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1.  Characterization of Pseudomonas aeruginosa Phage C11 and Identification of Host Genes Required for Virion Maturation 
Scientific Reports  2016;6:39130.
The underlying mechanisms of phage-host interactions largely remained to be elucidated. In this work, Pseudomonas aeruginosa phage C11 was first characterized as a Myoviridae virus having a linear dsDNA molecule of 94109 bp with 1173 bp identical terminal direct repeats (TDR). Then the mutants resistant to phage C11 were screened in a Tn5G transposon mutant library of P. aeruginosa PAK, including two mutants with decreased adsorption rates (DAR) and five mutants with wild-type adsorption rates (WAR). When the WAR mutants were incubated with phage C11, their growth rates were significantly inhibited; the replication of the phage genomic DNA was detected in all the WAR mutants with the real-time quantitative PCR analysis; and the synthesized phage genomic DNA was processed into monomers for packaging evidenced by the southern blot analysis. Moreover, with strain PAK as indicator, small quantities of phage C11 were synthesized in the WAR mutants. Taken together, these data suggested the identified genes of the WAR mutants are necessary for efficient synthesis of the infectious phage particles. Finally, the WAR mutants were detected sensitive to two other Pseudomonas phages closely related with C11, further implying the evolved diversity and complexity of the phage-host interactions in both sides.
PMCID: PMC5175280  PMID: 28000703
2.  Design and fabrication of six-volt vertically-stacked GaAs photovoltaic power converter 
Scientific Reports  2016;6:38044.
A six-volt vertically-stacked, high current GaAs photovoltaic power converter (PPC) has been designed and fabricated to produce output power over 1 W under monochromatic illumination. An N++-GaAs/P++-AlGaAs tunnel junctions (TJs) structure has been used for connecting each sub-cell in this vertically-stacked PPC device. The thickness of the each GaAs sub-cell has been derived based on the calculation of absorption depth of photons with a wavelength of 808 nm using absorption coefficient obtained from ellipsometry measurements. The devices were characterized under non-uniform CW laser illumination at 808 nm with incident power up to 4.1 W. A maximum conversion efficiency of 50.2% was achieved at 0.3 W under non-uniform (coupled in optical fiber) monochromatic illumination, dropping to 42.5% at 4.1 W. The operating voltage at the maximum power point is 5.5–6.0 V, depending on the incident laser power, and an output electrical power output of 1.3 W can be extracted at a laser power of 2.9 W and the maximum electrical power output amounts to 1.72 W. The external quantum efficiency (EQE) measurement indicates that the performance of PPC can be further improved by refining the design of the thickness of sub-cells and improving TJs.
PMCID: PMC5128921  PMID: 27901079
3.  Methyl salicylate 2-O-β-d-lactoside alleviates the pathological progression of pristane-induced systemic lupus erythematosus-like disease in mice via suppression of inflammatory response and signal transduction 
Systemic lupus erythematosus (SLE), with a high incidence rate and insufficient therapy worldwide, is a complex disease involving multiple organs characterized primarily by inflammation due to deposition of immunocomplexes formed by production of autoantibodies. The mechanism of SLE remains unclear, and the disease still cannot be cured. We used pristane to induce SLE in female BALB/c mice. Methyl salicylate 2-O-β-d-lactoside (MSL; 200, 400, and 800 mg/kg) was orally administered 45 days after pristane injection for 4.5 months. The results showed that MSL antagonized the increasing levels of multiple types of antibodies and cytokines in lupus mice. MSL was found to suppress joint swelling and have potent inhibitory effect on arthritis-like symptoms. MSL also significantly decreased the spleen index and expression of inflammatory markers in the lupus mice. MSL protected the kidneys of lupus mice from injury through inhibiting the expression of inflammatory cytokines and reducing the IgG and C3 immunocomplex deposits. Further Western blot assays revealed that the downregulation of the intracellular inflammatory signals of NFκB and JAK/STAT3 might be the potential molecular mechanisms of the pharmacological activity of MSL against SLE in vivo. These findings may demonstrate that MSL has the potential to be a useful and highly effective treatment for SLE.
