Distinct disulfide isomers of μ-Conotoxins KIIIA and KIIIB block voltage-gated sodium channels

In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1–C9,C2–C15,C4–C16] disulfide pattern based on homology with closely related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1–C15,C2–C9,C4–C16] disulfide connectivity, while the minor product adopts a [C1–C16,C2–C9,C4–C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd values of 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1–C15,C2–C9,C4–C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1–C9,C2–C15,C4–C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as μ-KIIIA, and both blocked NaV1.2 (Kd values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA and μ-KIIIB folded in vitro is 1–5/2–4/3–6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail’s repertoire of active peptides.

Exposure to a high-fat diet alters leptin sensitivity and elevates renal sympathetic nerve activity and arterial pressure in rabbits

The activation of the sympathetic nervous system through the central actions of the adipokine leptin has been suggested as a major mechanism by which obesity contributes to the development of hypertension. However, direct evidence for elevated sympathetic activity in obesity has been limited to muscle. The present study examined the renal sympathetic nerve activity and cardiovascular effects of a high-fat diet (HFD), as well as the changes in the sensitivity to intracerebroventricular leptin. New Zealand white rabbits fed a 13.5% HFD for 4 weeks showed modest weight gain but a 2- to 3-fold greater accumulation of visceral fat compared with control rabbits. Mean arterial pressure, heart rate, and plasma norepinephrine concentration increased by 8%, 26%, and 87%, respectively (P<0.05), after 3 weeks of HFD. Renal sympathetic nerve activity was 48% higher (P<0.05) in HFD compared with control diet rabbits and was correlated to plasma leptin (r=0.87; P<0.01). Intracerebroventricular leptin administration (5 to 100 μg) increased mean arterial pressure similarly in both groups, but renal sympathetic nerve activity increased more in HFD-fed rabbits. By contrast, intracerebroventricular leptin produced less neurons expressing c-Fos in HFD compared with control rabbits in regions important for appetite and sympathetic actions of leptin (arcuate: −54%, paraventricular: −69%, and dorsomedial hypothalamus: −65%). These results suggest that visceral fat accumulation through consumption of a HFD leads to marked sympathetic activation, which is related to increased responsiveness to central sympathoexcitatory effects of leptin. The paradoxical reduction in hypothalamic neuronal activation by leptin suggests a marked “selective leptin resistance” in these animals.

Retinal nerve fibre layer thinning associated with diabetic peripheral neuropathy

Aims
To investigate the relationship between retinal nerve fibre layer thickness and peripheral neuropathy in patients with Type 2 diabetes, particularly in those who are at higher risk of foot ulceration.

Methods
Global and sectoral retinal nerve fibre layer thicknesses were measured at 3.45 mm diameter around the optic nerve head using optical coherence tomography (OCT). The level of neuropathy was assessed in 106 participants (82 with Type 2 diabetes and 24 healthy controls) using the 0–10 neuropathy disability score. Participants were stratified into four neuropathy groups: none (0–2), mild (3–5), moderate (6–8), and severe (9–10). A neuropathy disability score ‡ 6 was used to define those at higher risk of foot ulceration. Multivariable regression analysis was performed to assess the effect of neuropathy disability scores, age, disease duration and retinopathy on RNFL thickness.

Results
Inferior (but not global or other sectoral) retinal nerve fibre layer thinning was associated with higher neuropathy disability scores (P = 0.03). The retinal nerve fibre layer was significantly thinner for the group with neuropathy disability scores ‡ 6 in the inferior quadrant (P < 0.005). Age, duration of disease and retinopathy levels did not significantly influence retinal nerve fibre layer thickness. Control participants did not show any significant differences in thickness measurements from the group with diabetes and no neuropathy (P > 0.24 for global and all sectors).

Conclusions
Inferior quadrant retinal nerve fibre layer thinning is associated with peripheral neuropathy in patients with Type 2 diabetes, and is more pronounced in those at higher risk of foot ulceration.

Permian fusuline fauna from the lower part of the lugu formation in the central qiangtang block and its geological implications

A Kubergandian (Kungurian) fusuline fauna from the lower part of the Lugu Formation in the Cuozheqiangma area, central Qiangtang Block is described. This fusuline fauna belongs to the Southern Transitional Zone in palaeobiogeography, and is characterised by the presence of the distinctive bi-temperate genus Monodiexodina and many genera common in lower latitude Tethyan areas such as Parafusulina and Pseudodoliolina. The occurrence of Monodiexodina in the fauna confirms that the seamount-type carbonates of the Lugu Formation did not originate from the Palaeotethys Ocean, but rather from a branch of the Neotethys Ocean after the rifting of the Qiangtang Block from the Tethys Himalaya area in the Artinskian. © 2014 Geological Society of China.

