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Apis cerana in Changbai Mountain is an ecological type of Apis cerana, which is an excellent breeding material with cold-resistant developed by long-term natural selection under the ecological conditions. However, the physiological and molecular mechanisms of Changbai Mountain population under cold stress are still unclear. In this study, the Nanopore sequencing was carried out for the transcriptome of Apis cerana in Changbai Mountain in the coldest period of overwintering, which will provide a reference to the cold-resistant mechanism. We determined 5,941 complete ORF sequences, 1,193 lncRNAs, 619 TFs, 10,866 SSRs and functional annotations of 11,599 new transcripts. Our results showed that the myosin family and the C2H2 zinc finger protein transcription factor family possibly have significant impacts on the response mechanism of cold stress during overwintering. In addition, the cold environment alters genes expression profiles in honeybees via different AS and APA mechanisms. These altered genes in Hippo, Foxo, and MARK pathways help them counter the stress of cold in overwinter period. Our results might provide clues about the response of eastern honeybees to extreme cold, and reflect the possible genetic basis of physiological changes., (Published by Elsevier B.V.)

Background: As of June 1, 2020, over 370000 coronavirus disease 2019 (COVID-19) deaths have been reported to the World Health Organization. However, the risk factors for patients with moderate-to-severe or severe-to-critical COVID-19 remain unclear., Aim: To explore the characteristics and predictive markers of severely and critically ill patients with COVID-19., Methods: A retrospective study was conducted at the B11 Zhongfaxincheng campus and E1-3 Guanggu campus of Tongji Hospital affiliated with Huazhong University of Science and Technology in Wuhan. Patients with COVID-19 admitted from 1 st February 2020 to 8 th March 2020 were enrolled and categorized into 3 groups: The moderate group, severe group and critically ill group. Epidemiological data, demographic data, clinical symptoms and outcomes, complications, laboratory tests and radiographic examinations were collected retrospectively from the hospital information system and then compared between groups., Results: A total of 126 patients were enrolled. There were 59 in the moderate group, 49 in the severe group, and 18 in the critically ill group. Multivariate logistic regression analysis showed that age [odd ratio (OR) = 1.055, 95% (confidence interval) CI: 1.099-1.104], elevated neutrophil-to-lymphocyte ratios (OR = 4.019, 95%CI: 1.045-15.467) and elevated high-sensitivity cardiac troponin I (OR = 10.126, 95%CI: 1.088 -94.247) were high-risk factors., Conclusion: The following indicators can help clinicians identify patients with severe COVID-19 at an early stage: age, an elevated neutrophil-to-lymphocyte ratio and high sensitivity cardiac troponin I., Competing Interests: Conflict-of-interest statement: None reported., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Clonorchiasis, which is caused by Clonorchis sinensis , is an important foodborne disease worldwide. The excretory-secretory products (ESPs) of C. sinensis play important roles in host-parasite interactions by acting as causative agents. In the present study, the ESPs and sera positive for C. sinensis were collected to identify proteins specific to the sera of C. sinensis (i.e., proteins that do not cross-react with Fasciola hepatica and Schistosoma japonicum ) at different infection periods. Briefly, white Japanese rabbits were artificially infected with C. sinensis , and their sera were collected at 7 days post-infection (dpi), 14 dpi, 35 dpi, and 77 dpi. To identify the specific proteins in C. sinensis , a co-immunoprecipitation (Co-IP) assay was conducted using shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) to pull down the sera roots of C. sinensis , F. hepatica , and S. japonicum . For the annotated proteins, 32, 18, 39, and 35 proteins specific to C. sinensis were pulled down by the infected sera at 7, 14, 35, and 77 dpi, respectively. Three proteins, Dynein light chain-1, Dynein light chain-2 and Myoferlin were detected in all infection periods. Of these proteins, myoferlin is known to be overexpressed in several human cancers and could be a promising biomarker and therapeutic target for cancer cases. Accordingly, this protein was selected for further studies. To achieve a better expression, myoferlin was truncated into two parts, Myof1 and Myof2 (1,500 bp and 810 bp), based on the antigenic epitopes provided by bioinformatics. The estimated molecular weight of the recombinant proteins was 57.3 ku (Myof1) and 31.3 ku (Myof2). Further, both Myof1 and Myof2 could be probed by the sera from rabbits infected with C. sinensis . No cross-reaction occurred with the positive sera of S. japonica , F. hepatica , and negative controls. Such findings indicate that myoferlin may be an important diagnostic antigen present in the ESPs. Overall, the present study provides new insights into proteomic changes between ESPs and hosts in different infection periods by LC-MS/MS. Moreover, myoferlin, as a biomarker, may be used to develop an objective method for future diagnosis of clonorchiasis., 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 Ma, Qiu, Chang, Gao, Jiang, Li, Wang and Chang.)

