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Calcineurin is a serine/threonine protein phosphatase that is highly conserved from yeast to human and plays a critical role in many physiological processes. Regulators of calcineurin (RCANs) are a family of endogenous calcineurin regulators, which are capable of inhibiting the catalytic activity of calcineurin in vivo and in vitro. In this study, we first characterized the biochemical properties of yeast calcineurin and its endogenous regulator Rcn1, a yeast homolog of RCAN1. Our data show that Rcn1 inhibits yeast calcineurin toward pNPP substrate with a noncompetitive mode; and Rcn1 binds cooperatively to yeast calcineurin through multiple low-affinity interactions at several docking regions. Next, we reinvestigated the mechanism underlying the inhibition of mammalian calcineurin by RCAN1 using a combination of biochemical, biophysical, and computational methods. In contrast to previous observations, RCAN1 noncompetitively inhibits calcineurin phosphatase activity toward both pNPP and phospho-RII peptide substrates by targeting the enzyme active site in part. Re-analysis of previously reported kinetic data reveals that the RCAN1 concentrations used were too low to distinguish between the inhibition mechanisms [Chan B et al. (2005) Proc Natl Acad Sci USA 102, 13075]. The results presented in this study provide new insights into the interaction between calcineurin and RCAN1/Rcn1., (© 2024 Federation of European Biochemical Societies.)

Analyzing the vibration features of a shipboard stabilized platform (SSP) is significant to the design or vibration compensation of a marine gravimetric survey vibration isolation system. Empirical Fourier decomposition (EFD) is a recently developed method for nonlinear and non-stationary signal decomposition. However, the spectral segmentation boundary needs to be set in advance according to sophisticated experience, and it is easy to be disturbed by noise, and the decomposition result is inaccurate. In order to accurately extract the nonlinear vibration characteristics of SSP, this paper proposes a new method called power spectrum envelope adaptive empirical Fourier decomposition (PSEEFD). Firstly, the number of selected modal decomposition is determined based on the mutual information to realize adaptive segmentation. Then, the improved power spectrum envelope segmentation method is adopted to effectively diminish the interference of noise since the segmentation boundary is formed by the minimum of the adjacent extreme points enveloped by the maximum value of the power spectrum. The spectrum segments obtained from segmentation contain less interference. Finally, the component signal in each frequency band is reconstructed by inverse fast Fourier transform, and the instantaneous frequency signal component with physical significance is obtained. Through the analysis of vibration simulation signals and measured data of SSP, the proposed method is compared with EMD, AFVMD, EWT and EFD. The results show that PSEEFD has a well suppression of noise interference and can effectively extract the characteristics of nonlinear vibration signals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 ISA. Published by Elsevier Ltd. All rights reserved.)

This study examined the role of intestinal microbiota in berberine (BBR)-mediated glucose (GLU) metabolism regulation in largemouth bass. Four groups of largemouth bass (133.7 ± 1.43 g) were fed with control diet, BBR (1 g/kg feed) supplemented diet, antibiotic (ATB, 0.9 g/kg feed) supplemented diet and BBR + ATB (1g/kg feed +0.9 g/kg feed) supplemented diet for 50 days. BBR improved growth, decreased the hepatosomatic and visceral weight indices, significantly downregulated the serum total cholesterol and GLU levels, and significantly upregulated the serum total bile acid (TBA) levels. The hepatic hexokinase, pyruvate kinase, GLU-6-phosphatase and glutamic oxalacetic transaminase activities in the largemouth bass were significantly upregulated when compared with those in the control group. The ATB group exhibited significantly decreased final bodyweight, weight gain, specific growth rates and serum TBA levels, and significantly increased hepatosomatic and viscera weight indices, hepatic phosphoenolpyruvate carboxykinase, phosphofructokinase, and pyruvate carboxylase activities, and serum GLU levels. Meanwhile, the BBR + ATB group exhibited significantly decreased final weight, weight gain and specific growth rates, and TBA levels and significantly increased hepatosomatic and viscera weight indices and GLU levels. High-throughput sequencing revealed that compared with those in the control group, the Chao one index and Bacteroidota contents were significantly upregulated and the Firmicutes contents were downregulated in the BBR group. Additionally, the Shannon and Simpson indices and Bacteroidota levels were significantly downregulated, whereas the Firmicutes levels were significantly upregulated in ATB and BBR + ATB groups. The results of in-vitro culture of intestinal microbiota revealed that BBR significantly increased the number of culturable bacteria. The characteristic bacterium in the BBR group was Enterobacter cloacae . Biochemical identification analysis revealed that E. cloacae metabolizes carbohydrates. The size and degree of vacuolation of the hepatocytes in the control, ATB, and ATB + BBR groups were higher than those in the BBR group. Additionally, BBR decreased the number of nuclei at the edges and the distribution of lipids in the liver tissue. Collectively, BBR reduced the blood GLU level and improved GLU metabolism in largemouth bass. Comparative analysis of experiments with ATB and BBR supplementation revealed that BBR regulated GLU metabolism in largemouth bass by modulating intestinal microbiota., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xia, Yang, Zhang, Tian, Li, Yu, Li, Gong, Xie, Wang and Xie.)

Objective: To investigate the related factors of radioiodine-refractory thyroid cancer (RAIR-DTC) and the increase of cumulative iodine treatment dose. Methods: The data of patients with papillary thyroid cancer who underwent surgery and iodine treatment for the first time in the Affiliated Cancer Hospital of Zhengzhou University from January 2015 to December 2017 were retrospectively analyzed. The related factors of RAIR-DTC and the increase of cumulative iodine treatment dose were explored. Results: A total of 650 patients were enrolled, including 217 males (33.4%) and 433 females (66.6%), aged 45 (34, 53) years. There were 123 patients (18.9%) over 55 years old, 171 patients (26.3%) with extranodal extension and 18 patients (2.8%) with distant metastasis. The median lymph node ratio was 0.22 (0.11, 0.33). Twenty patients (3.1%) had an accumulated iodine treatment dose>400 mCi and 19 patients (2.9%) had RAIR-DTC. Multivariate logistic regression analysis showed that extranodal extension ( OR =19.833, 95% CI : 6.057-73.325, P <0.001) was related factors for the increase of cumulative iodine treatment dose. Age>55 years old ( OR =3.322, 95% CI : 1.136-9.466, P =0.024), distant metastasis ( OR =10.059, 95% CI : 2.508-38.888, P <0.001), extranodal extension ( OR =5.278, 95% CI : 1.707-19.813, P =0.006) and lymph node ratio ( OR =34.724, 95% CI : 2.749-384.575, P =0.004) were related factors for RAIR-DTC. Conclusions: Extranodal extension and lymph node ratio are related factors for RAIR-DTC. In clinical practice, more attention should be paid to the influence of different lymph node metastasis characteristics on the occurrence of RAIR-DTC and the cumulative therapeutic dose of iodine.

