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The purpose of this research was to investigate the effects of replacing soybean meal (SBM) with a fermented corn gluten-wheat bran mixture (FCWM) on nutrient digestibility, lactation performance, plasma metabolites, ruminal fermentation, and bacterial communities in Holstein cows. Nine healthy multiparous (parity = 3) Holstein cows with similar body weights (624 ± 14.4 kg), days in milk (112 ± 4.2), and milk yields (31.8 ± 1.73 kg; all mean ± standard deviation) were used in a replicated 3 × 3 Latin square design with 3 periods of 28 d. Cows were fed 1 of 3 dietary treatments in which FCWM replaced SBM as follows: basal diet with no replacement (0FCWM); 50% replacement of SBM with FCWM (50%FCWM); and 100% replacement of SBM with FCWM (100%FCWM). The diets were formulated to be isocaloric and isonitrogenous. The results showed that the total-tract digestibility of dry matter and crude protein increased linearly with increased dietary FCWM, and we found a trend for increased total-tract neutral detergent fiber and potentially digestible NDF digestibility. Milk yield tended to increase in a linear manner as more FCWM was consumed, and energy-corrected milk production was significantly increased with FCWM supplementation as a result of increased milk protein and lactose yields. Plasma glucose and IgG concentrations increased linearly with increasing FCWM supplementation, but plasma malondialdehyde concentration decreased linearly. Concentrations of total volatile fatty acids and propionate showed a linear increase with increasing FCWM supplementation, leading to a linear decrease in pH. The relative abundance of ruminal Prevotellaceae, Veillonellaceae, and Prevotella 1 increased linearly with increasing FCWM supplementation, and the relative abundance of ruminal Succinivibrionaceae and Muribaculaceae decreased linearly. The relative abundance of fecal Ruminococcaceae, Prevotellaceae, and Ruminococcaceae UCG-005 increased linearly with increasing FCWM supplementation, but the relative abundance of fecal Peptostreptococcaceae decreased linearly. Overall, the replacement of SBM with FCWM altered the composition of the ruminal bacterial community and improved nutrient digestibility, lactation performance, and ruminal fermentation in cows, providing a data reference for the use of FCWM in dairy production.

A blastproof partition wall in civil air defense engineering is easily subjected to out-of-plane impact loads, such as object impacts, and structural collapse in peacetime. In this study, the pendulum impact test was carried out for the first time on six blastproof partition walls pasted with fiber-reinforced polymer (FRP). Using a method combining finite element simulation and experimental research, the influence of parameters such as the FRP type, layer number and width, and pendulum impact number on the impact resistance of the wall was systematically studied. The test results show that pasting FRP on the back of the blastproof partition wall can significantly improve the impact resistance and reduce the damage degree of the concrete. The impact recovery coefficient increased from 0.33 to 0.57, but with the increase in the number of impacts, large-rupture-strain FRP (LRS-FRP) can give full play to the advantages of a large tensile fracture strain and cooperate with the wall to deform, and the selection of FRP with a large tensile fracture strain is the key to resisting multiple impact loads. Finally, the experimental basis and an economical and reliable protection method are provided for the study of improving the impact resistance of the blastproof partition wall.

Little information is available regarding the effect of supplementing 25-hydroxyvitamin D3 during the transition period combined with a postpartum oral calcium bolus on Ca homeostasis. The objectives of the current study were to evaluate the effects of 25-hydroxyvitamin D3 combined with postpartum oral calcium bolus on lactation performance, serum minerals and vitamin D3 metabolites, blood biochemistry, and antioxidant and immune function in multiparous dairy cows. To evaluate the effects of 25-hydroxyvitamin D3 combined with oral calcium, 48 multiparous Holstein cows were randomly assigned to one of four treatments: (1) supplementing 240 mg/day vitamin D3 without a postpartum oral Ca bolus (control), (2) supplementing 240 mg/day vitamin D3 with an oral Ca bolus containing 90 g of Ca immediately post-calving (Ca + VitD), (3) supplementing 6 g/day 25-hydroxyvitamin D3 without an oral Ca bolus (25D), and (4) supplementing 6 g/day 25-hydroxyvitamin D3 with an oral Ca bolus containing 90 g of Ca immediately post-calving (Ca + 25D). Lactation performance during the first 21 days was measured. Blood was collected at the initiation of calving and then 1, 2, 7, 14, and 21 days relative to the calving date. The yield of milk (0.05 &lt, p &lt, 0.10), energy-corrected milk (p &lt, 0.05), 3.5% fat-corrected milk (p &lt, 0.05), and milk protein (p &lt, 0.05) were significantly higher in 25-hydroxyvitamin D3-treated groups within 3 weeks of lactation than in vitamin D3-treated cows. The iCa (p &lt, 0.05) and tCa (p &lt, 0.05) were higher in both Ca and 25D + Ca cows than in the control and 25D groups within 48 h. The concentrations of serum tCa (p &lt, 0.05), tP (p &lt, 0.05), and 25-hydroxyvitamin D3 (p &lt, 0.05) in 25D and 25D + Ca cows were higher than those in control and Ca cows within 21 days postpartum. Feeding 25-hydroxyvitamin D3 also showed a lower concentration of malondialdehyde (p &lt, 0.05), interleukin 6 (p &lt, 0.05), and tumor necrosis factor-alpha (TNF-α) (p &lt, 0.05), as well as a higher concentration of alkaline phosphatase (p &lt, 0.05), total antioxidant capacity (p &lt, 0.05), and immunoglobulin G (p &lt, 0.05) than vitamin D3. Supplementing Ca bolus also showed lower concentrations of alanine transaminase (p &lt, 0.05) and TNF-α (p &lt, 0.05). In conclusion, supplementing 25-hydroxyvitamin D3 during the transition period combined with a postpartum oral calcium bolus improved lactation performance, Ca homeostasis, and antioxidant and immune function of medium-production dairy cows within 21 days postpartum.