Temperature is a key environmental factor, and understanding how its fluctuations affect physiological and metabolic processes is critical for fish. The present study characterizes the energy response and fatty acid metabolism in Onychostoma macrolepis exposed to low temperature (10 °C). The results demonstrated that cold stress remarkably disrupted the energy homeostasis of O. macrolepis, then the AMP-activated protein kinase (AMPK) could strategically mobilize carbohydrates and lipids. In particular, when the O. macrolepis were faced with cold stress, the lipolysis was stimulated along with the enhanced fatty acid β-oxidation for energy, while the fatty acid synthesis was supressed in the early stage. Additionally, the fatty acid composition analysis suggested that saturated fatty acid (SFA) might accumulate while monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) in storage lipids (mainly containing non-polar lipid, NPL) could be utilized to supply energy during cold acclimation. Altogether, this study may provide some meritorious for understanding the cold-tolerant mechanism of fish in the viewpoint of energy balance combined with fatty acid metabolism, and thus to contribute to this species rearing in fish farms in the future., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Recombinant activated factor VIIa (rFVIIa) is a prohemostatic agent initially approved for use in hemophilia patients and has also been used for a diverse range of off-label indications in the context of massive uncontrolled blood loss; however, no convincing evidence exists regarding the optimal dose of rFVIIa to treat uncontrolled bleeding in surgical patients., Aim: To evaluate the effects and safety of a very low dose of rFⅦa in patients with uncontrolled perioperative bleeding in the surgical intensive care unit (ICU)., Methods: 55 patients from Beijing Hospital, who received rFⅦa between July 2004 and November 2018 for uncontrolled perioperative bleeding were included. The controls were matched for age, sex, severity, and operation type. The baseline demographics, survival, changes in bleeding and transfusion, coagulation parameters and complications were analyzed., Results: A low dose of rFⅦa (2.0∼3.6 mg, with a median dose of 39.02 μg/kg) appears to be effective in controlling massive hemorrhage (with an effective rate of 74.55%), and can reduce volume of red blood cell transfusion, improve coagulation status, while has a relatively low risk of thromboembolic complications (3.6%)., Conclusion: In patients with uncontrolled perioperative bleeding, a low dose of rFⅦa could be used when traditional methods are ineffective., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Considering the excessive lipid accumulation status caused by the increased dietary lipid intake in farmed fish, this study aimed to investigate the systemic effect of dietary lipid levels and α-lipoic acid supplementation on nutritional metabolism in zebrafish. A total of 540 male zebrafish (0.17 g) were fed with normal (CT) and high lipid level (HL) diets for 6 weeks, then fed on 1000 mg/kg α-lipoic acid supplementation diets for the second 6 weeks. HL diets did not affect whole fish protein content, but increased ASNS expression (P < 0.05). Dietary α-lipoic acid increased whole fish protein content, and decreased the expressions of protein catabolism-related genes in muscle of high lipid level groups (P < 0.05). Furthermore, HL diets increased the whole fish lipid content and the expressions of gluconeogenesis and lipogenesis-related genes (P < 0.05), and α-lipoic acid counteracted these effects and decreased the whole fish triglyceride and cholesterol contents and expressions of lipogenesis-related genes, with the enhanced expressions of lipolytic genes, especially in high lipid groups (P < 0.05). HL diets did not affect hepatocyte mitochondrial quantity or the mRNA expressions of mitochondrial biogenesis and electron transport chain-related genes; they were significantly increased by dietary α-lipoic acid (P < 0.05). These results indicated that high dietary lipid promotes lipid accumulation, while α-lipoic acid increases protein content in association of enhanced lipid catabolism. Thus, dietary α-lipoic acid supplementation could reduce lipid accumulation under high lipid, which provides a promising new approach in solving the problem of lipid accumulation in farmed fish.

Objective: To compare the effects of supplemental parenteral nutrition (SPN) and enteral nutrition (EN) on prognosis of critically ill patients in intensive care unit (ICU) using meta-analysis. Methods: Foreign language databases including PubMed, Embase, Cochrane Library, and Cochrane Central Register of Controlled Trials were retrieved with the search terms of " supplemental parenteral nutrition, parenteral nutrition, enteral nutrition, critically ill" , and Chinese database SinoMed database was retrieved with the search terms of ",,," to obtain the publicly published randomized controlled trials about the effects of SPN and EN supportive treatment on prognosis of critically ill patients in ICU from the establishment of each database to December 2018. The Google Scholar was retrieved for supplement. The outcome indexes included the infection rate, anti-infection time, antibiotic-free time, ICU overall mortality, overall mortality during hospitalization, mechanical ventilation time, length of ICU stay, and length of hospital stay. RevMan 5.3 and Stata 14.0 statistical software were used to conduct a meta-analysis of eligible studies. Results: A total of 794 patients were included in 8 studies, including 387 patients in SPN group who received SPN and EN and 407 patients in EN group who only received EN. The bias risks of the eight studies included were uncertain. Compared with that of EN group, the infection rate of patients in SPN group was significantly decreased (relative risk=0.79, 95% confidence interval=0.66-0.94, P <0.01). However, there were no statistically significant differences in ICU overall mortality, overall mortality during hospitalization, mechanical ventilation time, length of ICU stay, and length of hospital stay of patients between SPN group and EN group. The subgroup analysis showed that the risks of bias of studies and follow-up time might be sources of the heterogeneity of mechanical ventilation time. There was publication bias in ICU overall mortality ( P <0.05), while no publication bias in the other outcome indexes ( P >0.05). Conclusions: SPN supportive treatment can decrease the infection rate of critically ill patients in ICU, but it has no obvious influences on overall mortality, mechanical ventilation time, and length of hospital stay.