PMCID: PMC5047741  PMID: 27729775
methyl salicylate 2-O-β-d-lactoside; systemic lupus erythematosus; inflammatory response; lupus nephritis; signal transduction
4.  Study on Synergistic Mechanism of Inhibitor Mixture Based on Electron Transfer Behavior 
Scientific Reports  2016;6:33252.
Mixing is an important method to improve the performance of surfactants due to their synergistic effect. The changes in bonding interaction and adsorption structure of IM and OP molecules before and after co-adsorbed on Fe(001) surface is calculated by DFTB+ method. It is found that mixture enable the inhibitor molecules with higher EHOMO donate more electrons while the inhibitor molecules with lower ELUMO accept more electrons, which strengthens the bonding interaction of both inhibitor agent and inhibitor additive with metal surface. Meanwhile, water molecules in the compact layer of double electric layer are repulsed and the charge transfer resistance during the corrosion process increases. Accordingly, the correlation between the frontier orbital (EHOMO and ELUMO of inhibitor molecules and the Fermi level of metal) and inhibition efficiency is determined. Finally, we propose a frontier orbital matching principle for the synergistic effect of inhibitors, which is verified by electrochemical experiments. This frontier orbital matching principle provides an effective quantum chemistry calculation method for the optimal selection of inhibitor mixture.
PMCID: PMC5037402  PMID: 27671332
5.  Comparative genomic analysis identifies structural features of CRISPR-Cas systems in Riemerella anatipestifer 
BMC Genomics  2016;17(1):689.
Riemerella anatipestifer infection is a contagious disease that has resulted in major economic losses in the duck industry worldwide. This study attempted to characterize CRISPR-Cas systems in the disease-causing agent, Riemerella anatipestifer (R. anatipestifer). The CRISPR-Cas system provides adaptive immunity against foreign genetic elements in prokaryotes and CRISPR-cas loci extensively exist in the genomes of archaea and bacteria. However, the structure characteristics of R. anatipestifer CRISPR-Cas systems remains to be elucidated due to the limited availability of genomic data.
To identify the structure and components associated with CRISPR-Cas systems in R. anatipestifer, we performed comparative genomic analysis of CRISPR-Cas systems in 25 R. anatipestifer strains using high-throughput sequencing. The results showed that most of the R. anatipestifer strains (20/25) that were analyzed have two CRISPR loci (CRISPR1 and CRISPR2). CRISPR1 was shown to be flanked on one side by cas genes, while CRISPR2 was designated as an orphan. The other analyzed strains harbored only one locus, either CRISPR1 or CRISPR2. The length and content of consensus direct repeat sequences, as well as the length of spacer sequences associated with the two loci, differed from each other. Only three cas genes (cas1, cas2 and cas9) were located upstream of CRISPR1. CRISPR1 was also shown to be flanked by a 107 bp-long putative leader sequence and a 16 nt-long anti-repeat sequence. Combined with analysis of spacer organization similarity and phylogenetic tree of the R. anatipestifer strains, CRISPR arrays can be divided into different subgroups. The diversity of spacer organization was observed in the same subgroup. In general, spacer organization in CRISPR1 was more divergent than that in CRISPR2. Additionally, only 8 % of spacers (13/153) were homologous with phage or plasmid sequences. The cas operon flanking CRISPR1 was observed to be relatively conserved based on multiple sequence alignments of Cas amino acid sequences. The phylogenetic analysis associated with Cas9 showed Cas9 sequence from R. anatipestifer was closely related to that of Bacteroides fragilis and formed part of the subtype II-C subcluster.
Our data revealed for the first time the structural features of R. anatipestifer CRISPR-Cas systems. The illumination of structural features of CRISPR-Cas system may assist in studying the specific mechanism associated with CRISPR-mediated adaptive immunity and other biological functions in R. anatipestifer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-3040-4) contains supplementary material, which is available to authorized users.
