercapnic respiratory failure requiring mechanical ventilatory support. Respiratory failure as a complication of HTLV-I associated myopathy has not previously been described.

Adjuvant active specific immunotherapy of stage II and stage III colon cancer with an autologous tumor cell vaccine: first randomized phase III trials show promise.

We performed three multi-institutional, prospectively randomized, controlled clinical trials, assessing the therapeutic effect of post-resection adjuvant active specific immunotherapy in patients with stage II and stage III colon cancer. In each study four outcomes were considered: time-to-disease recurrence, overall survival intervals, disease-free survival intervals, and recurrence-free survival intervals using the Kaplan-Meir method for generating curves and the log-rank test used to compare efficacy distributions. In addition, a meta-analysis of the three phase III trials was performed since the trials had proven homogeneity. Two main analyses were performed: (1) the intent-to-treat colon cancer patients from all three studies; and (2) analyzable colon cancer patients in all three studies. The conclusion of these analyses is that adjuvant active specific immunotherapy provided significant clinical benefits in patients with stage II colon cancer and appears to be an important new adjuvant treatment for these patients.

Adjuvant active specific immunotherapy for stage II and III colon cancer with an autologous tumor cell vaccine: Eastern Cooperative Oncology Group Study E5283.

PURPOSE: A randomized phase III clinical trial of adjuvant active specific immunotherapy (ASI) with an autologous tumor cell-bacillus Calmette-Guérin (BCG) vaccine was conducted to determine whether surgical resection plus ASI was more beneficial than resection alone in stage II and III colon cancer patients. PATIENTS AND METHODS: Patients (n = 412) with colon cancer (297 with stage II disease, 115 with stage III disease) were randomly allocated to an observation arm or to a treatment arm in which they received three weekly intradermal vaccine injections of 10(7) irradiated autologous tumor cells beginning approximately 4 weeks after surgery. The first two weekly injections also contained 10(7) BCG organisms. Patients were observed for determination of time to recurrence and disease-free and overall survival. RESULTS: This was a negative study in that after a 7.6-year median follow-up period, there were no statistically significant differences in clinical outcomes between the treatment arms. However, there were disease-free survival (P =.078) and overall survival (P =.12) trends in favor of ASI when treatment compliance was evaluated, ie, patients who received the intended treatment had a delayed cutaneous hypersensitivity (DCH) response to the third vaccination (induration >/=5 mm). Also, the magnitude of the DCH response correlated with improved prognosis. The 5-year survival proportion was 84.6% for those with indurations greater than 10 mm, compared with 45.0% for those with indurations less than 5 mm. CONCLUSIONS: When all randomized patients were evaluated, no significant clinical benefit was seen with ASI in surgically resected colon cancer patients with stage II or III colon cancer. However, there was an indication that treatment compliance with effective immunization results in disease-free and overall survival benefits.

Increased risk of stroke in patients with the A12308G polymorphism in mitochondria.

Factors which increase the risk of stroke in patients with the A3243G (mitochondrial encephalomyopathy, lactic acidosis, and stroke [MELAS]) mutation in human mitochondrial DNA are unclear. Previous work on lung-cancer cells with an A3243G mutation showed that a mutation in the mitochondrial transfer gene for leucine tRNA(Leu(CUN)) was able to ameliorate the A3243G-induced biochemical phenotype. We analysed the tRNA(Leu(CUN)) gene in 48 unrelated A3243G cases. We showed that a polymorphism, A12308G, in tRNA(Leu(CUN)) increases the risk of developing stroke in patients with the A3243G mutation (relative risk=2.17). This may have implications for genetic counselling.