Fatty acid metabolism is crucial for maintaining energy homeostasis in aquatic vertebrates experiencing environmental stress. Both sterol regulatory element-binding protein 1 (SREBP-1) and peroxisome proliferator-activated receptor α (PPARα) are the key regulators of fatty acid metabolism. In this study, the coding sequences (CDS) of SREBP-1 and PPARα were firstly identified and characterized from Onychostoma macrolepis, encoding peptides of 1136 and 470 amino acids, respectively. The functional domains in O. macrolepis SREBP-1 and PPARα proteins retained the high similarity with those of other animals, at 74.69% and 77.29%, respectively. The mRNA encoding SREBP-1 was primarily expressed in the muscle and PPARα was highly expressed in the liver and intestine. Under thermal exposure, the content of non-esterified fatty acid (NEFA) decreased gradually after 1 h in the liver and muscle of O. macrolepis, which might be due to that the organism meet more energy expenditure via fatty acid β-oxidation. Furthermore, the mRNA expression level of SREBP-1 decreased, while the mRNA expression level of PPARα increased from 0 h to 6 h in the liver. And we found that the mRNA expression levels of both SREBP-1 and PPARα decreased significantly at 48 h (P < .05) in the muscle, which was in accordance with the significant decrease of target gene FAS and CPT1A mRNA expression levels, respectively. It might be the physiological adjustment that the fish adapted to thermal exposure at the end of experiment. These results illustrate that O. macrolepis SREBP-1 and PPARα-mediated fatty acid metabolism is a fundamental requirement for thermal adaptation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To explore the relevance between the expression of C-MYC gene and protein of patients with T lymphoblastic lymphoma and leukemia(T-LBL/ALL) and its effect on the prognosis., Methods: Paraffin specimens from 60 cases of T-LBL/ALL with detailed follow-up during May 2005 to May 2016 were selected as study group; at same time 20 cases of reactive hyperplasia (RH) of lymphonuedes were selected as control group. The immunohistochemical EnVision method was used to mark the terminal deoxynucleotidyl transferase (TDT), myeloperoxidase (MPO), Ki-67 and C-MYC immune tissue., Results: C-MYC gene rupture and copy number increase did not occur in 20 cases of RH.The expression of C-MYC protein did not correlate with C-MYC gene copy number increase. The expression rate of C-MYC protein was 66.7% (40/60), and 20 cases of lymph node RH was all negative (0/20), as compared with the positive expression rate of protein C-MYC, the difference was statistically significant (P<0.05). The Ki-67 positive index and mediastinal bloadening had influence on the expression of C-MYC protein (P<0.05), the sex, primary site, symptoms, age, AnnArbor stage and lactate dehydrogenase (LDH) level and bone marrow involvement have no influence on it, there was no statistically significant difference (P>0.05). The 8q24 chromosome breakage occurred in 6 cases (10%), and the number of copies increased in 11 cases (18.3%). C-MYC gene copy number increase and C-MYC gene rupture in a total 20 cases of reactive hyperplasia of lymph nodes did not occur., Conclusion: C-MYC gene may play an important role on the development of T-LBL/ALL. It can be an independent prognosis factor.

This study investigated the structure and thermal, electrical, optical, and adhesive properties of two magnetic CoFeB thin films with compositions of Co40Fe40B20 and Co60Fe20B20.The thin films were deposited on a glass substrate by using direct current (DC) magnetron sputtering at room temperature (RT) and ranged in thicknesses from 25 to 200 Å. X-ray diffraction (XRD) patterns indicated that the thin films were amorphous. The activation energy (Q) of the Co40Fe40B20 and Co60Fe20B20 thin films exhibited concave up and concave down trends, respectively. The critical thickness of the films was 75 Å. The 75-Å-thick Co60Fe20B20 thin film exhibited the highest Q value, indicating that transforming the amorphous structure into a crystalline structure is difficult. When the Co concentration ratio was increased, the stability of the amorphous state of CoFeB increased apparently. The 75-Å-thick Co60Fe20B20 thin film exhibited the highest resistivity, whereas the 75-Å-thick Co40Fe40B20 thin film exhibited the lowest resistivity. As the thickness of the Co40Fe40B20 and Co60Fe20B20 thin films was increased, the transmittance decreased and absorbance increased. The Co60Fe20B20 thin film exhibited a higher surface energy and stronger adhesion than did the Co40Fe40B20 thin film.

Factor forkhead box O1 (FoxO1) is a transcription factor and plays an important role in insulin-mediated lipid metabolism. In the present study, two distinct FoxO1 cDNAs, designated FoxO1a and FoxO1b, were firstly isolated and characterized from grass carp Ctenopharyngodon idella, encoding peptides of 654 and 631 amino acids, respectively. Phylogenetic and synteny analyses suggested that FoxO1a and FoxO1b were derived from paralogous genes that could be originated from teleost-specific genome duplication (TSGD) event. Analysis of the exon-intron structures clarified that grass carp FoxO1a and FoxO1b comprise 3 coding exons and contain a extra intron compared with human and mouse FoxO1. Both FoxO1a and FoxO1b mRNAs were expressed in a wide range of tissues, but the abundance of each FoxO1 mRNA showed the tissue- dependent expression patterns. Time-course analysis of FoxO1 expressions indicated that the level of FoxO1a mRNA reached almost maximal level at day 2, while that of FoxO1b mRNA reached almost maximal level at day 4 during grass carp primary preadipocyte differentiation. In insulin-inhibited adipocyte lipolysis, only FoxO1a showed a significant decrease in adipocyte, indicating that two FoxO1 isoforms may serve somewhat different roles in the regulation of lipolysis by insulin. These results suggested that grass carp FoxO1a and FoxO1b may play different roles in tissues, and their expression levels were differently modulated by insulin in adipocyte., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The carnitine palmitoyltransferase (CPT) gene family plays an essential role in fatty acid β-oxidation in the mitochondrion. We identified six isoforms of the CPT family in grass carp and obtained their complete coding sequences (CDS). The isoforms included CPT 1α1a, CPT 1α1b, CPT 1α2a, CPT 1α2b, CPT 1β, and CPT 2, which may have resulted from fish-specific genome duplication. Sequence analysis showed that the predicted protein structure was different among the CPT gene family members in grass carp. The N-terminal domain of grass carp CPT 1α1a, CPT 1α1b, CPT 1α2a, and CPT 1α2b contained two transmembrane region domains and two acyltransferase choActase domains that exist in human and mouse proteins also; however, only one acyltransferase choActase domain was found in grass carp CPT 1β. The grass carp CPT 2 had two acyltransferase choActase domains. The grass carp CPT 1α1b, CPT 1α2a, CPT 1α2b, and CPT 1β contained 18 coding exons, while CPT 1α1a and CPT 2 consisted of 17 coding exons and 5 coding exons, respectively. The mRNA of the six CPT isoforms was expressed in a wide range of tissues, but the mRNA abundance of each CPT showed tissue-dependent expression patterns. The expression of CPT 1α1a, CPT 1α2a, and CPT 1β at 48h post-feeding was significantly increased in the liver (P<0.01, P<0.05, and P<0.01, respectively). The diverse responses of multiple isoforms in the liver during nutritional limitation suggest that they may play different roles in fatty acid β-oxidation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