Background As a major disease affecting dairy cow production worldwide, bovine mastitis is caused by a variety of pathogenic microorganisms that eventually cause mammary gland inflammation. Acremonium terricola culture (ATC) is a new type of affordable feed additive produced by the solid fermentation of A. terricola isolated from Cordyceps gunnii and exerted its anti-inflammatory effect. Objectives To evaluate the protective effects of ATC on mastitis and investigate its active mechanism, a lipopolysaccharide (LPS)-induced rat mastitis model was used in two experiments. Design In Experiment 1, a total of 40 female Sprague-Dawley rats were used to determine the optimal supplementary dose of ATC via gavage trial. In Experiment 2, we examined the effects of an optimal dose of ATC on LPS-induced mastitis in rats. Results The results of Experiment 1 showed that administration of ATC improved growth performance and antioxidant functions in the serum and the liver, as well as immunoglobulin A, G, and M levels in rat serum, and it decreased the content of alanine aminotransferase, aspartate aminotransferase, triglyceride, cholesterol, low-density lipoprotein, and serum urea nitrogen in rat serum; a dosage of 250-1,250 mg/kg/day was shown to be high enough to be effective. The results of Experiment 2 indicated that ATC can relieve the inflammatory reaction of mammary glands in rats, and the LPS-induced expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and inducible nitric oxide synthase significantly decreased after ATC treatment. Moreover, our results demonstrated that ATC markedly enhanced the activity of antioxidase in this rat mastitis model. The results of Western blot analysis revealed that ATC could suppress the expression of toll-like receptor 4, phosphorylation of extracellular signal-regulated kinase, and activity of c-Jun N-terminal kinase in the LPS-stimulated mastitis model. Conclusion Taken together, ATC was shown to exert its anti-inflammatory effect by blocking mitogen-activated protein kinase signaling pathways. These results demonstrate that ATC exerts anti-inflammatory and antioxidant effects in mastitis prevention.

The effects of Acremonium terricola culture (ATC) on performance, rumen fermentation and microbiota, and antioxidant and immune functions in dairy cows were investigated in two experiments. In Experiment 1, thirty multiparous Holstein dairy cows were blocked for day in milk, somatic cell counts and milk production and were randomly assigned to 1 of 3 treatments in an 8-week trial: a basal diet plus 0, 15 or 30 g/d ATC. The result of supplementing dairy cows with ATC was a linear increase in the apparent digestibility of dry matter, crude protein, acid detergent fiber and neutral detergent fiber, milk production, 4% fat-corrected milk, energy-corrected milk, feed efficiency, milk fat and lactose yields, and milk fat percentage, but a linear decrease in the milk somatic cell count. In Experiment 2, five late-lactating dairy cows were fed the basal diet from d 1 to 14 (pre-trial period). From d 15 to 35 (trial period), the same cows were fed 30 g/d ATC. Supplementation with ATC was stopped from d 36 to 42 (post-trial period). Feed, rumen and blood samples were collected on d 13, 14, 34, 35, 41 and 42. Compared with the pre- and post-periods, the ruminal pH and molar proportion of butyrate decreased during the trial period. Additionally, the ammonia nitrogen and total volatile fatty acid concentrations as well as the molar proportion of acetate were increased. For most of the rumen bacteria that were measured, the relative expression level increased during the trial period, but that of Fibrobacter succinogenes decreased. Moreover, ATC significantly increased the total antioxidant capacity as well as the activities of glutathione peroxidase and total superoxide dismutase during the trial period. The concentrations of blood glucose and immunoglobulins A, M, and G also increased in the plasma. By contrast, ATC resulted in a significant reduction in the plasma concentrations of aspartate transaminase, non-esterified fatty acids, beta-hydroxybutyric acid, interleukin-1, interleukin-6, tumor necrosis factor-α and malondialdehyde. These results show that feeding ATC can increase performance, improve rumen fermentation, and increase antioxidant and immune functions in dairy cows, when fed up to 30 g/d.