Aquaculture is crucial for achieving the FAO's goal of a world without hunger and malnutrition. Recently, biofilm substratum has been proposed as an effective means to control waste pollution caused by excessive nutrient inputs from aquaculture, but key bacterial communities involved in the remediation remain unclear. Here we reported a freshwater mesocosm study where the addition of biofilm substrata with external carbon effectively controlled the total ammonia nitrogen and improved fish growth. 16S rRNA study and Weighted UniFrac analysis revealed that bacterial compositions were significantly different (999 permutations, p -value < 0.01) between the biofilm-substrata-added and biofilm-substrata-free systems. Planctomycetes were found, as key bacteria benefited from the biofilm substrata addition and exerted the major function of ammonia nitrogen control. Our study demonstrated that the addition of biofilm substrata and an external carbon source favored fish growth and improved the aquaculture environment by the formation of a unique bacteria community.

Object: The fluid management strategy in ARDS is not very clear. A secondary analysis of RCT data was conducted to identify patients with ARDS benefitting from a conservative strategy of fluid management. Methods: The data of this study were downloaded from the ARDS network series of randomized controlled trials (Conservative Strategy vs. Liberal Strategy in 2006). Based on the clinical feature of patients, within the first 24 h after admission, clustering was performed using the k -means clustering algorithm to identify the phenotypes of ARDS. Survival was analyzed using the Kaplan-Meier survival analysis to assess the effect of the two fluid management strategies on the 90-day cumulative mortality. Categorical/dichotomic variables were analyzed by the chi-square test. Continuous variables were expressed as the mean and standard deviation and evaluated through a one-way ANOVA. A P -value < 0.05 was defined as the statistically significant cut-off value. Results: A total of 1,000 ARDS patients were enrolled in this unsupervised clustering research study, of which 503 patients were treated with a conservative fluid-management strategy, and 497 patients were treated with a liberal fluid-management strategy. The first 7-day cumulative fluid balance in patients with the conservative strategy and liberal strategy were -136 ± 491 ml and 6,992 ± 502 ml, respectively ( P < 0.001). Four phenotypes were found, and the conservative fluid-management strategy significantly improved the 90-day cumulative mortality compared with the liberal fluid-management strategy (HR = 0.532, P = 0.024) in patients classified as "hyperinflammatory anasarca" phenotype (phenotype II). The characteristics of this phenotype exhibited a higher WBC count (20487.51 ± 7223.86/mm 3 ) with a higher incidence of anasarca (8.3%) and incidence of shock (26.6%) at baseline. The furthermore analysis found that the conservative fluid management strategy was superior to the liberal fluid management strategy in avoiding superinfection (10.10 vs. 14.40%, P = 0.037) and returned to assisted breathing (4.60 vs. 16.20%, P = 0.030) in patients classified as "hyperinflammatory anasarca" phenotype. In addition, patients with other phenotypes given the different fluid management strategies did not show significant differences in clinical outcomes. Conclusion: Patients exhibiting a "hyperinflammatory anasarca" phenotype could benefit from a conservative fluid management strategy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xing, Gong, Yang, Qi and Zhang.)

Fat accumulation in the mesenteric adipose tissue is a serious problem in grass carp (Ctenopharyngodon idella) culture. Lipid droplet-related proteins (LDRPs) are involved in the formation, degradation, and biological functions of lipid droplets. In this study, we aimed to provide reference proteomics data to study lipid droplet regulation in fish. We isolated LDRPs from the mesenteric adipose tissue of grass carp (1-year-old) after normal feeding and 7 days of starvation, and identified and analysed them using isobaric tags for relative and absolute quantitation (iTRAQ) technology. Short-term starvation had no significant effect on the body weight, condition factor, visceral index, hepatopancreas index, intraperitoneal fat index, adipose tissue triglyceride content, and adipocyte size of grass carp. Nine hundred and fifty proteins were identified and annotated using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; they are involved in a variety of metabolic and signalling pathways, including amino acid, lipid, and carbohydrate metabolism, and the PI3K-Akt signalling pathway. There were 296 differentially expressed proteins (DEPs), with 143 up-regulated and 153 down-regulated proteins. Three proteins involved in triglyceride and fatty acid syntheses and two proteins involved in autophagy were up-regulated, and six proteins involved in lipid catabolism were down-regulated. These results indicate that under short-term starvation, lipid droplets in the adipose tissue of grass carp may maintain their shape by promoting fat production and inhibiting lipolysis, and autophagy may be one of the main strategies for coping with short-term energy deprivation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Surface-enhanced Raman scattering (SERS) employing a non-noble substrate in comparison with conventional noble-metal ones offers advantages of low cost and rich selection of candidates; however, its application has been seriously hindered by its unsatisfactory detection sensitivity, poor uniformity, and undesirable modification of vibrational signals via changing the orientation and/or polarizability of probe molecules. Here, an unusually sensitive but nonselective enhancement was achieved by employing titanium carbide sheets modified with aluminum oxyanions in situ as active supports for Raman measurement. The analyte molecules adopted a conformation similar to what they adopt on a bare substrate, while closely interacting with the aluminum oxyanion surface, which leads to the rare observation of highly sensitive but nonselective enhancement with a detection limit close to the pM level. With the substrate surface roughness in the nanometer region, an outstanding uniformity with a relative standard deviation of less than 4.3% was achieved. In addition, the SERS effect on the modified titanium carbide sheets was shown to be applicable to a wide range of analyte molecules, including both organic dyes and trace harmful compounds. The success of the work demonstrates the feasibility of surface tuning to improve the SERS effect, and it introduces a new window for two-dimensional materials in SERS applications.