TRIP (Tumor Necrosis Factor (TNF) Receptor-Associated Factor (TRAF)-Interacting Protein), a member of the TNF superfamily, plays a crucial role in the modulation of inflammation in vertebrates. However, no information about TRIP is available in teleosts. In this study, the full-length cDNA of TRIP, containing a 5'UTR of 112 bp, an ORF of 1359 bp, and a 3'UTR of 29 bp before the poly (A) tail, was cloned from grass carp, Ctenopharyngodon idella. The TRIP gene encoded a protein of 452 amino acids with an estimated molecular mass of 51.06 KD and a predicted theoretical isoelectric point (pI) of 9.11. Quantitative real-time PCR analysis revealed that TRIP mRNA was expressed in all the tissues examined in grass carp, with the highest expression in the kidney, followed by the intestine and thymus. However, lower levels of expression were also detected in fat, spleen, liver, gonad and heart. Subcellular localization and two-hybrid analysis revealed that TRIP was located in the nucleus and that it interacted with TRAF1 and TRAF2 in HEK293T cells. Furthermore, similar to TNF-α, IL-10 and TRIP mRNA expression was upregulated in the spleen of fish fed high-fat or high-carbohydrate diets, suggesting that TRIP might be associated with the response to excessive energy intake. The mRNA relative expression of TRIP was significantly reduced (P < 0.05) after hepatocyte of C. idella was treated with 2 μg/mL lipopolysaccharide (LPS) for 4 h, while the expression levels of inflammatory cytokines TNF-α and IL-10 were significantly increased (P < 0.05). Taken together, these results indicate that TRIP might play important roles in immune defense and has the potential to be used as a anti-inflammation target in grass carp., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This investigation studies CoFeB/AlO x /Co magnetic tunneling junction (MTJ) in the magnetic field of a low-frequency alternating current, for various thicknesses of the barrier layer AlO x . The low-frequency alternate-current magnetic susceptibility (χ ac ) and phase angle (θ) of the CoFeB/AlO x /Co MTJ are determined using an c ac analyzer. The driving frequency ranges from 10 to 25,000 Hz. These multilayered MTJs are deposited on a silicon substrate using a DC and RF magnetron sputtering system. Barrier layer thicknesses are 22, 26, and 30 Å. The X-ray diffraction patterns (XRD) include a main peak at 2θ = 44.7° from hexagonal close-packed (HCP) Co with a highly (0002) textured structure, with AlO x and CoFeB as amorphous phases. The full width at half maximum (FWHM) of the Co(0002) peak, decreases as the AlO x thickness increases; revealing that the Co layer becomes more crystalline with increasing thickness. χ ac result demonstrates that the optimal resonance frequency ( f res ) that maximizes the χ ac value is 500 Hz. As the frequency increases to 1000 Hz, the susceptibility decreases rapidly. However, when the frequency increases over 1000 Hz, the susceptibility sharply declines, and almost closes to zero. The experimental results reveal that the mean optimal susceptibility is 1.87 at an AlO x barrier layer thickness of 30 Å because the Co(0002) texture induces magneto-anisotropy, which improves the indirect CoFeB and Co spin exchange-coupling strength and the χ ac value. The results concerning magnetism indicate that the magnetic characteristics are related to the crystallinity of Co.

This study determined the expression of genes involved in mitochondrial function and adipogenesis at mRNA and protein levels by transfecting rat differentiating preadipocytes with siRNA/Lipofectamine complex and pcDNA-PGC-1β (peroxisome proliferator-activated receptor-γ coactivator-1β)/Lipofectamine complex, respectively, to further elucidate the role of PGC-1β in white preadipocyte differentiation. The results showed that the transfection of PGC-1β siRNA inhibited the expressions of mitochondrial genes malate dehydrogenase, carnitine palmitoyltransferase 1, nuclear respiratory factor 1, ATP synthesis, adipocyte differentiation key transcription factor peroxisome proliferator-activated receptor-γ, sterol regulatory element binding protein 1c and fatty acid synthetase, whereas the triglyceride synthesis was retarded (p < 0.05). Furthermore, overexpression of PGC-1β up-regulated the expressions of adipogenic and mitochondrial biosynthetic marker genes and promoted triglyceride accumulation during 3T3-L1 adipocyte differentiation. These observations suggest that PGC-1β modulates the expression of mitochondrial function and adipogenesis-related genes and affects white preadipocyte differentiation., (© 2011 Blackwell Verlag GmbH.)

