The characteristics of high risk, high workload and high intensity make medical staff a high risk group of job burnout. The authors, by means of literature review, clarified the concept of job burnout, and analyzed the status quo and causes of medical staff job burnout.In view of the society, hospital and personal level, they put forward corresponding improvement countermeasures, in order to alleviate the current medical staff job burnout, create a harmonious doctor-patient relationship, and guarantee sustainable development of medical career.

Objective To study the effect of targeted inhibition of mitogen-activated protein kinase kinase kinase 1(Map3k1) gene on the cell proliferation and migration abilities of B6 mouse eyelid keratinocytes.Methods An artificial microRNA(amiRNA) interference vector targeting silent Map3k1 gene was constructed in vitro in the test.Lipofectamin 2000 was used to transfect the B6 mouse eyelid keratinocytes(the Ctrl Map3k1 group was transfected with empty vector,while the Map3k1 amiRNA-3 group was transfected with Map3k1 amiRNA-3 interference vector).The Map3k1 mRNA and protein expression levels were respectively de-tected by real-time PCR and Western-blot for determining the interference efficiency.The B6 mouse eyelid keratinocytes prolifera-tion level was detected by MTT.The migration ability of keratinocytes was detected by the scratch experiment.Results After the keratinocytes were transfected with Map3k1 amiRNA interference vector,the levels of Map3k1 mRNA and protein were effectively inhibited,and the interference efficiency was up to 70%(P<0.05).The proliferation level of keratinocytes in the Map3k1 amiRNA-3 group was lower than that in the Ctrl Map3k1 group(P<0.05).The migratory ability of keratinocytes in the Map3k1 amiRNA-3 group was also significantly lower than that in the Ctrl Map3k1 group(P<0.05).Conclusion Targeted inhibition of Map3k1 gene expression in B6 mouse eyelid keratinocytes significantly inhibits cell proliferation and migration,thus influence the cellular bi-ological behaviors.

Genes encoding thaumatin-like protein () are frequently found in fungal genomes. However, information ongenes inis still limited. In this study, threegenes were cloned from. The full-length coding sequence of,andwere 768, 759 and 561 bp long, respectively, encoding for 256, 253 and 187 amino acids. Phylogenetic trees showed that,andwere clustered with sequences fromand, respectively. The expression patterns of the threegenes were higher inwithinfection than in the sclerotia without. Furthermore, over-expression of three PuTLPs were carried out inBL21 (DE3) strain, and high quality proteins were obtained using Ni-NTA resin that can be used for preparation of specific antibodies. These results suggest that,andinmay be involved in the defense response toinfections.

Objective To compare the effects of gastric gavage and intramuscular injection of prednisone on the bone mineral density, skeletal biomechanical properties and bone metabolism in rats.Methods A total of 45 SPF rats were randomly divided into three groups:normal group, intragastric administration group, and intramuscular injection group.The normal group, as a control group, was administrated with normal saline 2 mL per day, both the intragastric administration group and i.m.injec-tion group received prednisone 0.5 mg/(kg.d) for 12 weeks.All rats were examined for bone mineral density (BMD) and the level of serum β-CTX and PINP.The femoral cortical biomechanical properties ( elastic load, maximal load, rupturing load) were measured by three point bending test.Results After 12 weeks, compared with the normal group, BMD and elastic load, maximal load, and rupturing load of the femur were significantly decreased.Compared with the intragastric gavage group, BMD was significantly decreased, while the elastic load, maximal load, and rupturing load of the femur were not significantly changed in the i.m.injection group (P＜0.05 for all).Compared with the normal group, the level of serum β-CTX was significantly raised (P＜0.05) and the level of serum PINP was significantly decreased (P＜0.05).Compared with the intragastric gavage group, the level of serumβ-CTX was also significantly raised (P＜0.05), the level of serum PINP was significantly decreased (P＜0.05), the bone trabecula and hemopoietic tissue were obviously decreased, while the adipose tissue increased obviously. Conclusions Both intragastric gavage and intramuscular injection of prednisone affect the level of BMD, skeletal biomechanical properties and bone metabolism.However, i.m.injection of prednisone decreases the BMD and bone strength more significantly, leading to a higher bone turnover with increased bone resorption, and leads to osteoporosis earlier.Our results may suggest that oral administration of prednisone is more safe in clinical treatment.