In this study, we aimed to explore the effects of faba bean ( Vicia faba L.) on the energy metabolism of grass carp ( Ctenopharyngodon idellus ). A total of 180 fish (∼2900 g) were randomly assigned to six tanks (2.5 × 2.5 × 1.2 m; 30 individuals per tank) and fed either faba bean ( Vicia faba L.) or a commercial diet for 120 days (3% body weight, twice per day). The results showed that faba bean-fed grass carp (FBFG) had significantly lower growth and higher fat accumulation in the mesenteric adipose tissue and hepatopancreas than commercial diet-fed grass carp (CDFG). Compared with CDFG, FBFG exhibited no significant difference in proximate composition of the muscle; however, an obvious decrease in muscle fiber size and significantly higher hardness, chewiness, and gumminess were observed. Transcriptome results showed that a total of 197 genes were differentially regulated in the dorsal muscle. Down-regulated genes included four genes annotated with myocyte development and 12 transcripts annotated with components of myofibrils. In addition, the FBFG group exhibited significantly lower expression of genes associated with oxygen transport, the mitochondrial respiratory chain, and creatine metabolism, suggesting reduced energy availability in the muscle of the FBFG. Moreover, using western-blotting and enzyme assays, we found decreased protein levels in the mitochondrial electron transport respiratory chain and creatine metabolism activities, as well as increased expression of autophagy marker protein levels, in the muscle of FBFG. Overall, our results suggest that an abnormal energy distribution may exist in grass carps after feeding with faba bean, which is reflected by a mass of fat deposition in the adipose tissue and hepatopancreas and subdued metabolic activity in the muscle., (Copyright © 2020 Tian, Fu, Yu, Li, Xia, Li, Zhang, Gong, Yu, Wang and Xie.)

With the ever-increasing growth in next-generation flexible and wearable electronics, fiber-shaped zinc-air batteries have attracted considerable attention due to their advantages of high energy density and low cost, though their development, however, has been seriously hampered by the unavailability of efficient electrocatalysts. In this work, we designed a trimetallic nitride electrocatalyst in an unusual molecular sheet form, which was stabilized by metallic titanium carbide sheets. Besides the expected elevation in catalytic activity toward the oxygen evolution reaction, the material simultaneously unlocked excellent catalytic activity for oxygen reduction reaction with the half-wave potential as small as 0.84 V. A flexible fiber-shaped zinc-air battery, employing the designed electrocatalyst as the air cathode and a gel as the electrolyte, demonstrated an enhanced and durable electrochemical performance, outputting a competitive energy density of 627 Wh kg zn -1 . This work opens new avenues for utilizing two-dimensional sheets in future wearable and portable device applications.

Lightweight, robust, and thin aerogel films with multifunctionality are highly desirable to meet the technological demands of current society. However, fabrication and application of these multifunctional aerogel films are still significantly underdeveloped. Herein, we demonstrate a multifunctional aerogel film composed of strong aramid nanofibers (ANFs), conductive carbon nanotubes (CNTs), and hydrophobic fluorocarbon (FC) resin. The obtained hybrid aerogel film exhibits large specific surface area (232.8 m 2 ·g -1 ), high electrical conductivity (230 S·m -1 ), and excellent hydrophobicity (contact angle of up to 137.0°) with exceptional Joule heating performance and supreme electromagnetic interference (EMI) shielding efficiency. The FC coating renders the hydrophilic ANF/CNT aerogel films hydrophobic, resulting in an excellent self-cleaning performance. The high electrical conductivity enables a low-voltage-driven Joule heating property and an EMI shielding effectiveness (SE) of 54.4 dB in the X-band at a thickness of 568 μm. The specific EMI SE is up to 33528.3 dB·cm 2 ·g -1 , which is among the highest values of typical metal-, conducting-polymer-, or carbon-based composites. This multifunctional aerogel film holds great promise for smart garments, electromagnetic wave shielding, and personal thermal management systems.

With the rapid development of portable devices and wireless protocols, miniaturized energy storage units have become an important prerequisite. Although in-plane microsupercapacitors are emerging as competitive candidate devices, their practical applications have been severely hindered by their low energy density. Here, employing pseudocapacitive active materials working in complementary voltage windows, namely, manganese oxide (MnO 2 ) and titanium carbide (Ti 3 C 2 ), both in the two-dimensional sheet morphology, a flexible asymmetric interdigitated solid-state microsupercapacitor was assembled. Profiting from the perfect voltage complementarity of the two types of sheets, the high exposure of electrochemically active sites and the maximized utilization of the sheets due to the planar ion transport, the designed device achieved excellent electrochemical performance even when using a gel electrolyte. In particular, the device obtained a high specific capacitance of 106 F g -1 (295 mF cm -2 ), a wide potential window (2 V), an ultrahigh rate performance (retaining 83% even with a 20-fold in current density to 20 A g -1 ), an excellent cycling stability (87% retention after 10 4 cycles at 10 A g -1 ), and a competitive energy density of 58 W h kg -1 (162 μW h cm -2 ) that are even comparable to those of some microbatteries, while maintaining a high power density of 985 W kg -1 (2.7 mW cm -2 ). Importantly, this outstanding electrochemical performance was also stably maintained under various bending conditions. These results indicate that two-dimensional pseudocapacitive sheet materials have a plethora of possibilities for constructing flexible and wearable devices.