A block-aware hybrid data dissemination with hotspot elimination in wireless sensor network

As a significant milestone in the data dissemination of wireless sensor networks (WSNs), the comb-needle (CN) model was developed to dynamically balance the sensor data pushing and pulling during hybrid data dissemination. Unfortunately, the hybrid push-pull data dissemination strategy may overload some sensor nodes and form the hotspots that consume energy significantly. This usually leads to the collapse of the network at a very early stage. In the past decade, although many energy-aware dynamic data dissemination methods have been proposed to alleviate the hotspots issue, the block characteristic of sensor nodes has been overlooked and how to offload traffic from hot blocks with low energy through long-distance hybrid dissemination remains an open problem. In this paper, we developed a block-aware data dissemination model to balance the inter-block energy and eliminate the spreading of intra-block hotspots. Through the clustering mechanism based on geography and energy, "similar" large-scale sensor nodes can be efficiently grouped into specific blocks to form the global block information (GBI). Based on GBI, the long-distance block-cross hybrid algorithms are further developed by effectively aggregating inter-block and intra-block data disseminations. Extensive experimental results demonstrate the capability and the efficiency of the proposed approach. © 2014 Elsevier Ltd.

Self-assembled multimicellar vesicles via complexation of a rigid conjugated polymer with an amphiphilic block copolymer

A viable method of encapsulating block copolymer micelles inside vesicles using a conjugated polymer is reported in this study. Self-assembly and complexation between an amphiphilic block copolymer poly(methyl methacrylate)-b-poly(acrylic acid) (PMMA-b-PAA) and a rod-like conjugated polymer polyaniline (PANI) in aqueous solution were studied using transmission electron microscopy, atomic force microscopy and dynamic light scattering. The complexation and morphology transformation were driven by electrostatic interaction between PANI and the PAA block of the block copolymer. Addition of PANI to PMMA-b-PAA induced the morphology transformation from micelles to irregular vesicles through vesicles, thick-walled vesicles (TWVs) and multimicellar vesicles (MMVs). Among the observed morphologies, MMVs were observed for the first time. Morphology transformation was studied as a function of aniline/acrylic acid molar ratio ([ANI]/[AA]). Micelles were observed for the pure block copolymer, while vesicles and TWVs were observed at [ANI]/[AA] = 0.1 and 0.3, respectively. MMVs were observed at [ANI]/[AA] = 0.5 and irregular vesicles were observed for molar ratios at 0.7 and above. Clearly, a conjugated polymer like polyaniline can induce a morphology transformation even at its lower concentrations and produce complex morphologies.

The effect of known cardiovascular risk factors on carotid-femoral pulse wave velocity in school-aged children: a population based twin study

Childhood cardiovascular risk factors affect vascular function long before overt cardiovascular disease. Twin studies provide a unique opportunity to examine the influence of shared genetic and environmental influences on childhood cardiovascular function. We examined the relationship between birth parameters, markers of adiposity, insulin resistance, lipid profile and blood pressure and carotid-femoral pulse wave velocity (PWV), a validated non-invasive measure of arterial stiffness in a healthy cohort of school-aged twin children. PWV was performed on a population-based birth cohort of 147 twin pairs aged 7-11 years. Fasting blood samples, blood pressure and adiposity measures were collected concurrently. Mixed linear regression models were used to account for twin clustering, within- and between-twin pair associations. There were positive associations between both markers of higher adiposity, insulin resistance, elevated triglycerides and PWV, which remained significant after accounting for twin birth-set clustering. There was a positive association between both diastolic and mean arterial blood pressure and PWV in within-pair analysis in dizygotic, but not monozygotic twins, indicating genetic differences evident in dizygotic not monozygotic twins may affect these associations. Increased blood pressure, triglycerides and other metabolic markers are associated with increased PWV in school-aged twins. These results support both the genetic and environmental contribution to higher PWV, as a marker of arterial stiffness, and reiterate the importance of preventing metabolic syndrome from childhood.

Large compound vesicles from amphiphilic block copolymer/rigid-rod conjugated polymer complexes

Morphology evolution in complexes of amphiphilic block copolymers poly(styrene)-b-poly(acrylic acid) (PS-b-PAA) and poly(styrene)-b-poly(ethylene oxide) (PS-b-PEO) in the presence of polyaniline (PANI) in aqueous solution is reported. Transmission electron microscopy, atomic force microscopy, and dynamic light scattering techniques were used to study the morphologies at various PANI contents [aniline]/[acrylic acid] ([ANI]/[AA]) ranging from 0.1 to 0.7. The interpolyelectrolyte complex formed between PAA and PANI plays a key role in the morphology transformation. Spherical micelles formed from pure block copolymers were transformed into large compound vesicles upon increasing PANI concentration due to internal block copolymer segregation. In addition to varying PANI content, the kinetic pathway of nanoparticle formation was controlled through different water addition methods and was critical in the formation of multigeometry nanoparticles.