The objective of this study was to evaluate the effect of supplementation of 25-hydroxyvitamin D3 (25(OH)VD3) instead of vitamin D3 on the quality and seminal plasma antioxidant indicators of fresh and cryopreserved semen, serum minerals, vitamin D3 status, hormone levels, blood biochemistry, antioxidant and immune function in different age of bulls. Thirty-two Holstein bulls (16 bulls aged 24–36 months and weighted 727 ± 99.0 kg; 16 bulls aged 12 months and weighted 332 ± 24.9 kg) randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement with 2 sources of vitamin D3 (vitamin D3 or 25(OH)VD3) fed at 300 IU/kg dry matter intake and two different age of bull (maturity or prepuberty) through a 60 day period. Bulls were tied in individual pens without access to sunlight. Bulls fed 25(OH)VD3 had greater semen concentration (P = 0.03), sperms motility (P = 0.02), viability (P

The objectives of this study were to evaluate the effects of milk replacers containing 25-hydroxyvitamin D3 [25(OH)D3] and enriched with vitamin D3 on the growth performance, fecal scores, vitamin D3 metabolites, biochemical blood parameters and antioxidant, inflammatory and stress-related parameters of Holstein male calves. Thirty Holstein male calves (body weight = 41.4 ± 2.79 kg) were randomly assigned to three groups for a 56-day experimental period. The control group received milk replacer containing 1.2 mg/kg vitamin D3, the VD group received milk replacer containing 13.2 mg/kg vitamin D3, and the 25D group received milk replacer containing 300 mg/kg 25(OH)D3 plus 1.2 mg/kg vitamin D3 until weaning at day 54. On days 52, 57 and 59, which were defined as days -2, 3 and 5 postweaning, blood samples were collected to measure biochemical blood parameters and antioxidant, inflammatory and stress-related indicators. Calves that received milk replacer containing 25(OH)D3 consumed more calf starter at one week postweaning (P 2 overall periods. Calves fed 25(OH)D3 had increased serum 25(OH)D3 during the preweaning (P

To improve the qualities of corn gluten meal, lactic acid bacteria (LAB) and acid protease were used for solid state fermentation and wheat bran was used as a carbon source supplement, and the nutritional value, antioxidant capacity and bioavailability were evaluated. The results showed that the contents of crude protein, ash, small peptide, free amino acids, total phenol and lactic acid in corn gluten-wheat bran mixture (CWM) significantly increased after fermentation, whereas the neutral detergent fiber, acid detergent fiber and starch contents in fermented CWM (FCWM) were lower than in CWM (P

The objectives of this study were to investigate the effects of different forage-to-concentrate ratios and sampling times on the genetic diversity of carbohydrate-active enzymes (CAZymes) and the taxonomic profile of rumen microbial communities in dairy cows. Six ruminally cannulated Holstein cows were arbitrarily divided into groups fed high-forage (HF) or low-forage (LF) diets. The results showed that, for glycoside hydrolase (GH) families, there were greater differences based on dietary forage-to-concentrate ratio than sampling time. The HF treatment group at 4 h after feeding (AF4h) had the most microbial diversity. Genes that encode GHs had the highest number of CAZymes, and accounted for 57.33% and 56.48% of all CAZymes in the HF and LF treatments, respectively. The majority of GH family genes encode oligosaccharide-degrading enzymes, and GH2, GH3, and GH43 were synthesized by a variety of different genera. Notably, we found that GH3 was higher in HF than LF diet samples, and mainly produced by Prevotella, Bacteroides, and unclassified reads. Most predicted cellulase enzymes were encoded by GH5 (the BF0h group under HF treatment was highest) and GH95 (the BF0h group under LF treatment was highest), and were primarily derived from Bacteroides, Butyrivibrio, and Fibrobacter. Approximately 67.5% (GH28) and 65.5% (GH53) of the putative hemicellulases in LF and HF treatments, respectively. GH28 under LF treatment was more abundant than under HF treatment, and was mainly produced by Ruminococcus, Prevotella, and Bacteroides. This study revealed that HF-fed cows had increased microbial diversity of CAZyme producers, which encode enzymes that efficiently degrade plant cell wall polysaccharides in the cow rumen.

Production of bioactive peptides from corn gluten meal (CGM) by solid-state fermentation (SSF) with Bacillus subtilis MTCC5480 (BS5480) and the antioxidant capacity in vivo were investigated in the present study. During the SSF process, CGM peptides (CGMP) production reached a maximum (369.4 mg/gdp) when the fermentation conditions were as follows: temperature, 36 °C; initial pH, 6.0; moisture, 46%; inoculation size, 5.8 × 109 spores/g; time, 5 d; peptone, 5 mg/g; and glucose, 10.7 mg/g. In addition, the d -galactose-induced aging rats fed CGMP (molecular weight

