[Implication of enhanced recovery after surgery in the surgical management of hypopharyngeal squamous cell carcinoma].

Objective: To investigate the safety and efficacy of enhanced recovery after surgery (ERAS) in the clinical management of hypopharyngeal squamous cell carcinoma (HSCC). Methods: In this retrospective study, a total of 168 patients with pyriform sinus carcinoma in Qilu Hospital of Shandong University from January 2015 to January 2019 were divided into two groups, based on the different perioperative interventions that patients received, i.e. the ERAS group (n=64) and the conventional group (n=104), including 164 males and 4 females, whose ages ranged from 42 to 84 years old. The difference between two groups in the operative time, postoperative nutritional status, incidences of postoperative complications and postoperative hospitalization time were compared using the student's t test, Chi-squared test or Fisher's exact test. Results: Compared with the conventional group, patients in the ERAS group had significantly shorter operative time [(166.8±58.2) min vs. (183.3±39.9) min,t=-2.72, P=0.031], higher levels of postoperative serum albumin [(38.3±4.2) µmol/L vs. (36.6±3.3) µmol/L, t=2.73, P=0.007] and more body weight [(65.4±9.4) kg vs. (62.1±9.4) kg, t=2.22, P=0.028], lower incidences of postoperative subcutaneous infection [7.8% (5/64) vs. 20.2% (21/104), χ²=4.64, P=0.03] and severe pneumonia [4.7% (3/64) vs. 15.4% (16/104), χ²=4.52, P=0.03], and shorter postoperative hospitalization time [(16.5±3.9) d vs. (18.2±4.3) d, t=-2.65, P<0.05]. Conclusion: ERAS is effective and safe in the surgical management of HSCC, with benefits in reducing the operative stress via saving operation time, shortening the hospitalization time, ameliorating nutritional status and decreasing the incidences of complications.

Development of the Forensically Important Beetle Creophilus maxillosus (Coleoptera: Staphylinidae) at Constant Temperatures.

Creophilus maxillosus (L., 1758) is a common and widely distributed beetle species found on corpses, and its development duration is far longer than species belonging to the genus Calliphoridae and Sarcophagidae. Therefore, C. maxillosus can be used as a supplementary indicator to estimate minimum postmortem interval (PMImin), and could greatly extend the range of PMImin when the primary colonizers are no longer associated with the corpse or have emerged from pupae. Better descriptions of C. maxillosus development are needed to apply this species for forensic investigations. In this study, the development of C. maxillosus at seven constant temperatures ranging from 17.5-32.5 °C was studied. Through regression analyses, the simulation equations of larval body length variation with time after hatching were obtained. Isomegalen diagrams of the changes of larval body length over time at specific temperatures, and the isomorphen diagrams on the duration of different developmental milestones at specific temperatures were generated. In addition, thermal summation models of different developmental stages and the overall development process of C. maxillosus were generated through regression analysis, by estimating the development threshold temperatures (D0) and the thermal summation constants (K). These results provide important tools for forensic investigations to generate a long-range of PMImin estimation based on the development of C. maxillosus.

Newly identified respiratory viruses associated with acute lower respiratory tract infections in children in Lanzou, China, from 2006 to 2009.

Nasopharyngeal aspirates were collected from 813 children ≤ 14 years old with acute lower respiratory tract infections in Lanzhou, China, from December 2006 to November 2009. PCR or RT-PCR was used to screen for the presence of 10 respiratory viruses. Viral agents were identified in 73.92% (601/813) of specimens, including RSV in 40.71%, hMPV in 6.15%, IFVA in 7.13%, IFVB in 0.98%, PIV1-3 in 7.87%, HCoV-HKU1 in 2.21%, HCoV-NL63 in 3.81%, HRV in 19.93%, AdV in 7.50% and HBoV in 11.56%. Two or more viruses were detected in 34.44% (280/813) of cases. The newly identified respiratory viruses, HBoV, hMPV, HCoV-HKU1 and HCoV-NL63, accounted for 22.01% of the detected viral pathogens. RSV and HRV were frequently detected in patients with bronchiolitis, and hMPV was frequently associated with pneumonia. HCoV-NL63 was found to be one of the causative agents of acute respiratory wheezing in young children. No seasonal variation was found in the incidence of detection of HCoV-HKU1, HCoV-NL63 or HBoV. This 3-year study demonstrated that viral pathogens play an important role in children with ALRTIs, and more attention should be paid to these newly identified viral agents.

N-doped SiO2/TiO2 mesoporous nanoparticles with enhanced photocatalytic activity under visible-light irradiation.