Lipid droplets (LDs) are increasingly being recognized as important immune modulators in mammals, in additional to their function of lipid ester deposition. However, the role of LDs in fish immunity remains poorly understood. In this study, the function of LDs in the innate immune response of Ctenopharyngodon idella kidney (CIK) cells, which are the equivalent of myeloid cells in vertebrates, was investigated. LD number and TG content significantly increased in the CIK cells following exposure to lipopolysaccharide (LPS), peptidoglycan (PGN), and polyriboinosinic-polyribocytidylic acid (Poly [I: C]) for 24 h, accompanied by increases in the relative expression of several innate immune genes. However, fatty acid compositions of the triglycerides were not changed after treatment with these three pathogenic mimics. LPS, PGN, and Poly (I: C) did not alter the relative expressions of lipogenic (FAS, SCD, and DGAT) and lipid catabolic (PPARα, ATGL, and CPT-1) genes. However, these treatments did increase the mRNA levels of lipid transportation genes (FATP/CD36, ACSL1, and ACSL4), and also decreased the non-esterified fatty acid level in the medium. To further explore the role of LDs in the immune response, CIK cells were incubated with different concentrations (0, 100, 200, 300, 400, 500 μM) of exogenous lipid mix (LM; oleic acid [OA]:linoleic acid [LA]:linolenic acid [LNA] = 2:1:1), and were then transferred to a lipid-free medium and incubated for 24 h. LD size and number increased with the increase in lipid levels, and this was accompanied by increased expression of innate immune genes, including MyD88, IRF3, and IL-1β, which were expressed at their highest levels in 300 μM exogenous lipid mix. Interestingly, after incubating with different fatty acids (LM, OA, LA, LNA, arachidonic acid [ARA], and docosahexaenoic acid [DHA]; 300 μM), ARA and DHA were more potent in inducing LD formation and innate immune gene expression in the CIK cells. Finally, atglistatin, an ATGL inhibitor, effectively attenuated the expression of most genes upregulated by ARA or DHA, suggesting that lipolysis may be involved in the regulation of immune genes at the transcriptional level. Overall, the findings of this study demonstrate that LDs are functional organelles that could act as modulators in the innate immune response of CIK cells. Additionally, long-chain polyunsaturated fatty acid enriched LDs play a unique role in regulating this process., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Feeding faba beans (Vicia faba L.) to grass carp (Ctenopharyngodon idellus) increases muscle compactness but decreases growth and motility. The lipid metabolism of grass carp was examined to assess potential effects of feeding faba beans on physiological properties using a total of 180 fish. The treatment group was fed faba beans for 120 days and a commercial diet for another 30 days. The control group received a commercial diet for 150 days. Fish were sampled every month. Weight gain was significantly lower in the treatment group than in the control. Hardness, springiness, chewiness, cohesiveness, and gumminess of the dorsal muscle increased significantly with the feeding faba beans from 30 to 120 days, which was not reversed by the subsequent feeding of commercial diet. Fat accumulation increased significantly in the treatment group as suggested by the condition factor, viscera index, hepatopancreatic index, and intraperitoneal fat index (IPFI), hepatopancreas, and muscle fat content but was not affected by subsequent feeding with the commercial diet. Serum triglyceride and total cholesterol levels were significantly reduced in the experimental diet group. In the hepatopancreas and intraperitoneal fat IPF, monounsaturated fatty acids showed significantly higher content in faba bean feeding fish, whereas polyunsaturated fatty acid content showed the reversed pattern. In the hepatopancreas, the activities of the lipogenic enzymes malate dehydrogenase and glucose 6-phosphate dehydrogenase were higher in the treatment than in the control group. Moreover, the treatment group showed lower mRNA levels of carnitine palmitoyltransferase-1. Overall, our results clearly demonstrate increasing lipid accumulation in the viscera of faba bean-fed grass carp.

Smad4 is the key regulator in the transforming growth factor β1 (TGF-β1)/Smads signal pathway, and is also the crux of the regulation of type I collagen expression in mammals. In fish, however, the relationship between Smad4 and type I collagen is still unknown. Given the widely accepted importance of type I collagen in fish muscle hardness, we seek to explore this issue by analyzing the expressions of the TGF-β1/Smads pathway molecules and type I collagen in the muscle of crisp grass carp fed with faba bean, which shows increased muscle hardness. The study found that (1) in the process of feeding the grass carp with faba bean, the mRNA and protein expressions of TGF-β1, Smad2 and Smad4 all increased along with the increase of type I collagen expression (Col1α1 and Col1α2); (2) one day after the injection of Smad4 over-expression vector, both mRNA and protein expressions of Col1α1 and Col1α2 significantly increased, reaching the maximum on the 2nd and 5th day, respectively; (3) one day after the injection of Smad4 RNAi interference vector, the mRNA and protein expressions of Col1α1 and Col1α2 decreased, reaching the minimum on the 5th day. These results revealed that Smad4 is the major regulator of type I collagen in the muscle of grass carp fed with faba bean. This study would provide an important mechanistic basis for nutritional regulation of type I collagen in the muscle of fish., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Polysaccharides are some of the most important bioactive compounds in Lentinus edodes, but little attention has been devoted to the genetic basis of polysaccharide content. Here, the polysaccharide content of fruiting body, and 11 agronomic traits related to morphologic characteristics of fruiting body, yield and precocity of 50 L. edodes strains were screened and analyzed. Results showed that polysaccharide content in L. edodes fruiting bodies is a quantitative trait controlled by the polygenic system. Several wild strains possess desirable polysaccharide contents, exhibiting the potential to improve shiitake cultivars by introducing the promising alleles. There were no close correlations between polysaccharide content and the 11 agronomic traits related with fruiting body morphology, yield and precocity, suggesting that polysaccharide content and the development of fruiting bodies may genetically be controlled independently. Moreover, based on polysaccharide content and the 11 agronomic traits, grey relational analysis (GRA) was employed for a comprehensive strain evaluation by calculating grey relational grades. The results demonstrate that weighted relational grade analysis is effective in the comprehensive evaluation of L. edodes strains. The ranking of most cultivars was higher than that of wild strains. Cultivar ZP9 was the best strain, concerning its comprehensive phenotypic performance. Wild strain YS1 with the highest polysaccharide content (6.60%) also ranked in the top seven. In our study, we developed the first multiple-trait strategy for L. edodes strain evaluation. The step-by-step procedures of GRA described here provide an important reference for strain evaluation and selection of other edible mushrooms.

