A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

Accuracy of Static Computer- Assisted Implant Placement in Posterior Edentulous Areas with Different Levels of Tooth- Support by Novice Clinicians.

Correct implant placement is necessary for satisfactory implant restoration. Therefore, the use of surgical guide is recommended. This study evaluated the accuracy of implant placement in posterior edentulous areas with different levels of tooth-support by novice clinicians according to fully-guided (FG), pilot-guided (PG), and freehand (FH) placement protocols. A mandibular model with missing first molars was designed. On one side, the model had a bound edentulous area (BEA), and on the other side, a free end edentulous area (FEA). Fourteen clinicians new to implant dentistry participated in the study, and each clinician inserted an implant in the BEA and FEA sites for every placement protocol. Angle, vertical and maximum horizontal platform and apex deviations were measured. The FG placement was more accurate than the PG and FH placements. This was significant for BEA angle deviation, BEA and FEA maximum horizontal platform deviations, and BEA maximum horizontal apex deviation. The PG placement was significantly more accurate than the FH placement for BEA and FEA maximum horizontal platform deviations. FG shows significantly greater angle, maximum horizontal platform and maximum horizontal apex deviations at FEA than BEA. This can be attributed to reduced guide support and the possibility of guide displacement during surgery.

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework.

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked ß-hairpins with opposing ß-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.

Antimicrobial peptides derived from the cartilage.-specific C-type Lectin Domain Family 3 Member A (CLEC3A) - potential in the prevention and treatment of septic arthritis.

OBJECTIVE: To investigate the antimicrobial activity of peptides derived from C-type Lectin Domain Family 3 Member A (CLEC3A), shed light on the mechanism of antimicrobial activity and assess their potential application in prevention and treatment of septic arthritis. DESIGN: We performed immunoblot to detect CLEC3A peptides in human cartilage extracts. To investigate their antimicrobial activity, we designed peptides and recombinantly expressed CLEC3A domains and used them to perform viable count assays using E.coli, P.aeruginosa and S.aureus. We investigated the mechanism of their antimicrobial activity by fluorescence and scanning electron microscopy, performed ELISA-style immunoassays and transmission electron microscopy to test for lipopolysaccharide binding and surface plasmon resonance to test for lipoteichoic acid (LTA) binding. We coated CLEC3A peptides on titanium, a commonly used prosthetic material, and performed fluorescence microscopy to quantify bacterial adhesion. Moreover, we assessed the peptides' cytotoxicity against primary human chondrocytes using MTT cell viability assays. RESULTS: CLEC3A fragments were detected in human cartilage extracts. Moreover, bacterial supernatants lead to fragmentation of recombinant and cartilage-derived CLEC3A. CLEC3A-derived peptides killed E.coli, P.aeruginosa and S.aureus, permeabilized bacterial membranes and bound lipopolysaccharide and LTA. Coating CLEC3A antimicrobial peptides (AMPs) on titanium lead to significantly reduced bacterial adhesion to the material. In addition, microbicidal concentrations of CLEC3A peptides in vitro displayed no direct cytotoxicity against primary human chondrocytes. CONCLUSIONS: We identify cartilage-specific AMPs originating from CLEC3A, resolve the mechanism of their antimicrobial activity and point to a novel approach in the prevention and treatment of septic arthritis using potent, non-toxic, AMPs.

Association between HbA1c and the development of cystic fibrosis-related diabetes.

AIMS: To examine HbA1c as a predictor of risk for future development of cystic fibrosis-related diabetes and to assess the association with the development of retinopathy in people with cystic fibrosis-related diabetes. METHODS: A 7-year retrospective longitudinal study was conducted in 50 adults with cystic fibrosis, comparing oral glucose tolerance test results with HbA1c values in predicting the development of cystic fibrosis-related diabetes. Retinal screening data were also compared with HbA1c measurements to assess microvascular outcome. RESULTS: An HbA1c value ≥37 mmol/mol (5.5%; hazard ratio 3.49, CI 1.5-8.1) was significantly associated with the development of dysglycaemia, as defined by the oral glucose tolerance test over a 7-year period. Severity of diabetic retinopathy was associated with a higher HbA1c and longer duration of cystic fibrosis-related diabetes. CONCLUSION: There is a link between HbA1c level and the future development of dysglycaemia in cystic fibrosis based on oral glucose tolerance test, as well as microvascular outcomes. Although current guidance does not advocate the use of HbA1c as a diagnostic tool in cystic fibrosis-related diabetes, it may be of clinical use in determining individuals at risk of future development of cystic fibrosis-related diabetes.