Mesoporous nanocrystalline N-doped SiO2/TiO2 visible-light photocatalysts were prepared by treating SiO2/TiO2 xerogels in a flow of nitrogen gas bubbled through concentrated ammonia solution. Structural characterization and performance analysis results revealed that the addition of SiO2 remarkably altered the phase composition, specific surface area, microstructure, as well as the photocatalytic activity of N-doped TiO2. The presence of SiO2 in N-doped TiO2 particles suppressed the formation of rutile phase and the crystal growth of N-doped TiO2 particles during thermal calcinations. When weight ratio of SiO2/TiO2 was in 0.05-0.20, the N-doped SiO2/TiO2 exhibited higher photocatalytic activity than the N-doped TiO2, and optimum ratio was found to be 0.05. The enhanced photocatalytic activity could be attributed to the higher specific area, larger pore volume, and more surface hydroxyl groups in the catalyst.

[Phenotype of peripheral blood mononuclear cells derived dendritic cells from patients with chronic hepatitis B.].

BACKGROUND: The aim of this study was to access phenotype changes of dendritic cells (DC) cultured from peripheral blood mononuclear cells (PBMC) in patients with chronic hepatitis B and to reveal the relationship between phenotype of DC and ALT or HBV DNA. METHODS: Indices of ALT and serum HBV DNA were measured in 37 patients with chronic hepatitis B and 21 healthy controls. Peripheral blood mononuclear cells were isolated from all patients and healthy controls, and cultured with granulocyte-macrophage colony-stumilating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor- (TNF-)in RPMI 1640 medium that contained 10% fetal calf serum. After culturing for 7 days, the DC was counted and the phenotypes were detected by FACS. Then the data were statistically analysed. RESULTS: The DC was significantly fewer (P less than 0.05) in patients with chronic hepatitis B than the controls. In particular, the expressive level of CD83 and CD86 on DC's surface from patients with chronic hepatitis B were also significantly lower (P less than 0.05) than that from the controls. In the patients with hepatitis B, the indices of DC had a significantly negative correlation with the level of serum HBV DNA (P less than 0.05), but no significant relationship was found between ALT and indices of DC (P greater than 0.05). CONCLUSION: The DC cultured from patients with chronic hepatitis B were few and had immature phenotype. These changes had a significantly negative correlation with the level of serum HBV DNA, but had not correlation with the inflammatory reaction levels in the liver. DC was associated with the clearance of HBV in patients with hepatitis B.

[Cloning of VEGF receptor KDR and its expression in insect cells].

The cDNA fragment of the first 3 loops of VEGF receptor, KDR, was cloned by PCR and inserted into a baculovirus expression plasmid pFASTBACI. The competent E. coli DH10BAC cell, which contain another plasmid with baculovirus genome in it, was transformed with pFASTBACI-KDRn3. Homologous recombination in the prokaryotic cells resulted in a recombinant plasmid containing KDRn3 in baculovirus genome. Transfection of the insect cell SF-9 with above plasmid generated a recombinant baculorvirus contain target gene fragment. SDS-PAGE and Western blot analysis of the supernatant of the infected SF-9 cell showed that KDRn3 was secreted in the medium. The recombinant protein was verified with Western blot and tested for their binding activity with VEGF. Its anti-angiogenic activity was assayed on chorionic allantoic membrane(CAM) of fertilized egg. The results showed that the recombinant protein could inhibit new vessel formation on CAM of fertilized eggs.

Isolation and complete nucleotide sequence of a Chinese Sindbis-like virus.

Infection with alphaviruses is common in the Chinese population. Here we report the isolation of a Sindbis-like virus from a pool of Anopheles mosquitoes collected in Xinjiang, China during an arbovirus survey. This virus, designated XJ-160, rapidly produced cytopathic effects on mosquito and hamster cells. In addition, it was lethal to neonatal mice if inoculated intracerebrally. Serologically, XJ-160 reacted with and was neutralized by an anti-Sindbis antibody. Anti-XJ-160 antibodies were found in several cohorts of Chinese subjects. The complete 11626-base nucleotide sequence of XJ-160 was determined. XJ-160 has diverged significantly from the prototype Sindbis virus, with an 18% difference in nucleotide sequence and an 8.6% difference in amino acids; there are 11 deletions and 2 insertions, involving 99 nucleotides in total. XJ-160 is most closely linked to Kyzylagach virus isolated in Azerbaijan. Both belong to the African/European genetic lineage of Sindbis virus, albeit more distantly related to other members.

Hepatitis C virus core protein-induced loss of LZIP function correlates with cellular transformation.

Hepatitis C virus (HCV) is the major etiological agent of blood-borne non-A non-B hepatitis and a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. HCV core protein is a multifunctional protein with regulatory functions in cellular transcription and virus-induced transformation and pathogenesis. Here we report on the identification of a bZIP nuclear transcription protein as an HCV core cofactor for transformation. This bZIP factor, designated LZIP, activates CRE-dependent transcription and regulates cell proliferation. Loss of LZIP function in NIH 3T3 cells triggers morphological transformation and anchorage-independent growth. We show that HCV core protein aberrantly sequesters LZIP in the cytoplasm, inactivates LZIP function and potentiates cellular transformation. Our findings suggest that LZIP might serve a novel cellular tumor suppressor function that is targeted by the HCV core.