MicroRNAs (miRNA) play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. However, the miRNAs from skin of Andrias davidianus have not been reported. In this study, a small-RNA cDNA library was constructed and sequenced from skin of A. davidianus. A total of 513 conserved miRNAs belonging to 174 families were identified. The remaining 108 miRNAs we identified were novel and likely to be skin tissue-specific but were expressed at low levels. The presence of randomly selected 15 miRNAs identified and their expression in eight different tissues from A. davidianus were validated by stem-loop qRT-PCR. For better understanding the functions of miRNAs, 129,791 predicated target genes were analyzed by GO and their pathways illustrated by KEGG pathway analyses. The results show that these identified miRNAs from A. davidianus skin are involved in a broad range of physiological functions including metabolism, growth, development, and immune responses. This study exhaustively identifies miRNAs and their target genes, which will ultimately pave the way for understanding their role in skin of A. davidianus and other amphibians. Further studies are necessary to better understand miRNA-mediated gene regulation.

Increasing yield is a principal goal when breeding Lentinus edodes. The detection of quantitative trait loci (QTLs) underlying yield and its related traits, precocity and the number of fruiting bodies (NFs), is important in order to breed high-yield cultivars. Using composite interval mapping (CIM), we mapped a total of 25 QTLs responsible for precocity, NFs, and yield in 2 segregating populations of L. edodes. QTLs for the 3 traits were mapped on 5 different linkage groups, contributing 5.9% to 15.4% of the phenotypic variation. Colocated QTLs were also found underlying multiple traits, implying the presence of a genic pleiotropic effect or tightly linked genes. This is, to our knowledge, the first report of the genetic dissection of precocity, NFs, and yield using QTL mapping in L. edodes. Findings of this study will facilitate marker-assisted breeding of high-yield cultivars of L. edodes.

Fish muscle growth is important for the rapidly developing global aquaculture industry, particularly with respect to production and quality. Changes in muscle fibre size are accomplished by altering the balance between protein synthesis and proteolysis. However, our understanding regarding the effects of different protein sources on fish muscle proteins is still limited. Here we report on the proteomic profile of muscle fibre hyperplasia in grass carp fed only with whole faba bean. From the results, a total of 99 significantly changed proteins after muscle hyperplasia increase were identified (p < 0.05, ratio <0.5 or >2). Protein-protein interaction analysis demonstrated the presence of a network containing 56 differentially expressed proteins, and muscle fibre hyperplasia was closely related to a protein-protein network of 12 muscle component proteins. Muscle fibre hyperplasia was also accompanied by decreased abundance in the fatty acid degradation and calcium signalling pathways. In addition, metabolism via the pentose phosphate pathway decreased in grass carp after ingestion of faba bean, leading to haemolysis. These findings could provide a reference for the prevention and treatment of human glucose-6-phosphate dehydrogenase deficiency ("favism").

To provide a better understanding of the genetic architecture of fruiting body formation of Lentinula edodes, quantitative trait loci (QTLs) mapping was employed to uncover the loci underlying seven fruiting body-related traits (FBRTs). An improved L. edodes genetic linkage map, comprising 572 markers on 12 linkage groups with a total map length of 983.7 cM, was constructed by integrating 82 genomic sequence-based insertion-deletion (InDel) markers into a previously published map. We then detected a total of 62 QTLs for seven target traits across two segregating testcross populations, with individual QTLs contributing 5.5 %-30.2 % of the phenotypic variation. Fifty-three out of the 62 QTLs were clustered in six QTL hotspots, suggesting the existence of main genomic regions regulating the morphological characteristics of fruiting bodies in L. edodes. A stable QTL hotspot on MLG2, containing QTLs for all investigated traits, was identified in both testcross populations. QTLs for related traits were frequently co-located on the linkage groups, demonstrating the genetic basis for phenotypic correlation of traits. Meta-QTL (mQTL) analysis was performed and identified 16 mQTLs with refined positions and narrow confidence intervals (CIs). Nine genes, including those encoding MAP kinase, blue-light photoreceptor, riboflavin-aldehyde-forming enzyme and cyclopropane-fatty-acyl-phospholipid synthase, and cytochrome P450s, were likely to be candidate genes controlling the shape of fruiting bodies. The study has improved our understanding of the genetic architecture of fruiting body formation in L. edodes. To our knowledge, this is the first genome-wide QTL detection of FBRTs in L. edodes. The improved genetic map, InDel markers and QTL hotspot regions revealed here will assist considerably in the conduct of future genetic and breeding studies of L. edodes.

The purpose of this study was to investigate the incidence of clinically common EDTA-dependent pseudo-thrombocytopenia (EDTA-PTCP) and methols for treating this diseese. A total of 1326 cases of thrombocytopenia found at blood routine examination were amalyzed anong 71 535 patients hospitalized in our hospital from January 2010 to May 2013, and 87 cases of PTCP caused EDTA-K anticoagulant were analyzed again by using sodium citrate auticoagulant, at the same time the platelet formation distribution was observed by microscopy of smear with Wright-Giemsa staining. The results showed that the platelet count detected by using EDTA-K anticoagulant in 87 cases was (56 ± 27)×10(9)/L, while the platelet count detected by using sodium citrate was (185 ± 39)×10(9)/L (t = 1.83,P < 0.01). The pseudo-thrombocytopenia incidence cansed by EDTA-K was 0.12%, it was 6.56% for the total number of thrombocytopenia. It is concluded that the incidence of PTCP cansed by EDTA-K is 0.12%, the PTCP is easily misdiagnosed. Therefore, the specimens of platelet count <100 10(9)/L should be tested again. When the platelet aggregation is found, the specimens should be examined again by using sodium citrate in order to avoid misdiagnosis.