Background: Aberrant expression of epidermal growth factor receptor (EGFR) has been detected in pancreatic cancer; however, the mechanisms of EGFR in inducing pancreatic cancer development have not been adequately elucidated. The objective of this study was to determine the role of EGFR in mediating epithelial-mesenchymal transition (EMT) in pancreatic cancer cells., Methods: Pancreatic cancer cell line PANC-1 was transfected with small interfering RNA of EGFR by use of a lentiviral expression vector to establish an EGFR-knockdown cell line (si-PANC-1). PANC-1 cells transfected with lentiviral vector expressing negative control sequence were used as negative control (NC-PANC-1). Scratch assay and transwell study were used to analyze cell migration and invasion. Real-time PCR and Western blotting were used to detect the expression of EMT markers E-cadherin, N-cadherin, vimentin, and fibronectin and transcription factors snail, slug, twist1, and sip1 in PANC-1, NC-PANC-1, and si-PANC-1 cells. Immunofluorescent staining with these antibodies and confocal microscopy were used to observe their cellular location and morphologic changes., Results: After RNA interference of EGFR, the migration and invasion ability of si-PANC-1 cells decreased significantly. The expression of epithelial phenotype marker E-cadherin increased and the expression of mesenchymal phenotype markers N-cadherin, vimentin, and fibronectin decreased, indicating reversion of EMT. We also observed intracellular translocation of E-cadherin. Expression of transcription factors snail and slug in si-PANC-1 cells decreased significantly., Conclusion: Suppression of EGFR expression can significantly inhibit EMT of pancreatic cancer PANC-1 cells. The mechanism may be related with the down-regulation of the expression of transcription factors snail and slug.

Objective: To explore the blocking effects of hedgehog signaling pathway on the processes of cell migration, invasion and epithelial-mesenchymal transition (EMT) in human pancreatic cancer cells and elucidate its possible mechanisms., Methods: The lentiviral expression vector for RNA interference of human Smoothened (SMO) gene was constructed to silence the expression of SMO. And RNAi against SMO was used to suppress the hedgehog signaling pathway in human pancreatic cancer Panc-1 cells. The in vitro invasion capacity in Panc-1 cells was assessed by Matrigel/Transwell chamber assay. Real-time PCR (polymerase chain reaction) and Western blot were used to detect the expressions of such EMT markers as E-cadherin, N-cadherin, β-catenin, vimentin and fibronectin and such transcription factors as Snail, Slug, Twist1 and Sip1., Results: The stable interference of SMO could suppress the hedgehog signaling activity in Panc-1 cells. The inhibition of hedgehog signaling reduced the in vitro invasion capacity significantly in Panc-1 cells. The expression of E-cadherin significantly increased while N-cadherin, vimentin and fibronectin were significantly down-regulated in the RNAi group. Compared to the control group, the expressions of Snail and Slug were significantly reduced in the SMO knock-down group., Conclusion: The inhibition of hedgehog signaling pathway reduces the in vitro invasion capacity in human pancreatic cancer cells. And the EMT process is significantly suppressed. The mechanism is partially correlated with the down-regulations of Snail and Slug.

Objective: To clone and express three VAR2CSA duffy antigen-binding ligand (DBL) domains (DBL4/ 5/6) encoded by var2csa gene of a Hainan isolate of Plasmodium falciparum, and study the difference of chondroitin sulfate A (CSA)-binding activity among them., Methods: Three DBL domains was amplified by PCR and cloned into the vector pMD18-T. The recombinant plasmids were identified by enzyme digestion and sequencing, and then subcloned into the prokaryotic expression vector pET-22b. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was purified with His GraciTrap kit and identified by SDS-PAGE and Western blotting. CSA-binding activity of the three recombinant DBL domains was assayed by ELISA., Results: The target genes were amplified with the length of 996 bp, 859 bp and 894 bp. The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. The recombinant proteins (DBL4/5/6) were purified, the relative molecular mass of DBLfA, DBL5 and DBL6 was Mr 439 800, Mr 34,500 and Mr 36,000, respectively. The purified protein has been confirmed with immunogenicity by Western blotting. The results of adhesion experiment indicated that A405 values of DBL5 domain with different concentration were significantly higher than that of DBLA and DBL6., Conclusion: The three recombinant proteins (DBLA/5/6) of VAR2CSA DBL domains were expressed, and DBL5 domain has high binding affinity with CSA.