Inducible overexpression of Bak sensitizes HCC-9204 cells to apoptosis induced by doxorubicin.

AIM: To investigate the role of overexpression of Bak in apoptotic pathways and drug susceptibility using doxorubicin and vinorelbine in human HCC-9204 cells. METHODS: An inducible system, MT-II regulatory system which allowed controlled expression of protein upon addition of ZnSO4(100 mumol/L) as an external inducer was used. Stable transfection of pMD-Bak gene was performed on HCC-9204 cells. Apoptotic cells were measured by morphological criteria, as well as by TUNEL assay and flow cytometry. The ability of Bak to decrease clonogenic cell survival was studied by colony-forming assays, while decrease in cell viability was assessed by MTT assay. RESULTS: Cells overexpressing Bak showed extensive cell death with nucleus fragmentation detected by TUNEL assay. FACS analyses showed that Bak could induce significant G1 accumulation and apoptosis in 19.29% cells 24 h after induction. Bak significantly decreased the clonogenic survival following exposure to adriamycin, but not vinorelbine. Furthermore, the time-course of cell viability rates following exposure of HCC-9204/Bak cells to adriamycin and vinorelbine was in agreement with the above findings. Bak selectively sensitized HCC-9204 cells to death induced by adriamycin while resisted to vinorelbine. CONCLUSION: Bak may prolong cell cycle in G1 phase, leading to apoptosis and decrease clonogenic survival of HCC-9204 cells in a drug-specific manner.

Identification of a single genotype of hepatitis G virus by comparison of one complete genome from a healthy carrier with eight from patients with hepatitis.

Different isolates of a putative hepatitis virus called hepatitis GB virus C or hepatitis G virus (HGV) have been cloned recently from patients with hepatitis. This virus has also been found commonly in healthy carriers. We have cloned and sequenced a complete HGV genome, designated HGVCN, from a healthy Chinese blood donor. HGVCN shares 85.8-90.0% nucleotide sequence identity and 95.4-97.5% amino acid identity with the eight available full-length HGV genomes. Furthermore, the majority (82.8%) of the nucleotide substitutions found in HGVCN were synonymous and a fairly uniform distribution of changes was found across the entire genome without identification of any hypervariable region. When compared with the African isolates GBVC and GBVC-EA, the HGVCN-encoded polyprotein contained a 31 amino acid N-terminal extension which was predicted to be a defective core-like sequence. The sequences of the HGV E1 and E2 proteins displayed unique motifs and were highly conserved. Phylogenetic analysis revealed that all nine complete HGV isolates were closely related and that HGVCN grouped with the other Chinese HGV isolate (HGVC964). Taken together, our findings suggest that there is one single genotype of HGV and that the HGV genome cloned from the healthy carrier is not significantly different from those derived from patient sera.

Bacterial scavengase p20 is structurally and functionally related to peroxiredoxins.

Scavengase p20 was recently identified as a novel family of bacterial antioxidant enzymes possessing thioredoxin-linked thiol peroxidase activity. In this study, the Escherichia coli gene coding for scavengase p20 was isolated from three different strains and the nucleotide sequence was determined. Multiple alignment of amino acid sequence revealed that a previously unidentified Cys-61 is most conserved among all bacterial p20 scavengases and corresponds to the active site in the well-characterized peroxiredoxins. Phylogenetic analysis further supported that scavengase p20 is a novel subfamily of peroxiredoxins. Site-directed mutagenesis studies demonstrated that Cys-61 is indispensable for the antioxidant activities of scavengase p20. Taken together, our findings strongly suggest that the p20 scavengases are structurally and functionally related to peroxiredoxins.

Scavengase p20: a novel family of bacterial antioxidant enzymes.

A novel antioxidant enzyme designated scavengase p20 was identified in various pathogenic bacteria through database searching for sequences strikingly homologous to a recently discovered Escherichia coli thiol peroxidase p20. The direct biochemical evidence for the existence of scavengase p20 in Haemophilus influenzae, Streptococcus pneumoniae and Helicobacter pylori was provided by protein microsequencing and by in vitro assays for antioxidant activities. Overlapping genes encoding scavengase p20 and superoxide dismutase were recognized in H. pylori and their functional implications are discussed.

Enhanced anti-tumor effect of an IFN-gamma-EGF fusion protein.