Grass carp (Ctenopharyngodon idellus) are important Chinese freshwater fish, and in China, the faba bean has been used as the sole food source for grass carp to transform them into crisp grass carp. Because of this, crisp grass carp has become an economically important fish because of its increased muscle hardness. To study the nutritional regulation of type I collagen in faba bean-fed grass carp, we isolated type I collagen alpha 2 (COL1A2) on the basis of our isolation of COL1A1. The COL1A2 cDNA was found to be 4899 bp in length and included a 4059-bp coding sequence (CDS) and encoded a polypeptide of 1352 AA. The protein peptide molecular weight was 127.39 kD, and the theoretical isoelectric point was 9.37. The COL1A2 protein possessed five α-helixes, eight β-sheets, 16 regions of triple helical repeats, 21 low-complexity regions, 10 function domains and two zinc-binding sites; however, no calcium-binding sites were observed. The mRNA expression of COL1A1 and COL1A2 was assessed in eight tissues (muscle, hepatopancreas, intestine, gills, skin, fin, kidney and spleen) from grass carp and crisp grass carp by semi-quantitative RT-PCR. Expression of COL1A1 in the muscle, intestines and skin of crisp grass carp was higher than that in grass carp, and expression of COL1A2 in the muscle, gills, fin and skin of crisp grass carp was higher than that in grass carp. In the muscle of crisp grass carp, expression of COL1A1 and COL1A2 was higher than that in grass carp, which was further confirmed by real-time PCR, and collagen content also was enhanced. These results demonstrated that type I collagen was closely related to the increased muscle hardness of faba bean-fed grass carp., (Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH.)

The most saturated linkage map for Lentinula edodes to date was constructed based on a monokaryotic population of 146 single spore isolates (SSIs) using sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), insertion-deletion (InDel) markers, and the mating-type loci. Five hundred and twenty-four markers were located on 13 linkage groups (LGs). The map spanned a total length of 1006.1 cM, with an average marker spacing of 2.0 cM. Quantitative trait loci (QTLs) mapping was utilized to uncover the loci regulating and controlling the vegetative mycelium growth rate on various synthetic media, and complex medium for commercial cultivation of L. edodes. Two and 13 putative QTLs, identified respectively in the monokaryotic population and two testcross dikaryotic populations, were mapped on seven different LGs. Several vegetative mycelium growth rate-related QTLs uncovered here were clustered on LG4 (Qmgr1, Qdgr1, Qdgr2 and Qdgr9) and LG6 (Qdgr3, Qdgr4 and Qdgr5), implying the presence of main genomic areas responsible for growth rate regulation and control. The QTL hotspot region on LG4 was found to be in close proximity to the region containing the mating-type A (MAT-A) locus. Moreover, Qdgr2 on LG4 was detected on different media, contributing 8.07 %-23.71 % of the phenotypic variation. The present study provides essential information for QTL mapping and marker-assisted selection (MAS) in L. edodes., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate the variants and quasispecies of reverse transcriptase region in polymerase gene of hepatitis B virus (HBV) during lamivudine treatment and their relationship with genotypes and viral loads., Methods: HBV DNA of 117 chronic hepatitis B patients treated with lamivudine were amplified by using PCR. The PCR products including the YMDD motif were sequenced by DNA sequencer, of which, HBV DNA viral loads of 99 patients were determined by real-time PCR and 64 samples were sequenced by Pyrosequencing., Results: In HBV YMDD variant group and no variant group, the HBV genotypes were 79.6% and 86.7% of type C, 18.5% and 12.7% of type B, 1.9% of A/B recombinant type and 2.6% of type D, respectively. The viral loads (log 10) were 6.5699 and 6.6165, respectively. There was no significant difference in HBV genotypes and viral loads between these two groups. The rtL180M variant was found in association with the rtM204I/V variant, HBV variants and wild-type in YMDD motif all existed together in these two groups., Conclusions: HBV variants (quasispecies) in YMDD motif could be quantified by pyrosequencing, which would be a feasible measure during nucleoside or nucleotide analogue therapy against chronic HBV infection.

The target region amplification polymorphism (TRAP) technique was utilized for assessing the genetic diversity of 55 wild strains and one cultivated strain of Lentinula edodes in China. From these strains, 932 DNA fragments were amplified using 12 primer combinations, 929 fragments (99.68%) of which were polymorphic between two or more strains. The average coefficient of pairwise genetic similarity was 0.696, within a range from 0.503 to 0.947. Cluster analysis and principal coordinate analysis separated the tested strains of L. edodes into two major groups. Group A was further divided into seven subgroups. In most cases, the strains from the same or adjoining regions could be preferentially clustered into small groups. The results from the average genetic similarity and the weighted average value of Shannon's Information Index among the tested strains of L. edodes from the same region revealed a vast genetic diversity in the natural germplasm found in China. Compared with the L. edodes strains from other regions, those found on the Yunnan Plateau, in the Hengduanshan Mountains, in Taiwan, South China, and Northeast China showed greater genetic diversity. The results of the present study indicated that the wild strains of L. edodes in China possessed abundant genetic variation, and the genetic relationships among them were highly associated with the geographic distribution. This is the first report demonstrating that TRAP markers were powerful for analyzing the genetic diversity of L. edodes, and the study lays the foundation for a further application of this remarkable technique to other fungi.

Objective: To assess the value of an intracavitary convex array probe in detecting internal carotid artery (ICA) disease., Methods: Eighty-six carotid arteries in 43 cases were examined with intracavitary convex array probe, low-frequency convex array probe and high-frequency linear probe to collect the data including the ICA visible length, peak systolic velocity (PSV), internal diameter, blood vessel shape; common carotid artery (CCA) intimae-medial thickness (IMT), PSV, and internal diameter., Results: Significant differences were noted in the visible length, PSV of ICA, and internal diameter detected by different frequency ultrasound probes. Intracavitary probe and high-frequency probe produced significantly different findings of the blood vessel shape., Conclusion: Intracavitary convex array probe has important clinical value in detecting of ICA disease.