Mitochondrial biogenesis is inherent to adipocyte differentiation. Mitochondrial dysfunction leads to abnormal lipid accumulation or the deterioration of the differentiation process. The aim of this study is to investigate the mitochondrial development during the differentiation of rat primary adipocytes and the effect of mitochondrial dysfunction on this process. We found, for the first time, that the number of mitochondria markedly increased during adipocyte differentiation by transmission electron microscopy. By immunofluorescence staining that the protein content of Cyt c increased in differentiated adipocyte in comparison with preadipocyte. The mRNA expression levels of mitochondrial gene including cytochromes c (Cyt c), malate dehydrogenases (MDH), and peroxisome proliferator activated receptor (PPAR) gamma coactivator-1beta (PGC-1beta) significantly increased along with the proceeding of adipocyte differentiation. The damage to mitochondrial respiratory chain function by rotenone caused significant decrease in gene expressions including mitochondrial MDH and PGC-1beta, and PPARgamma, CAAT/enhancer binding protein alpha (C/EBPalpha) and sterol regulatory element binding protein-1c (SREBP-1c), which are known as transcription factors of differentiation, and differentiation marker gene named fatty acid synthetase. Moreover, an apparent decrease was found in the synthesis of triglyceride and ATP due to the damage to mitochondria by rotenone. Based on the above results, our present study revealed that the density and oxidative capacity of mitochondrial markedly increased during primary adipocyte differentiation, and on the other hand, we suggested that mitochondria dysfunction might inhibit the differentiation process.

Objective: To compare the use of subclavian vein catheter and femoral vein catheter, in monitoring pulse indicator continuous cardiac output (PiCCO) monitoring data cardiac index (CI), extravascular lung water index (EVLWI), and global end-diastolic volume index (GEDVI) with central venous injection of the bolus cold saline injection, in order to determine whether the femoral vein access, which is not typically used, could be used to obtain reliable data., Methods: Thirteen patients in Beijing Hospital intensive care unit (ICU) were involved, from January 2007 to March 2009. Each patient was monitored with PiCCOplus device, after an injection of cold saline bolus via both femoral and subclavian venous catheter. Paired t-test and Bland-Altman analysis were used to compare CI, EVLWI and GEDVI values., Results: Data of 39 measurements were collected. The bias between femoral injection and subclavian injection were CI (0.28+/-0.46) L x min(-1) x m(-2), EVLWI (1.05+/-1.89) ml/kg, GEDVI (195.2+/-105.7) ml/m(2), and they were statistically significant (P values was 0.000 5, 0.001 3, <0.0001, respectively). The Bland-Altman analysis showed an clinically overestimation of GEDVI after femoral injection (limit of concordance was -11.9, 402.3), compared with that after subclavian injection., Conclusion: Measurements with a cold saline bolus via a femoral catheter, compared to those via a subclavian catheter, lead to overestimation of CI, EVLWI and GEDVI values, and a great bias of GEDVI should be taken into account in clinical work.

The aim of this study was to investigate if there were any effects on the electroencephalogram (EEG) of human brain by the manual stimulation of Neiguan (PC 6) acupuncture site. In this paper, two groups of six healthy male volunteers of ages 27.6 +/- 14.2 (mean +/- SD) and 28.5 +/- 13.0 (mean +/- SD) and no neurological disease participated in this study. A digital storage of 12-channel EEG recorder was used and spectral analyses of the data set of 18 trials were obtained before, during, and after sham/ manual acupuncture. To minimize artefacts, all data were collected with the subjects alert but eyes closed. No significant changes (P > 0.05) were obtained for the sham acupuncture group. As for the manual acupuncture group, the needle was inserted perpendicularly into the PC 6 acupuncture site and manually stimulated about 15 to 30 seconds to achieve De Qi sensation. Needles were left in place for 30 min and then removed. Analysis of the EEG data due to acupuncture was compared to the baseline data and changes were obtained. First, all trials had an increase in the amplitude and power of the alpha band during manual acupuncture (P < 0.05) when compared with the baseline data. Secondly, in the mean time, the frequency peaks in alpha band of 12-channels were all synchronized with much smaller standard deviation (P < 0.01). Thirdly, the manual acupuncture effects of higher power and synchronized frequencies persisted for at least 10 minutes after the experiment (P < 0.05) and did not disappear immediately for all 18 experiments. Finally, we hypothesized that the higher power and synchronized rhythms in brain oscillations may have to do with autonomic nervous system.

Objective: To explore the safety and clinical efficacy of intravenous infusion of concentrated potassium chloride using micro-pumps in critically ill patients with hypokalemia., Methods: One hundred and twenty-eight critically ill patients with hypokalemia, the endogenous creatinine clearance rate over 0.5 ml/second and the urine output over 50 ml/hour were randomly divided into the therapy group (n=64) and the control group (n=64). Patients in therapy group received 1,208 mmol/L (9%) KCl, while those in the control group received 201 mmol/L (1.5%) potassium chloride, intravenously with the aid of a micro-pump, with hourly equal quantity of KCl in both groups. Patients in both groups were monitored strictly, and the potassium infusion was stopped whenever the serum potassium exceeded or equal to 3.5 mmol/L., Results: It took (15.55+/-3.22) hours and (14.18+/-4.93) hours for the therapy group and the control group to correct the hypokalemia respectively, and there was no significant difference (P>0.05). Potassium infusion brought larger amount of fluid in the control group than the therapy group [(124.36+/-25.79) ml vs. (680.83+/-236.70) ml, P<0.01]. All patients tolerated the infusion without evidence of hemodynamic change, hyperkalemia or acute heart dysfunction. For all the patients, renal function did not throw significant influence on the potassium infusion time. An inverse correlation was observed between preinfusion potassium concentration and the quantity of potassium infused (r= -0.259, P<0.01)., Conclusion: Under meticulous monitoring, it is safe and effective to infuse concentrated potassium for the critically ill patients with hypokalemia. This strategy can also be followed in patients with mild renal dysfunction but without oliguria or anuria under careful monitoring.