Fluorescent color centers in laser ablated 4H-SiC nanoparticles.

Nanostructured and bulk silicon carbide (SiC) has recently emerged as a novel platform for quantum nanophotonics due to its harboring of paramagnetic color centers, having immediate applications as a single photon source and spin optical probes. Here, using ultra-short pulsed laser ablation, we fabricated from electron irradiated bulk 4H-SiC, 40-50 nm diameter SiC nanoparticles, fluorescent at 850-950 nm. This photoluminescence is attributed to the silicon vacancy color centers. We demonstrate that the original silicon vacancy color centers from the target sample were retained in the final nanoparticles solution, exhibiting excellent colloidal stability in water over several months. Our work is relevant for quantum nanophotonics, magnetic sensing, and biomedical imaging applications.

Dispersive Stiffness of Dzyaloshinskii Domain Walls.

It is well documented that subjecting perpendicular magnetic films that exhibit the interfacial Dzyaloshinskii-Moriya interaction to an in-plane magnetic field results in a domain wall (DW) energy σ, which is highly anisotropic with respect to the orientation of the DW in the film plane Θ. We demonstrate that this anisotropy has a profound impact on the elastic response of the DW as characterized by the surface stiffness σ[over ˜](Θ)=σ(Θ)+σ^{''}(Θ) and evaluate its dependence on the length scale of deformation. The influence of stiffness on DW mobility in the creep regime is assessed, with analytic and numerical calculations showing trends in σ[over ˜] that better represent experimental measurements of domain wall velocity in magnetic thin films compared to σ alone. Our treatment provides experimental support for theoretical models of the mobility of anisotropic elastic manifolds and makes progress toward a more complete understanding of magnetic domain wall creep.

Short communication: Measuring feed volume and weight by machine vision.

Individual dairy cow feed intake is closely related to the health and productive output of each cow, with healthy cows generally eating more feed than unhealthy cows. Incorporating the use of an automated system to monitor feed consumption for each cow may be beneficial for dairy farm management. This study examined the use of an inexpensive 3-dimensional video camera to measure feed volume, from which we derived feed weight. Proof-of-concept testing was conducted to determine the effectiveness and capability of the machine vision feed-scanning system and its possible use in feed intake monitoring. Such systems are ideal because they do not impede the workflow of the farm or interrupt feeding behavior. This is an improvement over existing systems that are labor and cost intensive. Our conducted experiments involve measuring feed volume at known weights, up to 22.68 kg, with the resulting volume and weight values analyzed by means of linear and quadratic least squares t-test regression analysis. The effects of feed positioning in the bin and near-range sensor limitations were also examined. The results showed that an estimation of feed weight from 3-dimensional scan of volume measurements could be made to within 0.5 kg of the physically measured feed weight using a digital scale. Future efforts will focus on extending this work to active bunks with multiple cows eating throughout the day and testing total mixed rations of varied composition.

Response to MAPK pathway inhibitors in BRAF V600M-mutated metastatic melanoma.

WHAT IS KNOWN AND OBJECTIVE: The management of metastatic melanoma has changed significantly in the past decade with the development of immunotherapies and targeted molecular therapies. Trials of targeted therapies have focused mainly on patients with the most common BRAF V600 mutations, namely V600E/K substitutions, with very little information available on the benefit of targeted therapies on less commonly occurring mutations such as V600R/D and M. CASE SUMMARY: We present a 54-year-old man with metastatic melanoma harbouring a rare BRAF V600M mutation, who experienced clinical and radiological response to combined therapy with the BRAF inhibitor dabrafenib and MEK inhibitor trametinib. WHAT IS NEW AND CONCLUSION: As our understanding of these therapies evolves and an increasing number of patients have mutational testing performed, there is a clear imperative--as highlighted by this case--to test for rarer mutations and facilitate their inclusion both in everyday practice and in future clinical trials.