Quasi-static and dynamic compressive tests are undertaken on the polyurethane (PU) foam and fumed silica reinforced polyurethane (PU/SiO2) foam experimentally. The ceramic microspheres with varying mass fractions are adopted to mix with the PU/SiO2 foam to fabricate the composite particle-reinforced foams. The effects of strain rate and particle mass fraction are discussed to identify and quantify the compressive response, energy-absorbing characteristic, and the associated mechanisms of the composite foams. The results show the initial collapse strength and plateau stress of the foams are improved significantly by reinforcing with the ceramic microsphere within 60 wt% at quasi-static compression. The rate sensitivity is observed on all the foams, but in different patterns due to the influence of ceramic microsphere. The compressive response affected by ceramic microsphere can be attributed to the particle cluster effect and stress wave propagation. Together with the deformation, the compressive characteristic experiences non-monotonic change from the low to high strain rates. The specific energy absorption (SEA) of the foam with 41 wt% ceramic microsphere show the largest magnitude at quasi-static compression. With the increasing strain rate, the ceramic reinforced foam exhibits superior energy absorption efficiency at high strain rates to that of the pure foams.

The ceramic particle reinforced foam-filled carbon fiber reinforced composites sandwich unit cell subjected to quasi-static and dynamic compressions are studied experimentally. The weight fraction of the ceramic particle is discussed to analyze its effect on the failure and energy absorption of the composite cells. The results show that the failure modes and energy absorption mechanisms of the cell are influenced significantly by the strain rate and ceramic particle reinforcement effects. The cluster of the particle makes the strut experience node failure under impulsive loadings, not the strut buckling and fracture shown at other cells. The mechanical property, especially the plateau stress, is enhanced noticeably as the foam is reinforced with the higher weight fraction of ceramic particles. The filling foams improve the impact resistance of the cells by transmitting less impulses and absorbing more energy. With a higher energy absorption efficiency, the foam-filled cell with higher weight fraction of ceramic particle has the highest energy absorption capacity under the quasi-static compression. Under the impulsive loading, the increasing ceramic particles can reduce the transmitted impulse, but cannot absorb additional energy.

The purpose of this study was to evaluate the effects of different forage-to-concentrate (F:C) ratios (7:3 high-forage, 3:7 high-concentrate) on rumen microflora and fiber degradation mechanism. Compared with the high-concentrate (HC) group, the high-forage(HF) group showed improved fiber degradation and a sustained high level of carboxymethyl cellulose (CMCase), β-glucosidase and β-xylosidase activities, but the total VFAs decreased. Among bacteria at the family level,LachnospiraceaeandSuccinivibrionaceaein HF groups were 2-fold and 4-fold more abundant than in the HC group, respectively. A KEGG analysis revealed that succinate-CoA synthetase (EC: 6.2.1.5) and propionate-CoA transferase (EC: 2.8.3.1) leading directly to propionate production were more abundant in HC group. Conversely, butyryl-CoA dehydrogenase (EC: 1.3.8.1) was directly related to butyrate production and was higher in the HF group. A gene expression analysis showed that the relative content ofFibrobacter succinogenesandButyrivibrio fibrisolvenswas higher in the HF group and contributed more to fiber degradation and VFA production.Prevotella ruminicola, Selenomonas ruminantium, andVeillonella alkalescenscontributed more to starch degradation and propionate production, which relative content was higher in the HC group. This research gave a further explanation of the fiber degradation parameters and microbiota under different F:C ration. The fiber-degrading bacteria in the roughage group have a high content level, and the corresponding cellulase activity is also high. These results supported the potential of diets for microbial manipulation, which can increase feed digestibility and explored new fibrinolytic bacteria.IMPORTANCEThe forage of the cow’s feed occupies a large proportion. The shortage of high-quality forage in cow breeding has become an important factor limiting the China’s dairy industry. The effective measure is to improve the utilization of low-quality forage. Based on traditional nutrient metabolism, the reasons for the effects of roughage on the growth and metabolism of dairy cows can be explored, but the metabolic mechanism is not well analyzed, and the further utilization of forage is also limited. Metagenomics has proven to be a powerful tool for studying rumen microbial structures and gene function. This experiment used metagenomics to study the metabolism of cellulose and volatile acids in the rumen. Our research showed that different forage-to-concentrate shifted the composition of microorganisms and the activity of enzymes, resulting in different metabolic pathways of volatile fatty acids. This work provides a background for microbial community composition and further use of forage.

TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG+) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs+, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K+, suggesting that K+ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG+ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K+, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.

Atrial fibrillation (AF) is the most prevalent nonfatal cardiac rhythm disorder associated with an increased risk of heart failure and stroke. Considering the ventricular side effects induced by anti-arrhythmic agents in current use, K(v)1.5 channel blockers have attracted a great deal of deliberation owing to their selective actions on atrial electrophysiology. Herein we report new chemotypes of K(v)1.5 channel blockers that were identified through a combination of structure-based virtual screening and in silico druglike property prediction including six scoring functions, as well as electrophysiological evaluation. Among them, five of the 18 compounds exhibited >50 % blockade ratio at 10 microM, and have structural features different from conventional K(v)1.5 channel blockers. These novel scaffolds could serve as hits for further optimization and SAR studies for the discovery of selective agents to treat AF.