Purpose: Because of its role in cell migration, the Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 has been implicated in cancer metastasis. Evidence to support such a role of WAVE2 in human cancer, however, is lacking. We thus examined the expression of WAVE2 in hepatocellular carcinoma (HCC) tissues to test whether the levels of WAVE2 expression correlated to the progression of HCC., Experimental Design: Samples of 112 HCC patients were determined immunohistochemically for WAVE2 expression and the correlation of WAVE2 levels with prognosis was analyzed. Among the 112 cases, 31 paired HCC and paracarcinomatous liver tissue specimens were analyzed for WAVE2 levels by reverse transcription-PCR and Western blotting, respectively., Results: Among 112 cases of HCCs, the immunohistochemistry data indicated significant increase of WAVE2 expression levels in 71 cases. Importantly, the increased WAVE2 expression correlated with the multiple tumor nodules (P = 0.008), the absence of capsular formation (P = 0.035), Edmondson-Steiner grade (P = 0.009), vein invasion (P = 0.023), and a shortened median survival time (326 versus 512 days; P = 0.003). Multivariable Cox regression analysis revealed the WAVE2 expression level was an independent factor for prognosis. The immunohistochemistry data were further confirmed by results of reverse transcription-PCR and Western analysis of 31 HCC cases, in which the WAVE2 mRNA and protein in HCC tissues were significantly elevated when compared with paracarcinomatous liver tissue (P < 0.001)., Conclusions: WAVE2 expression is elevated in HCC tissues, which correlates with a poor prognosis, suggesting WAVE2 as a candidate prognostic marker of HCC.

Aims: To establish a sustaining hepatitis B virus X protein expressed Chang liver cell line and to explore their biological behaviours of invasive potential induced by hepatitis B virus X protein., Methods: Polymerase chain reaction was used to amplify the HBx gene from the whole hepatitis B virus genome. The gene was then subcloned into the eukaryotic expression vector pcDNA3.1 to construct the pcDNA3.1-HBx plasmid. Gene transfection mediated by Lipofectamine was used to introduce the plasmid into the human liver cell line Chang, and stable expression of the HBx gene was detected., Results: HBx gene was cloned from the transfected Chang liver cells by reverse transcription-polymerase chain reaction, and confirmed by electrophoresis. The stably transfected Chang cells expressing HBx with malignant characteristics were verified and compared with control cells in terms of their growth curves, clonogenicity, wound healing abilities, migration and metastasis., Conclusion: The stabilising human liver cell lines Chang liver containing HBx gene expression have been established successfully. The invasive potential of Chang cells was conditionally enhanced by HBx transfection.

Our objective was to investigate the expression of HMGA1 mRNA and protein in hepatocellular carcinoma (HCC) and the correlation between its expression and clinical pathological characteristics and prognosis. HMGA1 expression was determined at both the mRNA level and the protein level in 30 HCC tissues and their corresponding paracancer liver tissues (PCLTs) and 2 normal liver tissues by RT-PCR and IHC. Follow-up study was done on the 30 patients involved in this research. HMGA1 mRNA was detected in nine cases of HCC tissues and two PCLTs, for a positivity rate of 30% and 6.7%, respectively (P < 0.05), whereas no HMGA1 mRNA expression was found in normal liver tissues. Clinicopathological analysis revealed that HMGA1 mRNA expression was significantly correlated with Edmondson's grade (P < 0.05). HMGA1 protein was detected in four HCC tissues by IHC and located mainly in the nuclei; no positive staining was found in PCLTs. Follow-up study showed that HMGA1 mRNA-positive patients had a higher risk of recurrence/metastasis and a shorter survival than negative cases (P < 0.05). Our findings indicate that HMGA1 may be involved in the carcinogenesis and invasiveness of HCC and the determination of HMGA1 can be of great value in predicting the prognosis of patients with HCC.

Objective: To analyze the risk factors for metastasis and recurrence of hepatocellular carcinoma (HCC) postoperatively., Methods: Data of 270 cases of postoperative HCC were analyzed by SPSS software retrospectively., Results: Out of the 270 cases, 162 got follow-up study and 136 showed metastasis and recurrence. Lots of risk factors induced the recurrence of HCC, such as AFP, tumor form, venous blood invasion, HBV infection, resection dimension and perioperative transfusion. There were different risk factors at different stages., Conclusion: The early recurrence of HCC may be mediated by macro- or micro-vessel blood invasion and metastasis, the late recurrence by multicentric carcinogenesis or introhepatic cacinoma de novo.

Aim: To identify the role of alpha-fetoprotein (AFP) mRNA expression in peripheral blood one week after surgery as a predictor for recurrence of hepatocellular carcinoma (HCC)., Methods: Published studies fulfilling the selection criteria were identified by searching several databases online. After a methodology assessment using a quality scale designed by European Lung Cancer Working Party, data in each research were aggregated by means of meta-analysis., Results: Altogether 368 cases were included in the 9 selected studies, which fulfilled the selection criteria. The quality scores ranged from 35% to 84% with a median score of 55%. The 'design' subscore had the lowest median value (38%). By aggregating the data, a high chi2 value (77.576) was presented. The fail-safe number was 136 and 64 for P = 0.05 and 0.01, respectively., Conclusion: AFP mRNA expression in peripheral blood 1 wk after surgery correlated with the recurrence of HCC and was a good predictor for tumor recurrence.