PMCID: PMC5006608  PMID: 27577199
Riemerella anatipestifer; CRISPR-Cas system; cas gene; repeat sequence; spacer sequence; phylogenetic analysis
6.  Molecular cloning, expression and the adjuvant effects of interleukin-8 of channel catfish (Ictalurus Punctatus) against Streptococcus iniae 
Scientific Reports  2016;6:29310.
Interleukin-8 (IL-8) as an important cytokine involving in inflammatory and immune response, has been studied as effective adjuvants for vaccines in mammals. However, there are fewer reports about the characterization and adjuvant effects of IL-8 in fish. In this study, cloning and sequence analysis of IL-8 coding region of channel catfish (Ictalurus punctatus) were conducted, mature IL-8(rtIL-8) was expressed and evaluated for its adjuvant effects on the immunoprotection of subunit vaccine encoding α-enolase (rENO) of Streptococcus iniae from several aspects in channel catfish. The results showed co-vaccination of rENO with rtIL-8 enhanced immune responses including humoral and cellular immunity, with higher relative percent survival(RPS,71.4%) compared with the moderate RPS of rENO alone(50%) against S. iniae infection at 4 week post vaccination. While rtIL-8 failed to maintain long-lasting immune protection, only with RPS of 26.67% in rENO + rtIL-8-vaccinated fish compared with that of rENO alone(20%) at 8 week, signifying that IL-8 hold promise for use as potential immunopotentiator in vaccines against bacterial infections in fish, whereas it is insufficient to extend the immunoprotection for long time, and further studies are required to understand the mechanisms of IL-8 used as an adjuvant and seek for more effective way to strengthen the adjuvanticity of IL-8.
PMCID: PMC4931690  PMID: 27373470
7.  Tubeimoside-1 induces oxidative stress-mediated apoptosis and G0/G1 phase arrest in human prostate carcinoma cells in vitro 
Acta Pharmacologica Sinica  2016;37(7):950-962.
Tubeimoside-1 (TBMS1), a triterpenoid saponin extracted from the Chinese herbal medicine Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae), has shown anticancer activities in various cancer cell lines. The aim of this study was to investigate the anticancer activity and molecular targets of TBMS1 in human prostate cancer cells in vitro.
DU145 and P3 human prostate cancer cells were treated with TBMS1. Cell viability and apoptosis were detected. ROS generation, mitochondrial membrane potential and cell cycle profile were examined. Western blotting was used to measure the expression of relevant proteins in the cells.
TBMS1 (5–100 μmol/L) significantly suppressed the viability of DU145 and P3 cells with IC50 values of approximately 10 and 20 μmol/L, respectively. Furthermore, TBMS1 dose-dependently induced apoptosis and cell cycle arrest at G0/G1 phase in DU145 and P3 cells. In DU145 cells, TBMS1 induced mitochondrial apoptosis, evidenced by ROS generation, mitochondrial dysfunction, endoplasmic reticulum stress, modulated Bcl-2 family protein and cleaved caspase-3, and activated ASK-1 and its downstream targets p38 and JNK. The G0/G1 phase arrest was linked to increased expression of p53 and p21 and decreased expression of cyclin E and cdk2. Co-treatment with Z-VAD-FMK (pan-caspase inhibitor) could attenuate TBMS1-induced apoptosis but did not prevent G0/G1 arrest. Moreover, co-treatment with NAC (ROS scavenger), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor) or salubrinal (ER stress inhibitor) significantly attenuated TBMS1-induced apoptosis.
TBMS1 induces oxidative stress-mediated apoptosis in DU145 human prostate cancer cells in vitro via the mitochondrial pathway.