Objective: To investigate the prevalence of antinuclear antibodies (ANA) and anti-liver/ kidney microsomal type 1 antibodies (anti-LKM1) in patients with chronic hepatitis C (CHC)and to explore the mechanism of production of these autoantibodies., Methods: Serum samples were collected from 360 patients with CHC (case group), 69 patients with chronic hepatitis B (CHB) and 69 patients with autoimmune hepatitis (AIH) (control group). Serum ANA and anti-LKM1 were detected by indirect immunofluorescence (HF) technique and enzyme-linked immunosorbent assay (ELISA), respectively. Multi-factor analysis was performed to explore the correlations of the production of autoantibodies with some factors such as age, sex, viral loads, HCV genotype, biochemical parameters and clinical characteristics., Results: Fifty-four (15%) of 360 patients infected with HCV were positive in autoantibodies. The prevalence of ANA and anti-LKM1 were 12.5% (45/360) and 2.5% (9/ 360), respectively. The positive rate of autoantibodies in patients with CHC was significantly higher than that in patients with CHB (15% vs 2.9%, P = 0.006), but significantly lower than that in patients with AIH (15% vs 47.9%, P < 0.001). Twenty-one (11.35%) of 185 male patients and 33 (18.86%) of 175 female patients were positive in autoantibodies, the difference in positive rate was significant (P < 0.05). HCV virus loads in the autoantibodies negative group were higher than that in the autoantibodies positive group (7.2 x 10(7) copies/L vs 1.23 x 10(7) copies/L, P < 0.05). There were not significant differences in age and genotype between the autoantibody positive group and the autoantibody negative group. The serum biochemical parameters of the autoantibody positive group were similar to those of the autoantibody negative group. The differences were not significant for the course of disease, clinical symptom, the incidence of cirrhosis between the autoantibody positive group and the autoantibody negative group. The prevalence of autoantibodies was not different for patients with or without interferon treatment (P > 0.05)., Conclusion: Autoantibodies related to AIH can be detected in CHC patients; interferon may not induce the production of autoantibodies; it is very likely that HCV infection induces the autoimmune reaction and the production of autoantibodies.

Objective: To investigate the feasibility of evaluating hepatic blood perfusion after liver transplantation using color Doppler and contrast-enhanced ultrasonography and the diagnostic value of hepatic blood perfusion for acute graft rejection., Method: In this before-after study with quantitative analysis, contrast-enhanced sonography was performed with simultaneous liver function monitoring in porcine models of allograft liver transplantation before and after the operation., Results: In pigs with acute graft rejection, the AUC decreased significantly after transplantation as compared with that of non-rejection transplant group (F=11.069, P=0.011), but the parameters including A, Alpha, C, t(0), DPI, or TTP showed no significant differences between the two groups., Conclusions: Pulse-inversion contrast harmonic imaging combined with acoustic quantitative technology can be an efficient means for evaluating blood perfusion in the liver allograft, and the TIC obtained using this method possesses high values for quantitative evaluation of liver allograft rejection.

Objective: To study the effect of Jiangzhuo Mixture (JZM) on glucose and lipid metabolism, free fatty acid (FFA) and insulin sensitivity index of sodium glutamate (MSG) induced obese rats., Methods: Sixty-four male MSG rats, 8-10 weeks old, were randomly divided into 4 groups equally, the low dose and high dose JZM group were treated respectively with 10 mL/(kg d) and 20 mL/(kg d) JZM, the rosiglitazone (RGZ) group with water solution of RGE 20 mg/(kg d), while the model group fed only with distilled water 10 mL/(kg d) for control, all by gastrogavage for 7 successive weeks. Levels of fasting blood glucose (FBG), triglyceride (TG), cholesterol (TC), and serum fasting insulin (Ins) were measured before and after treatment. Besides, the level of FFA and the proportion of fat weight to body weight (F/B) were measured and insulin sensitivity index (ISI) was calculated after treatment., Results: After being treated for 7 weeks, the indexes including TC, FBG, Ins, F/B and FFA were all lower than those in the model group. Compared with before treatment, TC level lowered, FBG and Ins level raised in the two JZM groups and the RGZ group. Comparisons between the three treated group showed a significant lower level of Ins in the RGZ group. Level of ISI was significantly lowered in the 3 groups after treatment (P <0.05), but still higher in the RGZ group than that in the model control group (P <0.05). As for level of FFA, it was 314.81 +/- 110.25 micromol/L in the high dose JZM group and 305.56 +/- 92.33 micromol/L in the RGZ group, which were lower than that in the low dose JZM group (375.00 +/- 219.95 micromol/L, P <0.05)., Conclusions: JZM could decrease the serum levels of TC, FBG, Ins and FFA in MSG rats, and decrease the fat content of organism in rats' growth process. The FFA decreasing action is dose-dependent. But its effect on ISI in MSG rats in the growth stage is insignificant. JZM can not reverse the forming processes of hyperglycemia, hyperinsulinemia, and insulin resistance in MSG rats.

Objective: To establish a new method to detect HBV cccDNA quantitatively and to apply it to detect cccDNA in liver needle biopsy specimens of chronic hepatitis B patients., Methods: The sequences of HBV DNA genotypes A through G were analyzed. According to the different sequence structure of cccDNA and rcDNA, primes and probe were designed in highly conservative region outside the nick of cccDNA in order to amplify cccDNA but not rcDNA. The best conditions of this method were found after testing experiments. Also we checked its specificity and sensitivity and reproducibility. The products of PCR were sequenced in order to ascertain if it was the right region expected. To amplify with standard plasmid ranged from 10(2) to 10(10) copies/ml to measure the sensitivity and amplify in parallel with standard plasmid of 10(6) copies/ml for 30 replicates so as to measure its reproducibility. DNA was extracted from 32 needle liver biopsy specimens of chronic hepatitis B patients. The cccDNA was quantitatively detected with this method. The data of cccDNA obtained before and after therapy and their relationship with total HBV DNA were analyzed. RESULTS Results of sequencing showed that the PCR product was from the right region. The sensitivity was 10(3)-10(10) copies/ml. The Ct value was 29.69+/-0.31 and the coefficient of variability was 1.04 percent calculated from the data of 30 PCR reactions with standard plasmid. The percentage of decrease in serum HBV DNA, total HBV DNA in liver and cccDNA in liver were 0.49+/-0.17, 0.22+/-0.18 and 0.16+/-0.28 respectively. There is 47 percent-98 percent cccDNA in total HBV DNA in liver and the mean is 81.5 percent., Conclusion: The method is good because of the simple and convenient operation, the high specificity, the wide linear detection range and the fine reproducibility. Therefore it can be used for both scientific research and clinical purpose. Lamividine can significantly inhibit serum HBV DNA by, but its inhibitory effect on cccDNA in liver was rather weak.