Phase I study of the halichondrin B analogue eribulin mesylate in combination with cisplatin in advanced solid tumors.

BACKGROUND: Eribulin mesylate is a synthetic macrocyclic ketone analogue of Halichondrin B that has demonstrated high antitumor activity in preclinical and clinical settings. This phase I study aimed to determine the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetics in combination with cisplatin (CP) in patients with advanced solid tumours. METHODS: Thirty-six patients with advanced solid tumours received eribulin mesylate 0.7-1.4 mg m(-2) and CP 60-75 mg m(-2). Eribulin mesylate was administered on days 1, 8, and 15 in combination with CP day 1 every 28-day cycle. The protocol was amended after dose level 4 (eribulin mesylate 1.4 mg m(-2), CP 60 mg m(-2)) when it was not feasible to administer eribulin mesylate on day 15 because of neutropenia; the treatment schedule was changed to eribulin mesylate on days 1 and 8 and CP on day 1 every 21 days. RESULTS: On the 28-day schedule, three patients had DLT during the first cycle: grade (G) 4 febrile neutropenia (1.0 mg m(-2), 60 mg m(-2)); G 3 anorexia/fatigue/hypokalemia (1.2 mg m(-2), 60 mg m(-2)); and G 3 stomatitis/nausea/vomiting/fatigue (1.4 mg m(-2), 60 mg m(-2)). On the 21-day schedule, three patients had DLT during the first cycle: G 3 hypokalemia/hyponatremia (1.4 mg m(-2), 60 mg m(-2)); G 4 mucositis (1.4 mg m(-2), 60 mg m(-2)); and G 3 hypokalemia (1.2 mg m(-2), 75 mg m(-2)). The MTD and recommended phase II dose was determined as eribulin mesylate 1.2 mg m(-2) (days 1, 8) and CP 75 mg m(-2) (day 1), on a 21-day cycle. Two patients had unconfirmed partial responses (PR) (pancreatic and breast cancers) and two had PR (oesophageal and bladder cancers). CONCLUSIONS: On the 21-day cycle, eribulin mesylate 1.2 mg m(-2), administered on days 1 and 8, in combination with CP 75 mg m(-2), administered on day 1 is well tolerated and showed preliminary anticancer activity.

A comparison of the assessment and management of cardiometabolic risk in patients with and without type 2 diabetes mellitus in Canadian primary care.

AIM: To investigate the cardiometabolic risk (CMR) assessment and management patterns for individuals with and without type 2 diabetes mellitus (T2DM) in Canadian primary care practices. METHODS: Between April 2011 and March 2012, physicians from 9 primary care teams and 88 traditional non-team practices completed a practice assessment on the management of 2461 patients >40 years old with no clinical evidence of cardiovascular disease and diagnosed with at least one of the following risk factor-T2DM, dyslipidaemia or hypertension. RESULTS: There were 1304 individuals with T2DM and 1157 without. Pharmacotherapy to manage hyperglycaemia, dyslipidaemia and hypertension was widely prescribed. Fifty-eight percent of individuals with T2DM had a glycated haemoglobin (HbA1c) ≤7.0%. Amongst individuals with dyslipidaemia, median low-density lipoprotein cholesterol (LDL-C) was 1.8 mmol/l for those with T2DM and 2.8 mmol/l for those without. Amongst individuals with hypertension, 30% of those with T2DM achieved the <130/80 mmHg target, whereas 60% of those without met the <140/90 mmHg target. The composite glycaemic, LDL-C and blood pressure (BP) target outcome was achieved by 12% of individuals with T2DM. Only 17% of individuals with T2DM and 11% without were advised to increase their physical activity. Dietary modifications were recommended to 32 and 10% of those with and without T2DM, respectively. CONCLUSIONS: Patients at elevated CMR were suboptimally managed in the primary care practices surveyed. There was low attainment of recommended therapeutic glycaemic, lipid and BP targets. Advice on healthy lifestyle changes was infrequently dispensed, representing a missed opportunity to educate patients on the long-term benefits of lifestyle modification.