The novel fusion proteins harboring human or mouse interferon combined with epidermal growth factor receptor binding domain were constructed using methods of genetic and protein engineering. The fusion proteins were assayed to retain complete antiviral activities. The EGF receptor binding moiety of the fusion proteins exhibited competitive binding against 125I-EGF for EGF receptors on A431 cells. The fusion proteins were shown to be more potent in inhibiting the growth of cultured target carcinoma cells than interferon-gamma alone. Experimental data derived from mouse B16 malignant melanoma models indicates that the weight of tumor in mice treated with IFN fusion proteins was significantly smaller than that of mice treated with interferon-gamma alone. The work here is unprecedented in the world and provides a reliable evidence to support the upcoming clinical employment of a class of interferons that specifically target tumor cell.

Cloning, sequencing and expression in E. coli of interferon-omega 1 gene.

Human interferon omega 1 (huIFN-omega 1) gene was isolated and cloned from chromosome DNA derived from a Chinese fetal liver via polymerase chain reaction (PCR). By determining its nucleotide sequence we proved that the 88th codon should be GGA, coding for Gly. After engineering the original IFN-omega 1 gene clone to a form that may be expressed as a nonfused protein, we also took the IFN-omega 1 gene under the control of the PRPL promoter with an expression vector pBV220 in E. coli. The antivirus activity of the recombinant IFN-omega 1 is about 6.5 x 10(7) units/L CULTURE (OD600 = 0.75). Since IFN-omega 1 not only has antivirus activity but also shows considerably high homology with animal trophoblast proteins which have been proved antiluteolysins as a maternal recognition signal for pregnancy, we believe that study on it will be practically and theoretically significant.

Synthesis and expression in Escherichia coli of a human neutrophil activating protein-1/interleukin-8 gene.

The complete gene coding for human neutrophil activating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the PRPL tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to > 95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of < 10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP-1/IL-8 was determined using the Edman method and was shown to be identical to that of the native protein.

Molecular cloning of a new variant of human papillomavirus type 6 isolated from a Chinese woman and expression of its L1 gene in E. coli--molecular cloning & expression of HPV6 gene.

A human papillomavirus genome DNA of 7.9 kb from a Chinese woman with genital condyloma acuminata was cloned in BamHI site of pAT153. According to the results obtained from Southern blotting, restriction mapping as well as partial DNA sequencing, the isolated genome (HPV6BV) had obvious variance and was referred to as a new variant of HPV6 found in China the first time. HPV6BV L1 gene was successfully expressed in E. coli as a fusion protein with pUR288. The beta-galactosidase/L1 fusion protein reacted with both beta-galactosidase antiserum and HPV antibody using Western blot technique. The E. coli-produced fusion protein, possessing HPV antigenicity, may provide a reagent for clinical diagnosis and epidemiological survey.

A novel variant of human interferon alpha 1 gene.

A 520-base pair human IFN-alpha gene was isolated by PCR method twice from chromosome DNA of a Chinese (Han Nationality) fetal liver. The nucleotide sequences were determined. These two separately amplified DNA fragments shared the completely identical nucleotide sequence but possessed C and G at positions 410 and 541, respectively, which differ from those of IFN-alpha 1 and IFN-alpha D previously described. Therefore the deduced amino acid sequence would have an Ala at position 114 and a Val at position 158. At all other sites it has the same amino acids as those in IFN-alpha 1 and IFN-alpha D. We recommend that IFN-alpha D gene, IFN-alpha I gene and IFN-alpha I/158V gene found in our laboratory, be named IFN-alpha 1a gene, IFN-alpha 1b gene and IFN-alpha 1c gene.

A new human interferon-gamma mutant harboring EGF receptor-interfering sequence possesses high antiproliferative activity.

An interferon gamma (IFN-gamma) mutant gene harboring an additional epidermal growth factor (EGF) sequence at its 3' terminal was constructed and highly expressed in E. coli by using pBV220 as a vector. The new fusion protein-IFN-gamma-EGF possesses not only high antiviral activity, but also high antiproliferative effect as demonstrated by 3H-TdR incorporation method using CN II cells which are rich in EGF receptors.

Evaluation of the purity of recombinant proteins and detection of residual protein contaminants via N-terminal microsequencing and database searching.

The N-terminal amino acid sequences of purified recombinant human gamma-interferon, alpha 2a-interferon and interleukin-2 expressed in E. coli were determined on an Applied Biosystems 477A Protein/Peptide Sequencer and 120A PTH Amino Acid Analyzer. From the raw chromatographic data of these samples, the identity, heterogeneity, amount of methionine-plus species remaining in the final products, and the probable process contaminants were evaluated with the help of computer methods including database searching. General methods to characterize trace contaminants in protein samples were also discussed. Among the sequenced samples, only gamma-interferon was shown to be N-terminal homogeneous. Methionine-containing species were found in interleukin-2 and alpha 2a-interferon. Chicken eggwhite lysozyme was detected in very small amounts in one batch of samples. These results provide valuable information for the development and improvement of preparation methods as well as regulatory responses to recombinant products.