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.

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.

Taxonomic Status and Population Oscillations of Aphidius colemani Species Group (Hymenoptera: Braconidae) in Southern Brazil.

Aphidius colemani (Viereck) was reported in Brazil before the Biological Control Program of Wheat Aphids (BCPWA) when Mediterranean genotypes were introduced from France and Israel. This species was re-described as a complex called A. colemani group composed of three species. Consequently, uncertainty remains about which parasitoid of the group is occurring in southern Brazil. This study has two main objectives: (i) re-examine the species status of A. colemani group collected during the introduction of parasitoids and from a 10-year (2009-2018) monitoring program in wheat fields in northern Rio Grande do Sul (RS), Brazil; (ii) describe the variation in the population density of parasitoids and its association with meteorological factors during this period. We examined 116 specimens from the Embrapa Wheat entomological collection, and those collected in Moericke traps in Coxilha, RS. All the parasitoids of the A. colemani group from the BCPWA period were identified as Aphidius platensis (Brèthes). In traps, 6541 cereal aphid parasitoids were collected, of which 61.9% (n = 4047) were from A. colemani group and all those were identified as A. platensis. Temperature was the factor that effected population density with the highest number of parasitoids recorded in the winter months. Sex ratio changed between years varying from 0.50 to 0.97. The parasitoid A. platensis was the only species in the A. colemani group sampled during 10 years of monitoring.

Alginate Trisaccharide Binding Sites on the Surface of ß-Lactoglobulin Identified by NMR Spectroscopy: Implications for Molecular Network Formation.

ß-lactoglobulin (BLG) is a promiscuous protein in terms of ligand interactions, having several binding sites reported for hydrophobic biomolecules such as fatty acids, lipids, and vitamins as well as detergents. BLG also interacts with neutral and anionic oligo- and polysaccharides for which the binding sites remain to be identified. The multivalency offered by these carbohydrate ligands is expected to facilitate coacervation, an electrostatically driven liquid-liquid phase separation. Using heteronuclear single quantum coherence NMR spectroscopy and monitoring chemical shift perturbations, we observed specific binding sites of modest affinity for alginate oligosaccharides (AOSs) prepared by alginate lyase degradation. Two different AOS binding sites (site 1 and site 2) centered around K75 and K101 were identified for monomeric BLG isoform A (BLGA) at pH 2.65. In contrast, only site 1 around K75 was observed for dimeric BLGA at pH 4.0. The data suggest a pH-dependent mechanism whereby both the BLGA dimer-monomer equilibrium and electrostatic interactions are exploited. This variability allows for control of coacervation and particle formation of BLGA/alginate mixtures via directed polysaccharide bridging of AOS binding sites and has implication for molecular network formation. The results are valuable for design of polyelectrolyte-based BLG particles and coacervates for carrying nutraceuticals and modulating viscosity in dairy products by use of alginates.

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.

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.

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.

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.

Magnetic field-induced enhancement of the nitrogen-vacancy fluorescence quantum yield.

The nitrogen-vacancy (NV) centre in diamond is a unique optical defect that is used in many applications today and methods to enhance its fluorescence brightness are highly sought after. We observed experimentally an enhancement of the NV quantum yield by up to 7% in bulk diamond caused by an external magnetic field relative to the field-free case. This observation is rationalised phenomenologically in terms of a magnetic field dependence of the NV excited state triplet-to-singlet transition rate. The theoretical model is in good qualitative agreement with the experimental results at low excitation intensities. Our results significantly contribute to our fundamental understanding of the photophysical properties of the NV defect in diamond.

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.

Bright and photostable nitrogen-vacancy fluorescence from unprocessed detonation nanodiamond.

Bright and photostable fluorescence from nitrogen-vacancy (NV) centers is demonstrated in unprocessed detonation nanodiamond particle aggregates. The optical properties of these particles is analyzed using confocal fluorescence microscopy and spectroscopy, time resolved fluorescence decay measurements, and optically detected magnetic resonance experiments. Two particle populations with distinct optical properties are identified and compared to high-pressure high-temperature (HPHT) fluorescent nanodiamonds. We find that the brightness of one detonation nanodiamond particle population is on the same order as that of highly processed fluorescent 100 nm HPHT nanodiamonds. Our results may open the path to a simple and up-scalable route for the production of fluorescent NV nanodiamonds for use in bioimaging applications.

Long-term lamivudine therapy in chronic hepatitis B.