Trends in the use of implantable cardioverter defibrillators in Australia: a 10-year nationwide study from 2000-2009.

BACKGROUND: Implantable cardioverter defibrillators (ICD) have been demonstrated to reduce mortality in survivors of life-threatening arrhythmias (secondary prevention) and in patients at increased risk of sudden cardiac death (primary prevention). Other nations have reported significant increases in ICD use in recent years. AIM: To investigate Australian nationwide trends of ICD procedures over a 10-year period (2000-2009). METHODS: A retrospective analysis of the Australian Institute of Health and Welfare's National Hospital Morbidity Database was performed to determine the annual number of ICD implantation and replacement procedures between 2000 and 2009. Rates were calculated using Australian Bureau of Statistics data on the annual estimated population. Time trends in the yearly procedure number and rate were analysed using negative binomial regression models with comparisons made by age and sex. RESULTS: The number of new ICD implantations increased from 708 to 3198 procedures between 2000 and 2009. Replacement procedures increased from 290 to 1378. The implantation rate (per million) increased from 37.0 to 145.6 and the replacement rate from 15.1 to 62.7. When rates were adjusted for age and sex, the implantation rate increased annually by 15.8% and the replacement rate by 16.6% (P < 0.0001). Procedures occurred most commonly in men (implantations: 80.1%; replacements: 78.0%) between ages 70-79. CONCLUSIONS: ICD procedures increased significantly in Australia between 2000-2009. Despite these increases, other studies have suggested ICD devices are currently under-utilised. During the study period, males accounted for the majority of ICD procedures. While there are numerous reasons for this, it is not known if device under-use is more common in females.

Adipocytokine zinc α2 glycoprotein (ZAG) as a novel urinary biomarker for normo-albuminuric diabetic nephropathy.

OBJECTIVE: A substantial proportion of diabetic nephropathy individuals are non-albuminuric. Using a proteomic approach, we searched for novel urinary biomarkers. METHODS: We studied three groups (n = 6 per group) of males with Type 2 diabetes: (1) normal renal function; (2) classical diabetic nephropathy (urinary albumin-creatinine ratio > 1000 mg/g and glomerular filtration rate < 60 ml/min.1.73 m(2) ) and (3) non-albuminuric diabetic nephropathy (glomerular filtration rate < 60 ml/min.1.73 m(2) and urinary albumin-creatinine ratio < 30 mg/g). We used two-dimensional fluorescence differential gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide identification and western blot validation in the study. RESULTS: Sixty protein spots were differentially abundant between the non-albuminuric and macro-albuminuric subjects (> 2.5-fold, P < 0.05). In the non-albuminuric subjects, in addition to previously reported α(1) -microglobulin, the next most interesting spot (upregulated 3.44-fold, P = 0.0026) was human zinc-α(2) -glycoprotein, a novel adipose-cytokine associated with glomerular injury. This was confirmed by western blot and replicated in female diabetic nephropathy subjects. CONCLUSIONS: From our preliminary results, human zinc-α(2) -glycoprotein may be a novel urinary biomarker for non-albuminuric diabetic nephropathy.

HLA-G polymorphisms, genetic susceptibility, and clinical outcome in childhood neuroblastoma.

Neuroblastoma is the most common solid tumor in children less than 5 years of age. The early onset of neuroblastoma suggests that genes involved in fetal development and pregnancy may have a putative role in the etiology of neuroblastoma. The human leukocyte antigen subtype G (HLA-G) molecule plays an important role in immune response regulation and appears to regulate immune tolerance during early pregnancy as well as tumor immunosurveillance. Elevated levels of soluble HLA-G (sHLA-G) have been detected in a number of malignancies including serum samples from neuroblastoma and have been reported to be predictive of tumor relapse in neuroblastoma. In light of previous investigations suggesting that single nucleotide polymorphisms in the HLA-G gene may impact on protein expression levels and isoform production, we examined the influence of HLA-G polymorphisms on the susceptibility and clinical outcome of neuroblastoma in 163 neuroblastoma patients and 404 healthy controls. The distribution of HLA-G polymorphisms, alleles, or allelic groups did not differ between children diagnosed with neuroblastoma and healthy controls. Our analyses did not detect an association between common HLA-G polymorphisms and clinical outcome in patients treated for neuroblastoma.