Long QT interval syndrome (LQTS) type 1 (LQT1) has been reported to arise from mutations in the S3 domain of KCNQ1, but none of the seven S3 mutations in the literature have been characterized with respect to trafficking or biophysical deficiencies. Surface channel expression was studied using a proteinase K assay for KCNQ1 D202H/N, I204F/M, V205M, S209F, and V215M coexpressed with KCNE1 in mammalian cells. In each case, the majority of synthesized channel was found at the surface, but mutant I(Ks) current density at +100 mV was reduced significantly for S209F, which showed approximately 75% reduction over wild type (WT). All mutants except S209F showed positively shifted V(1/2)'s of activation and slowed channel activation compared with WT (V(1/2) = +17.7 +/- 2.4 mV and tau(activation) of 729 ms at +20 mV; n = 18). Deactivation was also accelerated in all mutants versus WT (126 +/- 8 ms at -50 mV; n = 27), and these changes led to marked loss of repolarizing currents during action potential clamps at 2 and 4 Hz, except again S209F. KCNQ1 models localize these naturally occurring S3 mutants to the surface of the helices facing the other voltage sensor transmembrane domains and highlight inter-residue interactions involved in activation gating. V207M, currently classified as a polymorphism and facing lipid in the model, was indistinguishable from WT I(Ks). We conclude that S3 mutants of KCNQ1 cause LQTS predominantly through biophysical effects on the gating of I(Ks), but some mutants also show protein stability/trafficking defects, which explains why the kinetic gain-of-function mutation S209F causes LQT1.

We have investigated the role of the kinesin I isoform Kif5b in the trafficking of a cardiac voltage-gated potassium channel, Kv1.5. In Kv1.5-expressing HEK293 cells and H9c2 cardiomyoblasts, current densities were increased from control levels of 389 ± 50.0 and 317 ± 50.3 pA pF−1, respectively, to 614 ± 74.3 and 580 ± 90.9 pA pF−1 in cells overexpressing the Kif5b motor. Overexpression of the Kif5b motor increased Kv1.5 expression additively with several manipulations that reduce channel internalization, suggesting that it is involved in the delivery of the channel to the cell surface. In contrast, expression of a Kif5b dominant negative (Kif5bDN) construct increased Kv1.5 expression non-additively with these manipulations. Thus, the dominant negative acts by indirectly inhibiting endocytosis. The increase in Kv1.5 currents induced by wild-type Kif5b was dependent on Golgi function; a 6 h treatment with Brefeldin A reduced Kv1.5 currents to control levels in Kif5b-overexpressing cells but had little effect on the increase associated with Kif5bDN expression. Finally, expression of the Kif5bDN prior to induction of Kv1.5 in a tetracycline inducible system blocked surface expression of the channel in both HEK293 cells and H9c2 cardiomyoblasts. Thus, Kif5b is essential to anterograde trafficking of a cardiac voltage-gated potassium channel.

Little is known about the postinternalization trafficking of surface-expressed voltage-gated potassium channels. Here, for the first time, we investigate into which of four major trafficking pathways a voltage-gated potassium channel is targeted after internalization. In both a cardiac myoblast cell line and in HEK293 cells, channels were found to internalize and to recycle quickly. Upon internalization, Kv1.5 rapidly associated with Rab5-and Rab4-positive endosomes, suggesting that the channel is internalized via a Rab5-dependent pathway and rapidly targeted for recycling to the plasma membrane. Nevertheless, as indicated by colocalization with Rab7, a fraction of the channels are targeted for degradation. Recycling through perinuclear endosomes is limited; colocalization with Rab11 was evident only after 24 h postsurface labelling. Expression of dominant negative (DN) Rab constructs significantly increased Kv1.5 functional expression. In the myoblast line, Rab5DN increased Kv1.5 current densities to 1305 ± 213 pA pF−1 from control 675 ± 81.6 pA pF−1. Rab4DN similarly increased Kv1.5 currents to 1382 ± 155 pA pF−1 from the control 522 ± 82.7 pA pF−1 at +80 mV. Expression of the Rab7DN increased Kv1.5 currents 2.5-fold in HEK293 cells but had no significant effect in H9c2 myoblasts, and, unlike the other Rab GTPases tested, over-expression of wild-type Rab7 decreased Kv1.5 currents in the myoblast line. Densities fell to 573 ± 96.3 pA pF−1 from the control 869 ± 135.5 pA pF−1. The Rab11DN was slow to affect Kv1.5 currents but had comparable effects to other dominant negative constructs after 48 h. With the exception of Rab11DN and nocodazole, the effects of interference with microtubule-dependent trafficking by nocodazole or p50 overexpression were not additive with the Rab dominant negatives. The Rab GTPases thus constitute dynamic targets by which cells may modulate Kv1.5 functional expression.