Mitogen-activated protein kinases (MAPKs) are important signaling transduction components well conserved in eukaryotes and play essential roles in various physiological, developmental and hormonal responses in plant. In the present study, a MAPK gene, designated as DoMPK4 (GenBank accession No. JX297597), is identified from a rare endangered medicinal orchid species D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK4 is 1 518 bp in length and encoded a 369 aa protein with a molecular weight of 42.42 kD and an isoelectric point of 5.55. DoMPK4 protein contained a serine/threonine protein kinase active site (158-170), a MAP kinase site (71-174), and eight conserved motifs. DoMPK4 had a transmembrane (214-232) but no signal peptide. Multiple sequence alignment showed that DoMPK4 shared high identities (74.9%-80.6%) with MAPK proteins from various plants. Phylogenetic analysis demonstrated that DoMPK4 belonged to group A of the MAPK evolutionary tree, and is closely related to monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK4 is differentially expressed among the five organs including leaf, stem, root, seed, and protocorm-like body (PLB). The transcription level of DoMPK4 is the highest in the PLBs with 17.65 fold, followed by seeds, roots, and stems with 5.84, 2.28, and 1.64 fold, respectively. The progressive enhancement of DoMPK4 transcripts in the developing PLBs compared to that in the germinating seeds, suggests a role of DoMPK4 during the development of embryogenic PLBs formation in D. officinale.

HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.

A total of 52 endophytic fungi were isolated from roots and stems of Tibetan medicinal plant Phlomis younghusbandii Mukerjee. These fungal isolates were molecularly identified based on ITS sequnces and 28S sequences distributed to 12 genera, including Phoma, Chaetosphaeronema, Fusarium and Leptosphaeria, etc. Among them, the dominant genus was Phoma. Extracts of all strains were evaluated for anti-HIV-1 integrase activity by using soluable integrase expressed in E. coli BL21 (DE3). The results showed that seven samples from five fungal endophytes PHY-24, PHY-38, PHY-40, PHY-51, PHY-53, which belonged to genus Chaetosphaeronema, inhibited strand transfer reaction catalyzed by HIV-1 integrase with IC50 values, of 6.60, 5.20, 2.86, 7.86, 4.47, 4.56 and 3.23 microg x mL(-1) respectively. In conclusion, the endophytic fungi of Phlomis younghusbandii Mukerjee are valuable for further screening anti-HIV-1 integrase agents.

S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.

Calcium-dependent protein kinases (CDPKs) play an important regulatory role in the plantarbuscular mycorrhiza/rhizobium nodule symbiosis. However, the biological action of CDPKs in orchid mycorrhiza (OM) symbiosis remains unclear. In the present study, a CDPK encoding gene, designated as DoCPK1 (GenBank accession No. JX193703), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoCPK1 was 2137 bp in length and encoded a 534 aa protein with a molecular weight of 59.61 kD and an isoelectric point (pI) of 6.03. The deduced DoCPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain and four Ca2+ binding EF hand motifs. Multiple sequence alignment demonstrated that DoCPK1 was highly homologous (85%) to the Panax ginseng PgCPK1 (ACY78680), followed by CDPKs genes from wheat, rice, and Arabidopsis (ABD98803, ADM14342, Q9ZSA2, respectively). Phylogenetic analysis showed that DoCPK1 was closely related to CDPKs genes from monocots, such as wheat, maize and rice. Real time quantitative PCR (qPCR) analysis revealed that DoCPK1 was constitutively expressed in the included tissues and the transcript levels were in the order of roots > stems > seeds > leaves. Furthermore, DoCPK1 transcripts were significantly accumulated in roots 30 d after fungal infection, with 5.16 fold compared to that of the mock roots, indicating involvement of DoCPK1 during the early interaction between D. officinale and Mycena sp., and a possible role in the symbiosis process. This study firstly provided important clues of a CDPK gene associated with OM symbiosis, and will be useful for further functional determination of the gene involving in D. officinale and Mycena sp. symbiosis.