PMCID: PMC4933758  PMID: 27292614
TBMS1; human prostate cancer; apoptosis; G0/G1 phase arrest; oxidative stress; mitochondrial dysfunction; ER stress; Z-VAD-FMK; NAC; SB203580; SP600125; salubrinal
8.  The Complete Chloroplast Genome Sequences of the Medicinal Plant Pogostemon cablin 
Pogostemon cablin, the natural source of patchouli alcohol, is an important herb in the Lamiaceae family. Here, we present the entire chloroplast genome of P. cablin. This genome, with 38.24% GC content, is 152,460 bp in length. The genome presents a typical quadripartite structure with two inverted repeats (each 25,417 bp in length), separated by one small and one large single-copy region (17,652 and 83,974 bp in length, respectively). The chloroplast genome encodes 127 genes, of which 107 genes are single-copy, including 79 protein-coding genes, four rRNA genes, and 24 tRNA genes. The genome structure, GC content, and codon usage of this chloroplast genome are similar to those of other species in the family, except that it encodes less protein-coding genes and tRNA genes. Phylogenetic analysis reveals that P. cablin diverged from the Scutellarioideae clade about 29.45 million years ago (Mya). Furthermore, most of the simple sequence repeats (SSRs) are short polyadenine or polythymine repeats that contribute to high AT content in the chloroplast genome. Complete sequences and annotation of P. cablin chloroplast genome will facilitate phylogenic, population and genetic engineering research investigations involving this particular species.
PMCID: PMC4926354  PMID: 27275817
Pogostemon cablin; sequencing; chloroplast genome; SSR; phylogenetic analysis
9.  Survey of the genome of Pogostemon cablin provides insights into its evolutionary history and sesquiterpenoid biosynthesis 
Scientific Reports  2016;6:26405.
Pogostemon cablin (Blanco) Benth. (Patchouli) is an important traditional Chinese medicinal plant that has both essential oil value and a broad range of therapeutic effects. Here we report the first de novo assembled 1.15-Gb draft genome sequence for P. cablin from next-generation sequencing technology. Our assembly, with a misassembly rate of <4 bp per 100 kb, is ~73% of the predicted genome size (1.57 Gb). Analysis of whole-genome sequences identified 3,147,333 heterozygous single-nucleotide polymorphisms and 490,407 insertions and deletions, giving an estimated heterozygosity rate of 0.274%. A comprehensive annotation pipeline indicated that repetitive sequences make up 58.55% of the assemblies, and that there are estimated 45,020 genes. Comparative genomics analysis showed that the Phrymaceae and Lamiaceae family split ~62.80 Mya, and the divergence between patchouli and sesame occurred ~52.42 Mya, implying a potentially shared recent whole-genome duplication event. Analysis of gene homologs involved in sesquiterpenoid biosynthesis showed that patchouli contains key genes involved in more sesquiterpenoid types and has more copies of genes for each sesquiterpenoid type than several other related plant species. The patchouli genome will facilitate future research on secondary metabolic pathways and their regulation as well as potential selective breeding of patchouli.
PMCID: PMC4873823  PMID: 27198881
10.  Neuroendocrine-like cells -derived CXCL10 and CXCL11 induce the infiltration of tumor-associated macrophage leading to the poor prognosis of colorectal cancer 
Oncotarget  2016;7(19):27394-27407.
Our previous study revealed that neuroendocrine differentiation in colorectal cancer is one of the important factors leading to worse prognosis. In this study, we apply immunohistochemical staining, Western-blot, RT-PCR and ELISA to investigate the underlying mechanism that how the neuroendocrine differentiation to affect the prognosis of colorectal cancer. The interaction of colorectal cancer cells, neuroendocrine-like cells and tumor-associated macrophages in colorectal cancer progress is also investigated. By analyzing 82 cases of colorectal cancer patients treated in our institution, we found that colorectal adenocarcinoma with neuroendocrine differentiation had increasing number of tumor-associated macrophages and worse prognosis. Further evaluation of cytology showed that neuroendocrine cells have the ability to recruit tumor-associated macrophages to infiltrate the tumor tissue, and the tumor-associated macrophages enhance the proliferation and invasion abilities of the colon cancer cells. Moreover, we confirmed that CXCL10 and CXCL11 are the key chemokines in neuroendocrine-like cells and they promote the chemotaxis activity of tumor-associated macrophages. The secretion of CXCL10 and CXCL11 by neuroendocrine-like cells can recruit tumor-associated macrophages to infiltrate in tumor tissues. The latter enhances the proliferation and invasion of colorectal cancer cell and lead to poor prognosis.