Based on the least absolute criteria, the image reconstruction problem is transformed into an optimization one by constructing the objective function for optimization. The optimization problem is transformed into the separable optimization problem to be solved by using the alternating direction method. At the same time, a new CT image reconstruction algorithm is got by combining with ART algorithm. Experimental results show that the algorithm is better than ART algorithm in obviously improving reconstruction quality, and as a result, the image signal to noise ratio has increased significantly.

In the actual scanning process, the projection data is inevitably influenced by various noises, thus it affects the precision of reconstructed image, which brings certain troubles for subsequent image processing. In order to improve the quality of the image reconstruction, this paper proposes a new method of image reconstruction. Firstly, we use the wiener filtering to deal with the iteration result after each iteration by SART algorithm, and adopt the steepest gradient descent method to adjust the total variation of the filtered results. The experimental results show that this method is very effective, and the quality of image is improved obviously compared with the traditional reconstruction methods.

Objective Current study was conducted to investigate the seed physiological characteristics of Tulipa edulis and improve germination rate. Method Anatomical characteristics was observed. Seed water absorption curve was tested by soaking method. Dynamic of embryo development and germination rate as well as germination index under different conditions were recorded. And the biological test of cabbage seed was used for detecting the germination inhibitors. Result The embryo rate of newly matured seeds was about 10%, and there was no obstacle of water absorption on testa of T. edulis. The optimum method for embryo development was exposure to 300 mg x L(-1) gibberellin solution for 24 hours, and stratification at 25 degrees C for 70 days followed by stratification at 5 degrees C for 40 days. The germintion rate and germination index of dormancy-broken seeds under the dark environment at 10 degrees C and 15 degrees C were significantly higher than those under other conditions. Additionally, there were some germination inhibitory substances in dry seeds. Conclusion The seed of T. edulis can be classified as having complex morphophysiological dormancy, and the morphological embryo dormancy played a leading role. Warm and cold stratification resulted in a fast dormancy breaking effect, and a high germination rate more than 90% could be obtained under the optimum conditions.

The dried over-ground parts of the plant Glechoma longituba are one of the important ingredients used in traditional Chinese medicine (TCM). In this study, five different watering treatments, namely 95 to100% (T1), 80 to 85% (T2), 65 to 70% (T3), 50 to 55% (T4) and 35 to 40% (T5) of field capacity were used to investigate the effects of water deficiency on the growth, physiological characteristics and total flavonoid content of G. longituba. The study found that epidermis, palisade tissue, spongy tissue and vascular cylinder thickness peaked in the T2 treatment and then decreased in the other treatments in anatomy structure of leaf. And anatomical structure analysis of the stem revealed that the thickness of the cortex and the vascular cylinder were the highest in the T2 treatment, too. Water stress increased the thickness of the vessel walls in the xylem and the size of the parenchyma cells in the cortex, whereas it decreased the size of the parenchyma cells in the pith. As the water stress intensified, the resulting changes found in physiological indices suggested that water stress stimulated the level of redox activity. In addition, the yield of total flavonoids in the T2 treatment was higher than in the other treatments. Our results suggested that 80 to 85% of field capacity is the most suitable watering treatment for the growth of G. longituba. This procedure would be suitable for the domestication of G. longituba. Key words: Glechoma longituba, water treatment, physiological characteristics, total flavonoids.

OBJECTIVE Current study on the pollination biology of Tulipa edulis was conducted to investigate its pollination characteristics and to provide references for artificial domestication and breeding of T. edulis. METHOD Flowering dynamics, pollinators, morphology and structure of flower were observed. Different methods were adopted to evaluate the pollen vitality, and benzidine-H2O2 method was used for estimation of the stigma receptivity. Breeding system was evaluated based on out-crossing index (OCI) , pollen-ovule ratio (P/O) and the results of emasculation, bagging and artificial pollination studies. RESULT The flower of T. edulis showed typical characteristics of Liliaceae. The pollen remained viable to some extent during all the anthesis and peaked within three days after blossoming. Stigma acceptability peaked in the first day of blossom and dwindled away in the next four days. The type of breeding system of T. edulis was facultative xenogamy, three species of Halictus are the main pollination insects. CONCLUSION The type of breeding system of T. edulis was facultative xenogamy, withal cross-pollination give priority to self-pollination, and the insects play a main role on the pollination of T. edulis, further validation are needed to judge if the wind is helpful to pollination.

Summary The transport of calcium ions (Ca 2+ ) to the cytosol is essential for immunoreceptor signaling, regulating lymphocyte differentiation, activation, and effector function. Increases in cytosolic-free Ca 2+ concentrations are thought to be mediated through two interconnected and complementary mechanisms: the release of endoplasmic reticulum Ca 2+ "stores" and "store-operated" Ca 2+ entry via plasma membrane channels. However, the identity of molecular components conducting Ca 2+ currents within developing and mature T cells is unclear. Here, we have demonstrated that the L-type "voltage-dependent" Ca 2+ channel Ca V 1.4 plays a cell-intrinsic role in the function, development, and survival of naive T cells. Plasma membrane Ca V 1.4 was found to be essential for modulation of intracellular Ca 2+ stores and T cell receptor (TCR)-induced rises in cytosolic-free Ca 2+ , impacting activation of Ras-extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) pathways. Collectively, these studies revealed that Ca V 1.4 functions in controlling naive T cell homeostasis and antigen-driven T cell immune responses.

