RNA sequencing identifies key genes involved in intramuscular fat deposition in chickens at different developmental stages.

BACKGROUND: Intramuscular fat (IMF) is an important factor in meat quality, and triglyceride (TG) and Phospholipids (PLIP), as the main components of IMF, are of great significance to the improvement of meat quality. RESULTS: In this study, we used 30 RNA sequences generated from the transcriptome of chicken breast muscle tissues at different developmental stages to construct a gene expression matrix to map RNA sequence reads to the chicken genome and identify the transcript of origin. We used weighted gene co-expression network analysis (WGCNA) and identified 27 co-expression modules, 10 of which were related to TG and PLIP. We identified 150 highly-connected hub genes related to TG and PLIP, respectively, which were found to be mainly enriched in the adipocytokine signaling pathway, MAPK signaling pathway, mTOR signaling pathway, FoxO signaling pathway, and TGF-beta signaling pathway. Additionally, using the BioMart database, we identified 134 and 145 candidate genes related to fat development in the TG-related module and PLIP-related module, respectively. Among them, RPS6KB1, BRCA1, CDK1, RPS3, PPARGC1A, ACSL1, NDUFAB1, NDUFA9, ATP5B and PRKAG2 were identified as candidate genes related to fat development and highly-connected hub genes in the module, suggesting that these ten genes may be important candidate genes affecting IMF deposition. CONCLUSIONS: RPS6KB1, BRCA1, CDK1, RPS3, PPARGC1A, ACSL1, NDUFAB1, NDUFA9, ATP5B and PRKAG2 may be important candidate genes affecting IMF deposition. The purpose of this study was to identify the co-expressed gene modules related to chicken IMF deposition using WGCNA and determine key genes related to IMF deposition, so as to lay a foundation for further research on the molecular regulation mechanism underlying chicken fat deposition.

Formulation and Performance of NBR/CR-Based High-Damping Rubber Composites for Soundproof Using Orthogonal Test.

Multiple functional-material-filled nitrile butadiene rubber/chloroprene rubber (NBR/CR) acoustic composites were extensively studied and prepared. According to the orthogonal test table L25 (56), 25 groups of samples were prepared by using a low-temperature one-time rubber mixing process. With tensile strength, average transmission loss, and damping peak as indexes, the influence degree of different factors and levels on the properties of acoustic composites was quantitatively discussed and analyzed. The matrix weight analysis was employed to optimize the material formula of rubber composites, and the corresponding influence weight was given. Results showed that the acoustic composite with blending ratio of 70/30 for NBR/CR matrix had preferable mechanical and acoustic properties; adding mica powder (MP) and montmorillonite (MMT) in matrix contributed to improve all above three indexes owing to their specific lamellar structures; hollow glass beads (HGB) had a positive influence on improving acoustic property due to its hollow microcavities, however, it had a negative impact on damping property because of the smooth spherical surfaces. Accordingly, the optimal formulation was found to be NBR/CR blending ratio of 70/30, MP of 10 phr (per hundred rubber), HGB of 4 phr, and MMT of 10 phr.

[Yin-yang balance penetrating acupuncture combined with rehabilitation training on upper limb spasticity in stroke hemiplegia].

OBJECTIVE: To compare the therapeutic effect of yin-yang balance penetrating acupuncture combined with rehabilitation training and single rehabilitation training on upper limb spasticity in patients with stroke hemiplegia. METHODS: A total of 60 patients with upper limb spasticity of stroke hemiplegia were randomized into an observation group and a control group, 30 cases in each one. On the basis of conventional western medication, rehabilitation training was adopted in the control group. On the basis of treatment in the control group, yin-yang balance penetrating acupuncture was applied from Jianyu (LI 15) to Binao (LI 14), Quchi (LI 11) to Shaohai (HT 3), Yanglingquan (GB 34) to Yinlingquan (SP 9), Xuanzhong (GB 39) to Sanyinjiao (SP 6), etc. of the affected side in the observation group. The treatment was given once a day, 5 days were as one course, with a 2-day interval between two courses, 4 courses were required in both groups. The classification of modified Ashworth spasticity scale (MAS), surface integrated electromyogram (iEMG) of affected upper limb and the scores of National Institute of Health stroke scale (NIHSS), Fugl-Meyer assessment (FMA) of upper limb and modified Barthel index (MBI) before and after treatment were observed, the therapeutic effect was evaluated in both groups. RESULTS: ①After treatment, the MAS classification reduced in both groups (P<0.05), the cases of grade 0 to Ⅰ+ in the observation group were more than those in the control group (P<0.05); iEMG values of the maximum isometric voluntary contraction of affected usculus biceps brachii, musculus triceps brachii, musculus flexor carpi, musculus extensor carpi, extensor digitorum, aductor pollicis brevis were increased in both groups (P<0.05), and the variations of iEMG of above muscles on the affected side in the observation group were larger than those in the control group (P<0.05). ②After treatment, the scores of NIHSS were decreased (P<0.05), the scores of FMA, MBI were increased in both groups (P<0.05), and the variations of NIHSS, FMA and MBI scores were larger than those in the control group (P<0.05). ③The total effective rate was 93.3% (28/30) in the observation group, which was superior to 70.0% (21/30) in the control group (P<0.05). CONCLUSION: Yin-yang balance penetrating acupuncture combined with rehabilitation training can improve upper limb spasticity, heighten the motor function of upper limb and daily self care in patients with stroke hemiplegia, its therapeutic effect is superior to single rehabilitation training.

Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome†P450 Monooxygenase Mutants.

Steroidal C7ß alcohols and their respective esters have shown significant promise as neuroprotective and anti-inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C-H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450-BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and ß stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil-Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high-resolution X-ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio- and stereoselectivity.