A rapid infusion protocol is safe for total dose iron polymaltose: time for change.

BACKGROUND: Intravenous correction of iron deficiency by total dose iron polymaltose is inexpensive and safe, but current protocols entail prolonged administration over more than 4 h. This results in reduced patient acceptance, and hospital resource strain. We aimed to assess prospectively the safety of a rapid intravenous protocol and compare this with historical controls. METHODS: Consecutive patients in whom intravenous iron replacement was indicated were invited to have up to 1.5 g iron polymaltose by a 58-min infusion protocol after an initial 15-min test dose without pre-medication. Infusion-related adverse events (AE) and delayed AE over the ensuing 5 days were also prospectively documented and graded as mild, moderate or severe. RESULTS: One hundred patients, 63 female, mean age 54 (range 18-85) years were studied. Thirty-four infusion-related AE to iron polymaltose occurred in a total of 24 patients--25 mild, 8 moderate and 1 severe; higher than previously reported for a slow protocol iron infusion. Thirty-one delayed AE occurred in 26 patients--26 mild, 3 moderate and 2 severe; similar to previously reported. All but five patients reported they would prefer iron replacement through the rapid protocol again. The presence of inflammatory bowel disease (IBD) predicted infusion-related reactions (54% vs 14% without IBD, P < 0.001) and the serum C-reactive protein was higher in those with reactions (P = 0.043). CONCLUSION: Iron polymaltose can be successfully administered using a rapid total dose infusion protocol and was well accepted by patients. It offers significant cost, resource utilization and time benefits for the patient and hospital system.

New role for L-arginine in regulation of inducible nitric-oxide-synthase-derived superoxide anion production in raw 264.7 macrophages.

Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O(2)(∙-) formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O(2)(∙-) and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O(2)(∙-) soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O(2) (∙-) formation in inflammatory macrophages.

DisCoTune: versatile auxiliary plasmids for the production of disulphide-containing proteins and peptides in the E. coli T7 system.

Secreted proteins and peptides hold large potential both as therapeutics and as enzyme catalysts in biotechnology. The high stability of many secreted proteins helps maintain functional integrity in changing chemical environments and is a contributing factor to their commercial potential. Disulphide bonds constitute an important post-translational modification that stabilizes many of these proteins and thus preserves the active state under chemically stressful conditions. Despite their importance, the discovery and applications within this group of proteins and peptides are limited by the availability of synthetic biology tools and heterologous production systems that allow for efficient formation of disulphide bonds. Here, we refine the design of two DisCoTune (Disulphide bond formation in E. coli with tunable expression) plasmids that enable the formation of disulphides in the highly popular Escherichia coli T7 protein production system. We show that this new system promotes significantly higher yield and activity of an industrial protease and a conotoxin, which belongs to a group of disulphide-rich venom peptides from cone snails with strong potential as research tools and pharmacological agents.

Contribution of gut bacteria to the metabolism of the spleen tyrosine kinase (Syk) inhibitor R406 in cynomolgus monkey.

The spleen tyrosine kinase (Syk) inhibitor R406 is orally administered as the prodrug R788. Following administration of R788 (12.5 mg kg(-1), 20 microCi kg(-1 14)C-R788) to intact and bile duct-cannulated cynomolgus monkeys, drug-related radioactivity was rapidly observed in plasma. No R788 was observed in plasma, while R406 was the major radioactive peak observed at all time points. Only low levels of metabolites were observed in plasma. The half-life for plasma radioactivity was 2.0-2.8 h. The majority (68.9%) of drug-related radioactivity was eliminated into bile. No intact R406 was observed in excreta. Biliary and urinary metabolites consisted of glucuronide and sulfate conjugates of the para-O-demethylated metabolite of R406 (R529), and a direct N-glucuronide of R406. The major metabolite in faeces from intact and bile duct-cannulated monkeys was a unique 3,5-benzene diol metabolite of R406. This metabolite was formed following the sequential O-demethylation and para-dehydroxylation of R529 by anaerobic gut bacteria.