PMCID: PMC5053658  PMID: 27034164
colorectal cancer; neuroendocrine differentiation; tumor-associated macrophages; prognosis
11.  Optimized selection of three major EGFR-TKIs in advanced EGFR-positive non-small cell lung cancer: a network metaanalysis 
Oncotarget  2016;7(15):20093-20108.
Background: To answer which epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is the best choice for advanced non-small cell lung cancer (NSCLC) EGFR mutants.
Results: 16 phase III randomized trials involving 2962 advanced NSCLC EGFR mutants were enrolled. Multiple treatment comparisons showed different EGFR-TKIs shared equivalent curative effect in terms of all outcome measures among the overall, chemo-naïve and previously treated patients. Rank probabilities showed that erlotinib and afatinib had potentially better efficacy compared with gefitinib in both of the overall and chemo-naïve patients. Potentially survival benefit of erlotinib was also observed in previously treated patients compared with gefitinib. Additionally, EGFR-TKI showed numerically greater survival benefit in 19 Del compared with chemotherapy, while it was opposite in 21 L858R. Furthermore, afatinib, erlotinib and gefitinib had high, moderate and low risk of rash & diarrhea, respectively, while the occurrence of elevated liver transaminase was more common in gefitinib.
Methods: Data of objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and adverse events were extracted from included studies. Efficacy and toxicity of all included treatments were integrated by network meta-analyses.
Conclusion: Our study indicated a high efficacy-high toxicity pattern of afatinib, a high efficacy-moderate toxicity pattern of erlotinib and a medium efficacy-moderate toxicity pattern of gefitinib. Recommended EGFR-TKI should be suggested according to patients' tolerability and therapeutic efficacy in clinical practice. Moreover, the treatment for advanced EGFR-positive NSCLC might be different between 19 Del and 21 L858R.
PMCID: PMC4990517  PMID: 26933807
EGFR-TKI; NSCLC; gefitinib; erlotinib; afatinib
12.  Genome Sequence of the Fish Pathogen Yersinia ruckeri SC09 Provides Insights into Niche Adaptation and Pathogenic Mechanism 
Yersinia ruckeri is the etiologic agent of enteric red mouth disease (ERM), a severe fish disease prevailing in worldwide aquaculture industries. Here we report for the first time the complete genome of Y. ruckeri (Yersinia ruckeri) SC09, a highly virulent strain isolated from Ictalurus punctatus with severe septicemia. SC09 possesses a single chromosome of 3,923,491 base pairs, which contains 3651 predicted protein coding sequences (CDS), 19 rRNA genes, and 79 tRNA genes. Among the CDS, we have identified a Ysa locus containing genes encoding all the components of a type III secretion system (T3SS). Comparative analysis suggest that SC09-Ysa share extensive similarity in sequence, gene content, and gene arrangement with Salmonella enterica pathogenicity island 1 (SPI1) and chromosome-encoded T3SS from Yersinia enterocolitica biotype 1B. Furthermore, phylogenetic analysis shown that SC09-Ysa and SPI1-T3SS belong on the same branch of the phylogenetic tree. These results suggest that SC09-Ysa and SPI1-T3SS appear to mediate biological function to adapt to specific hosts with a similar niche, and both of them are likely to facilitate the development of an intracellular niche. In addition, our analysis also indicated that a substantial part of the SC09 genome might contribute to adaption in the intestinal microenvironment, including a number of proteins associated with aerobic or anaerobic respiration, signal transduction, and various stress reactions. Genomic analysis of the bacterium offered insights into the pathogenic mechanism associated with intracellular infection and intestinal survivability, which constitutes an important first step in understanding the pathogenesis of Y. ruckeri.
PMCID: PMC4849013  PMID: 27089334
Yersinia ruckeri; whole-genome sequencing; type III secretion system; niche adaptation; pathogenicity
13.  Intracorporeal esophagojejunostomy after totally laparoscopic total gastrectomy: A single-center 7-year experience 
World Journal of Gastroenterology  2016;22(12):3432-3440.