Biosynthesis of plant tetrahydroisoquinoline alkaloids through an imine reductase route.

Herein, we report a biocatalytic approach to synthesize plant tetrahydroisoquinoline alkaloids (THIQAs) from dihydroisoquinoline (DHIQ) precursors using imine reductases and N-methyltransferase (NMT). The imine reductase IR45 was engineered to significantly expand its substrate specificity, enabling efficient and stereoselective conversion of 1-phenyl and 1-benzyl 6,7-dimethoxy-DHIQs into the corresponding (S)-tetrahydroisoquinolines (S-THIQs). Coclaurine N-methyltransferase (CNMT) was able to further efficiently convert these (S)-THIQ intermediates into (S)-THIQAs. By assembling IRED, CNMT, and glucose dehydrogenase (GDH) in one reaction, we effectively constituted two artificial biosynthetic pathways in Escherichia coli and successfully applied them to the production of five (S)-THIQAs. This highly efficient (100% yield from DHIQs) and easily tailorable (adding other genes) biosynthetic approach will be useful for producing a variety of plant THIQAs.

Pyrazolopyrimidines as Potent Stimulators for Transient Receptor Potential Canonical 3/6/7 Channels.

Transient receptor potential canonical 3/6/7 (TRPC3/6/7) are highly homologous receptor-operated nonselective cation channels. Despite their physiological significance, very few selective and potent agonists are available for functional examination of these channels. Using a cell-based high throughput screening approach, a lead compound with the pyrazolopyrimidine skeleton was identified as a TRPC6 agonist. Synthetic schemes for the lead and its analogues were established, and structural-activity relationship studies were carried out. A series of potent and direct agonists of TRPC3/6/7 channels were identified, and among them, 4m-4p have a potency order of TRPC3 > C7 > C6, with 4n being the most potent with an EC50 of <20 nM on TRPC3. Importantly, these compounds exhibited no stimulatory activity on related TRP channels. The potent and selective compounds described here should be suitable for evaluation of the roles of TRPC channels in the physiology and pathogenesis of diseases, including glomerulosclerosis and cancer.

Identification and optimization of 2-aminobenzimidazole derivatives as novel inhibitors of TRPC4 and TRPC5 channels.

BACKGROUND AND PURPOSE: Transient receptor potential canonical (TRPC) channels play important roles in a broad array of physiological functions and are involved in various diseases. However, due to a lack of potent subtype-specific inhibitors the exact roles of TRPC channels in physiological and pathophysiological conditions have not been elucidated. EXPERIMENTAL APPROACH: Using fluorescence membrane potential and Ca(2+) assays and electrophysiological recordings, we characterized new 2-aminobenzimidazole-based small molecule inhibitors of TRPC4 and TRPC5 channels identified from cell-based fluorescence high-throughput screening. KEY RESULTS: The original compound, M084, was a potent inhibitor of both TRPC4 and TRPC5, but was also a weak inhibitor of TRPC3. Structural modifications of the lead compound resulted in the identification of analogues with improved potency and selectivity for TRPC4 and TRPC5 channels. The aminobenzimidazole derivatives rapidly inhibited the TRPC4- and TRPC5-mediated currents when applied from the extracellular side and this inhibition was independent of the mode of activation of these channels. The compounds effectively blocked the plateau potential mediated by TRPC4-containing channels in mouse lateral septal neurons, but did not affect the activity of heterologously expressed TRPA1, TRPM8, TRPV1 or TRPV3 channels or that of the native voltage-gated Na(+) , K(+) and Ca(2) (+) channels in dissociated neurons. CONCLUSIONS AND IMPLICATIONS: The TRPC4/C5-selective inhibitors developed here represent novel and useful pharmaceutical tools for investigation of physiological and pathophysiological functions of TRPC4/C5 channels.

Synthesis, biological evaluation and molecular modeling of substituted 2-aminobenzimidazoles as novel inhibitors of acetylcholinesterase and butyrylcholinesterase.

A series of novel 2-aminobenzimidazole derivatives were synthesized under microwave irradiation. Their biological activities were evaluated on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). A number of the 2-aminobenzimidazole derivatives showed good inhibitory activities to AChE and BuChE. Among them, compounds 9, 12 and 13 were found to be >25-fold more selective for BuChE than AChE. No evidence of cytotoxicity was observed by MTT assay in PC12 cells or HepG2 cells exposed to 100µM of the compounds. Molecular modeling studies indicate that the benzimidazole moiety of compounds 9, 12 and 13 forms a face-to-face π-π stacking interaction in a 'sandwich' form with the indole ring of Trp82 (4.09Å) in the active gorge, and compounds 12 and 13 form a hydrogen bond with His438 at the catalytic site of BuChE. In addition, compounds 12 and 13 fit well into the hydrophobic pocket formed by Ala328, Trp430 and Tyr332 of BuChE. Our data suggest the 2-aminobenzimidazole drugs as promising new selective inhibitors for AChE and BuChE, potentially useful to treat neurodegenerative diseases.

[Construction of rabbit hemorrhagic disease virus replicons and its replication in RK-13 cells].

To provide an efficient and safe technology platform for studying the replication and pathogenesis mechanisms of RHDV, the interaction between the RHDV and its host cells, a replicon system of RHDV, was constructed based on the infectious cDNA clone of RHDV, in which VP60 gene encoding the capsid protein was deleted, but all the necessary protease coding regions and non-coding regions were retained. Results from RT-PCR, IFA and qRT-PCR confirmed that the replicon RNA could efficiently replicate in RK-13 cells. Besides, the results also suggested that the capsid protein which is the structural protein of RHDV is necessary for maintaining the viral infectivity.