Aim: To comprehend the risk factors of recurrence of hepatocellular carcinoma (HCC) and its relationship with the infection patterns of hepatitis B virus (HBV)., Methods: All materials of 270 cases of postoperative HCC were statistically analyzed by SPSS software. Recurrence and metastasis were classified into early (< or =2 years) and late phase (>2 years). Risk factors for recurrence and metastasis after surgery in each group were analyzed., Results: Out of 270 cases of HCC, 162 cases were followed up in which recurrence and metastasis occurred in 136 cases. There were a lot of risk factors related to recurrence and metastasis of HCC; risk factors contributing to early phase recurrence were serum AFP level, vascular invasion, incisal margin and operative transfusion, gross tumor classification and number of intrahepatic node to late phase recurrence. The HBV infective rate of recurrent HCC was 94.1%, in which "HBsAg, HBeAb, HbcAb" positive pattern reached 45.6%. The proportion of HBV infection in solitary large hepatocellular carcinoma (SLHCC) evidently decreased compared to nodular hepatocellular carcinoma (NHCC) (P<0.05)., Conclusion: The early and late recurrence and metastasis after hepatectomy of HCC were associated with different risk factors. The early recurrence may be mediated by vascular invasion and remnant lesion, the late recurrence by tumor's clinical pathology propert, as multicentric carcinogenesis or intrahepatic carcinoma de novo. HBV replication takes a great role in this process. From this study, we found that SLHCC has more satisfactory neoplasm biological behavior than NHCC.

Objectives: To describe and assess the effectiveness of a smoking cessation clinic., Design: Single group, unblinded study., Setting: Seven chain (mass-merchandise) community pharmacies in Virginia., Participants: Forty-eight patients who were smokers when admitted to the study., Interventions: Fifteen community chain pharmacists were trained using a smoking cessation training manual developed at the Virginia Commonwealth University School of Pharmacy. From April 1, 1997, through December 31, 1999, interested patients met individually with a study pharmacist during scheduled clinic times in semiprivate counseling areas. A patient chart was maintained at the site and updated after each visit. The first 30 patients completed a questionnaire after the third visit to the smoking cessation clinic to assess satisfaction with pharmacists and the overall program., Main Outcome Measure: Rate of long-term smoking cessation., Results: Of the 48 patients, 12 (25.0%) abstained from smoking cigarettes for 12 months or more beyond their predetermined quit dates. Abstinence rates for 1, 3, and 6 months were 43.8%, 31.3%, and 25.0%, respectively. Women were nearly five times more successful in attaining long-term abstinence than were men (33.3% versus 6.7%; P= .047). No significant differences were observed in cessation rates related to age, number of cigarettes smoked per day, level of nicotine dependence, number of previous quit attempts, or method of cessation. Increased appetite and weight gain were the most frequently reported withdrawal symptoms (16.7% of patients). Patients expressed satisfaction with the pharmacists and the smoking cessation program., Conclusion: Compared with other types of previously reported interventions, a community pharmacist-managed smoking cessation clinic achieved greater long-term smoking cessation rates.

Considered to have immunostimulating activity, echinacea is a widely used phytomedicinal for treatment of the common cold and upper respiratory tract infections (URTIs). We reviewed the literature from the MEDLINE database (January 1966-July 1999), International Pharmaceutical Abstracts (IPA) online database, Cambridge Scientific Abstracts Biological Sciences online database, Alt-Health Watch online database, EMBase CD-ROM database, and references from published articles, reviews, and letters to evaluate evidence from clinical trials of echinacea's purported efficacy for treating or preventing URTIs. Twelve clinical studies published from 1961-1997 concluded that echinacea was efficacious for treating the common cold, but the results are unclear due to inherent flaws in study design. Five trials were published since 1997; two showed that echinacea lacked efficacy for treating and preventing URTI symptoms, and three concluded that it was effective in reducing the frequency, duration, and severity of common cold symptoms. Again, these results are unclear because of methodologic uncertainties, such as small populations and use of noncommercially available, nonstandardized dosage forms. Although evidence for echinacea's efficacy is inconclusive, it appears to be safe. Patients without contraindications to it may not be dissuaded from using an appropriate preparation to treat the common cold.

Objective: The use and sales of herbal medications have increased dramatically over the past several years. Pharmacists are in an ideal position to educate patients about herbal medicines. This study was intended to determine the knowledge and attitudes of pharmacists regarding herbal medications., Methods: A survey was distributed to pharmacists at several state and regional meetings in Virginia and North Carolina between August and October 1998. The survey evaluated demographic data, attitudinal scales, and a 15-item herbal medicine knowledge test. Pharmacists immediately returned the surveys to the distributor on completion., Results: Of the 217 surveys distributed, 164 met the inclusion criteria for further evaluation. Of the pharmacists surveyed, 68.0% practiced in a community pharmacy, 45.1% had previous continuing education on herbal medications, and 73.6% sold herbal medications in their practice settings. The average score on the herbal knowledge test was 6.3 (maximum score of 15). Pharmacists with previous continuing education scored significantly higher than those without prior continuing education (p < 0.001). Of the 15 questions, the five that pharmacists were most likely to answer correctly assessed the uses of herbal medications. Additionally, pharmacists with prior continuing education or with access to herbal medication information at their practice site were more likely to agree that providing information about herbal medication is a pharmacist's professional responsibility (p = 0.02 and p = 0.01, respectively)., Conclusions: The findings from this study demonstrate that pharmacists were more likely to answer correctly about the uses of herbal medications than about drug interactions, adverse drug effects, and precautions of herbal medications. Additionally, pharmacists with previous continuing education on herbal medications were more knowledgeable about these products. With the increasing use of herbal medications, there is a greater need for pharmacy training programs in this area.