BACKGROUND: One to 5 years of therapy of chronic hepatitis B with oral nucleoside analogues result in significant clinical improvements, but effects of more prolonged therapy are not well defined. AIM: To describe outcomes of chronic hepatitis B with long-term lamivudine therapy. METHODS: Forty-two patients with chronic hepatitis B treated with lamivudine were followed for 3.2-19.5 (median = 16.1) years. Therapy was switched to other agents (n = 16) if patients developed lamivudine resistance and relapse of disease. RESULTS: Among 22 HBeAg-positive patients, 17 (77%) became HBeAg negative, of whom 5 (23%) subsequently cleared HBsAg. Among 20 HBeAg-negative patients, 10 (50%) cleared HBsAg. The time to HBsAg clearance ranged from 0.9 to 16.8 (median = 9.3) years. Lamivudine resistance arose in 24 patients (57%) of whom 6 (25%) lost HBsAg. HBsAg clearance was not always accompanied by seroconversion; anti-HBs appearing concurrently in only five patients (33%). Nevertheless, HBsAg loss allowed for stopping therapy in all patients, none re-developing HBsAg or suffering relapse; all having normal alanine aminotransferase levels and no (n = 13) or unquantifiable HBV DNA levels (n = 2) when last seen. In contrast, seven of 27 patients (26%) who remained HBsAg-positive died of liver disease or liver cancer or underwent liver transplantation, all of whom had cirrhosis. CONCLUSIONS: Long-term viral suppression with nucleoside analogues leads to HBsAg loss in a substantial proportion of patients, particularly if HBeAg-negative. Serious outcomes during the first 10-20 years of treatment occur largely among patients with pre-existing cirrhosis who do not clear HBsAg with therapy.

Green lasers are beyond power limits mandated by safety standards.

There has been an increasing number of reports of people losing vision from laser exposure from pocket laser pointers despite the safety limit of 1 milliwatt (1mW) imposed by the Australian government. We hypothesize that this is because commercially available red and green laser pointers are exceeding their labeled power outputs. We tested the power outputs of 4 red and 4 green lasers which were purchased for less than AUD$30 each. The average of 10 measurements was recorded for each laser. We found that 3 out of 4 red lasers conformed to the 1mW safety standard; in contrast, all of the green lasers exceeded this limit, with one of the lasers recording an output of 127.9 mW. This contrast in compliance is explained by the construction of these lasers - green lasers are typically Diode Pumped Solid State (DPSS) lasers that can emit excessive infrared (IR) radiation with poor workmanship or inconsistent adherence to practices of safe design and quality control; red lasers are diode lasers which have limited power outputs due to `Catastrophic Optical Damage' (COD). Relevant professional bodies ought to advocate more strongly for stringent testing, quality control and licensing of DPSS lasers with a view towards government intervention to banning green laser pointer use.

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.

Retinal screening of patients with cystic fibrosis-related diabetes in Wales -- a real eye opener.

BACKGROUND: The National Screening Committee recommends annual retinal screening for all diabetic patients over the age of twelve years. Currently, patients on insulin therapy for Cystic Fibrosis-Related Diabetes Mellitus (CFRD) at the All Wales Adult Cystic Fibrosis Service, UK are invited for annual screening. METHODS: Age, sex, FEV(1)%, BMI, HbA1c, duration of diabetes, duration of insulin therapy, presence of retinopathy and attendance at retinal screening in 2012 were examined for patients on insulin therapy for CFRD. RESULTS: 67 of the 228 (29%) patients attending the CF centre were receiving insulin therapy. 24 of the 67 (36%) did not attend retinal screening in 2012. Of the 43 who had retinal scans, 18 (42%) had evidence of retinopathy. Patients with retinopathy had a higher mean HbA1c (p=0.04), mean duration of diabetes and mean duration on insulin (p<0.001). CONCLUSIONS: Almost half of the patients screened had evidence of retinopathy but over a third of the patients with CFRD did not attend screening appointments. Improving patient uptake of retinal scans will become increasingly important in an ageing CF population.

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.

Hyperpharmacotherapy in ageing cystic fibrosis patients: The first report of an atypical hip fracture.

Cystic fibrosis (CF) is a common autosomal recessive disorder in Caucasian populations with respiratory, gastrointestinal and endocrine manifestations. Thanks to recent advances in medical therapies and infection control, life expectancy of a patient with CF has significantly increased from less than 5 years in the mid-1900s to almost 50 years nowadays. However, as CF patients are living longer, multimorbidity and Hyperpharmacotherapy are becoming more common. This case illustrates a cascade of problems that ensued from medication side-effects, highlighting the increasing challenge of managing an ageing CF population.