Vernakalant (RSD1235) is an investigational drug recently shown to convert atrial fibrillation rapidly and safely in patients as an intravenous formulation (Roy et al., 2004) and to maintain sinus rhythm when taken orally (Savelieva and Camm, 2008). In the present study, the modeling software AutoDock4 was used to explore potential binding modes of vernakalant to the open state of the Kv1.2 model, which is 100% homologous in the binding region with Kv1.5. Docking simulations were run with a maximum number of evaluations of 25,000,000 and a maximum number of generations of 27,000. Point mutations were made in the channel model based on earlier patch-clamp studies (Eldstrom et al., 2007) and the docking simulations re-run to evaluate the ability of the docking software to predict changes in drug-channel interactions. Each AutoDock run predicted a binding conformation with an associated value for free energy of binding (FEB) in kcal/mol and an estimated inhibitory concentration (Ki). Increasing the number of evaluations and thus the time allowed for the program to look for an optimal binding site decreased average FEB and Ki values, and resulted in two front runner binding conformations. The most favored conformation had a FEB of -7.12 kcal/mol and a predicted Ki of 6.08 μM. This conformation makes contact with all four T480 residues and when examined from the side view and from above appears to be clearly positioned to block the channel as it directly occludes the pore.

Voltage-clamp fluorimetry (VCF) has been used to examine voltage sensor movement and the mechanism of coupling of activation to fast inactivation in the hERG potassium channel. Here, we used both tetramethylrhodamine maleimide (TMRM) and PyMPO maleimide as experimental fluorophores, as it is hypothesized that PyMPO experiences less steric hindrance than TMRM and is able to access cysteine residues with a greater sensitivity. Prior to examining constructs with strategically engineered cysteine residues, we looked at the fluorescence report from wild-type channels. We observed a robust fluorescence signal with a voltage-dependence that overlaid the conductance relationship. The cysteine residues readily accessible to labeling by thiol-reactive fluorophores in the wild-type channel, based on the accepted homology model, are C445 and C449; located on the S1-S2 linker. hERG channels with both C445V and C449V mutations show an attenuation of fluorescence signal. Consistent with prior reports, wild-type hERG channels treated with TMRM did not show detectable voltage-dependent changes in fluorescence. However, pre-treatment with dithiothreitol (DTT), a compound that reduces disulfide bridges, prior to labeling with TMRM resulted in a resolvable voltage-dependent signal in oocytes with high levels of protein expression. The time- and voltage- dependent kinetics of the PyMPO fluorescence report from the native cysteines was investigated in terms of channel gating. The correlation between the F-V and the G-V relationship suggest that the environmental changes sensed by PyMPO attached to C445 and C449 reflect gating movements and channel opening. The contribution of these cysteine residues as a background signal must be considered when evaluating fluorescent reports from cysteine residues engineered into other domains of the channel designed to track voltage sensor movement and coupling of activation to fast inactivation.

Little is known about the postinternalization trafficking of surface-expressed voltage-gated potassium channels. Here, for the first time, we investigate into which of four major trafficking pathways a voltage-gated potassium channel is targeted after internalization. In both a cardiac myoblast cell line and in HEK293 cells, channels were found to internalize and to recycle quickly. Upon internalization, Kv1.5 rapidly associated with Rab5-and Rab4-positive endosomes, suggesting that the channel is internalized via a Rab5-dependent pathway and rapidly targeted for recycling to the plasma membrane. Nevertheless, as indicated by colocalization with Rab7, a fraction of the channels are targeted for degradation. Recycling through perinuclear endosomes is limited; colocalization with Rab11 was evident only after 24 h postsurface labelling. Expression of dominant negative (DN) Rab constructs significantly increased Kv1.5 functional expression. In the myoblast line, Rab5DN increased Kv1.5 current densities to 1305 +/- 213 pA pF(-1) from control 675 +/- 81.6 pA pF(-1). Rab4DN similarly increased Kv1.5 currents to 1382 +/- 155 pA pF(-1) from the control 522 +/- 82.7 pA pF(-1) at +80 mV. Expression of the Rab7DN increased Kv1.5 currents 2.5-fold in HEK293 cells but had no significant effect in H9c2 myoblasts, and, unlike the other Rab GTPases tested, over-expression of wild-type Rab7 decreased Kv1.5 currents in the myoblast line. Densities fell to 573 +/- 96.3 pA pF(-1) from the control 869 +/- 135.5 pA pF(-1). The Rab11DN was slow to affect Kv1.5 currents but had comparable effects to other dominant negative constructs after 48 h. With the exception of Rab11DN and nocodazole, the effects of interference with microtubule-dependent trafficking by nocodazole or p50 overexpression were not additive with the Rab dominant negatives. The Rab GTPases thus constitute dynamic targets by which cells may modulate Kv1.5 functional expression.