Peripheral injected cholecystokinin-8S modulates the concentration of serotonin in nerve fibers of the rat brainstem

Serotonin and cholecystokinin (CCK) play a role in the short-term inhibition of food intake. It is known that peripheral injection of CCK increases c-Fos-immunoreactivity (Fos-IR) in the nucleus of the solitary tract (NTS) in rats, and injection of the serotonin antagonist ondansetron decreases the number of c-Fos-IR cells in the NTS. This supports the idea of serotonin contributing to the effects of CCK. The aim of the present study was to elucidate whether peripherally injected CCK-8S modulates the concentration of serotonin in brain feeding-regulatory nuclei. Ad libitum fed male Sprague-Dawley rats received 5.2 and 8.7 nmol/kg CCK-8S (n = 3/group) or 0.15 M NaCl (n = 3-5/group) injected intraperitoneally (ip). The number of c-Fos-IR neurons, and the fluorescence intensity of serotonin in nerve fibers were assessed in the paraventricular nucleus (PVN), arcuate nucleus (ARC), NTS and dorsal motor nucleus of the vagus (DMV). CCK-8S increased the number of c-Fos-ir neurons in the NTS (mean ± SEM: 72 ± 4, and 112 ± 5 neurons/section, respectively) compared to vehicle-treated rats (7 ± 2 neurons/section, P < 0.05), but did not modulate c-Fos expression in the DMV or ARC. Additionally, CCK-8S dose-dependently increased the number of c-Fos-positive neurons in the PVN (218 ± 15 and 128 ± 14, respectively vs. 19 ± 5, P < 0.05). In the NTS and DMV we observed a decrease of serotonin-immunoreactivity 90 min after injection of CCK-8S (46 ± 2 and 49 ± 8 pixel/section, respectively) compared to vehicle (81 ± 8 pixel/section, P < 0.05). No changes of serotonin-immunoreactivity were observed in the PVN and ARC. Our results suggest that serotonin is involved in the mediation of CCK-8's effects in the brainstem. © 2014 Elsevier Inc.

Nerve guidance conduits from aligned nanofibers: improvement of nerve regeneration through longitudinal nanogrooves on a fiber surface

A novel fibrous conduit consisting of well-aligned nanofibers with longitudinal nanogrooves on the fiber surface was prepared by electrospinning and was subjected to an in vivo nerve regeneration study on rats using a sciatic nerve injury model. For comparison, a fibrous conduit having a similar fiber alignment structure without surface groove and an autograft were also conducted in the same test. The electrophysiological, walking track, gastrocnemius muscle, triple-immunofluorescence, and immunohistological analyses indicated that grooved fibers effectively improved sciatic nerve regeneration. This is mainly attributed to the highly ordered secondary structure formed by surface grooves and an increase in the specific surface area. Fibrous conduits made of longitudinally aligned nanofibers with longitudinal nanogrooves on the fiber surface may offer a new nerve guidance conduit for peripheral nerve repair and regeneration.

Graphene/tri-block copolymer composites prepared via RAFT polymerizations for dual controlled drug delivery via pH stimulation and biodegradation

A novel tri-block copolymer poly(oxopentanoate ethyl methacrylate)-block-poly(pyridyl disulfide ethyl acrylate)-block-poly(ethylene glycol acrylate) [poly(OEMA-b-PDEA-b-PEGA)], retaining active keto groups and pyridyl disulfide (PDS) side functionalities, was synthesized as a drug delivery vehicle using reversible addition-fragmentation chain transfer (RAFT) polymerization method. One mimic drug pyridine-2-thione (PT) was introduced into the monomer, PDEA for copolymerization. The other mimic drug O-benzylhydroxylamine (BHA) was conjugated with tri-block copolymer via efficient oxime coupling chemistry, followed by the attachment onto graphene via π-π stacking interaction to obtain a graphene/tri-block copolymer composite. 1H NMR, UV-vis absorption spectroscopy, fluorescence spectroscopy, gel permeation chromatography (GPC), atomic force microscope (AFM) and transmission electron microscope (TEM) were used to verify the successful step-wise preparation of the tri-block copolymer and drug loaded composite. In vitro release behaviors of BHA and PT from graphene/tri-block copolymer composite via dual drug release mechanisms were investigated. BHA can be released under acid environment, while PT will be released in the presence of reducing agents, such as dithiothreitol (DTT) or glutathione (GSH). It can be envisioned that this novel composite could be exploited as a novel intracellular drug delivery system via dual release mechanisms.