Objective: To determine the distribution and virologic characteristics of HBV genotypes, sub-type and possible association with the severity of liver disease., Methods: 884 patients infected with HBV were enrolled from 8 provinces in China. HBV genotype and sub-type was determined, using PCR-RFLP method., Results: The most common HBV genotypes were B (20.77% ) and C (78.22 % ) but only 1 patient showed genotypes D. We found sub-type Ba in patients with genotype B, C1 and C2 sub-type in patients with genotype C. Genotype C (83.62%) and sub-type C2 (90.32%) were predominant in northern China. Patients with genotype B were much younger than those with genotype C. There was no significant difference between patients with sub-type C1 and C2. There was no significant difference in liver function and serum HBV-DNA load between patients with genotype B and C,or between patients with sub type C1 and C2. However, hepatic inflammation and fibrosis score in patients with genotype B were significantly lower than those with genotype C., Conclusion: There were no significantly differences in liver function and HBV-DNA load between patients with genotype B and C, or between patients with sub-type C1 and C2. Hepatic inflammation and fibrosis score in patients with genotype B were significantly lower than those with genotype C. Genotype C/sub-type C2 were preponderance in northern China.

Aim: To investigate the clinical significance and presence of mutations in the surface (S) and overlapping polymerase gene of hepatitis B patients with coexisting HBsAg and anti-HBs., Methods: Twenty-three patients with chronic hepatitis B were studied. Of the 23 patients, 11 were both positive for hepatitis B virus (HBV) surface antigen (HBsAg) and antibody to HBV surface antigen (anti-HBs), 12 were negative for anti-HBs while positive for HBsAg. DNA was extracted from 200 muL serum of the patients. Nucleotide of the surface and overlapping polymerase gene from HBV-infected patients was amplified by PCR, and the PCR products were sequenced., Results: Forty-one mutations were found within the surface gene protein of HBV in 15 patients (10 with coexisting HBsAg and anti-HBs). Six (14.6%) out of 41 mutations were located at "alpha " determinant region in 5 patients (4 positive for HBsAg and anti-HBs). Eleven mutations (26.8%) occurred in the downstream or upstream of "alpha " determinant region. Lamivudine (LMV)-selected mutations were found in three patients who developed anti-HBs, which occurred in amino acid positions (196, 198, 199) of the surface protein and in YMDD motif (M204I/V) of the polymerase protein simultaneously. Presence of these mutations did not relate to changes in ALT and HBV DNA levels., Conclusion: Besides mutations in the "alpha " deter-minant region, mutations at downstream or upstream of the "alpha " determinant region may contribute to the development of anti-HBs. These mutations do not block the replicating competency of HBV in the presence of high titer of anti-HBs.

The 2.2.15 cells-derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids and virions (Proc Natl Acad Sci U S A 1987; 84: 1005-1009). The latter elicit acute hepatitis in chimpanzees (Proc Natl Acad Sci U S A 1987; 84: 4641-4644). We studied the presence of intracellular and extracellular HBV covalently closed circular (ccc) DNA in this culture system by polymerase chain reaction (PCR), kinetically analysed HBsAg and hepatitis B e antigen (HBeAg) released in the culture media by quantitative enzyme-linked immunosorbent assay and quantitated by real-time PCR but HBV DNA from intracellular and extracellular HBV-DNA. HBV cccDNA was found both intracellularly and extracellularly. A significant correlation was seen between the extracellular HBV DNA levels and virus antigens (r = 0.833; P = 0.01 and r = 0.939; P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no statistical correlation between intracellular HBV DNA levels and virus antigen levels (r = 0.024; P = 0.955 and r = 0.177; P = 0.625 for HBsAg and HBeAg, respectively). These data would be valuable in studies of the HBV life cycle and of potential anti-viral agents.

Objective: To assess the value of intermittent contrast second harmonic imaging (SHI) incorporating acoustic densitometry in the diagnosis of acute renal allograft rejection in comparison with color Doppler flow imaging (CDFI) for determining the resistance index (RI)., Methods: Eight canine models of acute renal allograft rejection were established, subjected subsequently to examinations with SHI and acoustic densitometry to determine the acoustic density. Color flow Doppler was also performed to determine RI, and serum creatinine (Cr) levels were measured., Results: The time-intensity curve (TIC) showed that the area under the curve, the peak intensity (PI) and RI all had linear correlation with serum Cr levels, with the correlation coefficient gamma of 0.978, 0.972 and 0.708 respectively (by SPSS10.0)., Conclusion: Intermittent contrast SHI is effective to evaluate the perfusion of renal allograft, and when combined with acoustic densitometry, the resultant TIC parameters are in closer correlation with acute renal allograft rejection than RI.

LC1 is a type of novel antibacterial polypeptide secreted by a Bacillus subtilis strain. It consists of 47 residues. Using bioengineering, LC1 was expressed in E.coli DH5alpha by using recombinant plasmid PBVAB16. By means of two-dimensional DQF-COSY, TOCSY and NOESY spectroscopies, protons of all 47 residues are identified. The studies show that the secondary structures of LC1 are principally anti-parallel beta sheets and extended conformations. It was speculated that there may be a hydrophobic core around Trp(23) in its three-dimensional structure.

LCI is a type of novel antibacterial polypeptide secreted by a Bacillus subtilis strain. It consists of 47 residues with a molecular weight of 5468 Da. Using bioengineering, LCI was expressed in Escherichia coli DH5alpha with recombinant plasmid pBVAB16. It was crystallized using PEG 4000 as a precipitant. The crystal belongs to space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 29.30, c = 187.09 A, and diffracts to 2.44 A. A set of diffraction data to 2.8 A was collected.