Vernakalant (RSD1235) is an investigational drug recently shown to convert atrial fibrillation rapidly and safely in patients (J Am Coll Cardiol 44:2355-2361, 2004). Here, the molecular mechanisms of interaction of vernakalant with the inner pore of the Kv1.5 channel are compared with those of the class IC agent flecainide. Initial experiments showed that vernakalant blocks activated channels and vacates the inner vestibule as the channel closes, and thus mutations were made, targeting residues at the base of the selectivity filter and in S6, by drawing on studies of other Kv1.5-selective blocking agents. Block by vernakalant or flecainide of Kv1.5 wild type and mutants was assessed by whole-cell patch-clamp experiments in transiently transfected human embryonic kidney 293 cells. The mutational scan identified several highly conserved amino acids, Thr479, Thr480, Ile502, Val505, and Val508, as important residues for affecting block by both compounds. In general, mutations in S6 increased the IC50 for block by vernakalant; I502A caused an extremely local 25-fold decrease in potency. Specific changes in the voltage-dependence of block with I502A supported the crucial role of this position. A homology model of the pore region of Kv1.5 predicted that, of these residues, only Thr479, Thr480, Val505, and Val508 are potentially accessible for direct interaction, and that mutation at additional sites studied may therefore affect block through allosteric mechanisms. For some of the mutations, the direction of changes in IC50 were opposite for vernakalant and flecainide, highlighting differences in the forces that drive drug-channel interactions.

TheDmca1Dgene encodes aDrosophilacalcium channel α1subunit. We describe the first functional characterization of a mutation in this gene. This α1subunit mediates the dihydropyridine-sensitive calcium channel current in larval muscle but does not contribute to the amiloride-sensitive current in that tissue. A mutation, which changes a highly conserved Cys to Tyr in transmembrane domain IS1, identifies a residue important for channel function not only inDrosophilamuscle but also in mammalian cardiac channels. In both cases, mutations in this Cys residue slow channel activation and reduce expressed currents. Amino acid substitutions at this Cys position in the cardiac α1subunit show that the size of the side chain, rather than its ability to form disulfide bonds, affects channel activation.

Hereditary Long QT syndrome (LQTS) is characterized by a prolonged QT interval in the electrocardiogram and an increased likelihood of serious ventricular arrhythmias. It has been reported to arise from missense mutations in the S3 segment of KCNQ1′s S3. This is not surprising as tetramers of KCNQ1 subunits co-assemble with KCNE1 accessory subunits to form the slowly activating and deactivating IKs in the heart, which modulates repolarization of cardiac action potentials, particularly during sympathetic activation. However, the means by which these mutations, specifically in the S3 segment, cause LQTS remains poorly understood. The cause is often assumed to be decreased protein folding and trafficking to the cell membrane, but here we have investigated the properties of reported S3 mutations in a heterologous system to understand the potential biophysical basis for inherited LQTS. Functional channel properties were studied in transfected mouse ltk- cells utilizing whole cell patch clamp. Specifically, the voltage dependence of activation and deactivation was determined by tail current analysis at 35°C. Additionally, cardiac ionic current generated by an applied ventricular action potential clamp was measure at 1 and 3 Hz. Wild type KCNQ1 with KCNE1 subunits for IKs were compared with the KCNQ1 mutants. All mutants were expressed and trafficked to the cell surface. The majority of these mutations caused a dramatic depolarizing voltage shift in voltage dependence of activation, coupled with an acceleration of channel deactivation which would suppress IKs. This is expected to impair the physiological ability of this IKs channel current to summate in response to a rapid heart rhythm and lead to a reduction of the overall repolarization reserve in affected hearts

The voltage gated potassium channel family contains several functionally distinct isoforms which serve to shape the duration, frequency and timing of action potential firing in electrically excitable cells. Several mechanistic elements that contribute to the function of the voltage sensing apparatus are not yet fully described, especially in channels other than the prototypical Shaker potassium channel. We have studied voltage sensor rearrangements of Kv1.2 channels using two-electrode voltage clamp fluorometry and gating current recordings in mammalian cells. Fluorescence measurements reporting on voltage sensor movement revealed a transition into a relaxed state upon prolonged depolarization, causing a left shift in the fluorescence-voltage relationship. Gating current measurements of wild type Kv1.2 channels recorded in permeant ion free solutions (NMDG+int//TEA+ext) also displayed a left shifted Q-V after depolarizing pre-pulses, reflecting a stabilization of the activated state of the voltage sensor. To enable examination of the effects of different cations and processes of inactivation in Kv1.2 a non-conducting double mutant channel mimicking the permanently slow inactivated and non-conducting Shaker W434F, Kv1.2(W366F, V381T) was created. Off-gating currents recorded from this channel in the presence of permeant ions displayed less voltage sensor stabilization in the activated state than the wild type channel. These data suggest that cations play a specific role in regulating voltage sensor dynamics in the Kv1.2 channel.