AIM: To assess the efficacy and safety of intracorporeal esophagojejunostomy in patients undergoing laparoscopic total gastrectomy (LTG) for gastric cancer.
METHODS: A retrospective review of 81 consecutive patients who underwent LTG with the same surgical team between November 2007 and July 2014 was performed. Four types of intracorporeal esophagojejunostomy using staplers or hand-sewn suturing were performed after LTG. Data on clinicopatholgoical characteristics, occurrence of complications, postoperative recovery, anastomotic time, and operation time among the surgical groups were obtained through medical records.
RESULTS: The average operation time was 288.7 min, the average anastomotic time was 54.3 min, and the average estimated blood loss was 82.7 mL. There were no cases of conversion to open surgery. The first flatus was observed around 3.7 d, while the liquid diet was started, on average, from 4.9 d. The average postoperative hospital stay was 10.1 d. Postoperative complications occurred in 14 patients, nearly 17.3%. However, there were no cases of postoperative death.
CONCLUSION: LTG performed with intracorporeal esophagojejunostomy using laparoscopic staplers or hand-sewn suturing is feasible and safe. The surgical results were acceptable from the perspective of minimal invasiveness.
PMCID: PMC4806201  PMID: 27022225
Gastric cancer; Total gastrectomy; Esophagojejunostomy; Laparoscopy; Hand-sewn
14.  Comparing the short-term outcomes of intracorporeal esophagojejunostomy with extracorporeal esophagojejunostomy after laparoscopic total gastrectomy for gastric cancer 
BMC Surgery  2016;16:13.
Totally laparoscopic distal gastrectomy (TLDG) using intracorporeal anastomosis has gradually developed due to advancements in laparoscopic surgical instruments. However, totally laparoscopic total gastrectomy (TLTG) with intracorporeal esophagojejunostomy (IE) is still uncommon because of technical difficulties. Herein, we evaluated various types of IE after TLTG in terms of the technical aspects. We compared the short-term operative outcomes between TLTG with IE and laparoscopy-assisted total gastrectomy (LATG) with extracorporeal esophagojejunostomy (EE).
Between March 2006 and December 2014, a total of 213 patients with gastric cancer underwent TLTG and LATG. Overall, 92 patients underwent TLTG with IE, and 121 patients underwent LATG with EE. Generally, there are two methods of IE: mechanical staplers (circular or linear staplers) and hand-sewn sutures. Surgical efficiencies and outcomes were compared between two groups. We also described various types of IE using a subgroup analysis.
The mean operation times were similar in the two groups, as was the number of retrieved lymph nodes. However, the mean estimated blood loss of TLTG was statistically lower than LATG. There were no significant differences in time to first flatus, the time to restart oral intake, the length of the hospital stay after operation, and postoperative complications. Four types of IE have been applied after TLTG, including 42 cases of hand-sewn IE. The overall mean operation time and the mean anastomotic time in TLTG were 279.5 ± 38.4 min and 52.6 ± 18.9 min respectively. There was no case of conversion to open procedure. Postoperative complication occurred in 16 patients (17.4 %) and no postoperative mortality occurred.
IE is a feasible procedure and can be safely performed for TLTG with the proper laparoscopic expertise. It is technically feasible to perform hand-sewn IE after TLTG, which can reduce the cost of the laparoscopic procedure.
PMCID: PMC4802707  PMID: 27000746
Laparoscopic gastrectomy, Intracorporeal anastomosis, Hand-sewn; Stomach neoplasms
15.  Risk Factors for Life-Threatening Complications of Maxillofacial Space Infection 
The purpose of this study was to review the clinical features of maxillofacial space infection (MSI), and to identify the potential risk factors predisposing to life-threatening complications. A retrospective review of the medical charts of patients with MSI treated at Peking University School and Hospital of Stomatology from August 2008 to September 2013 was conducted. A total of 127 patients [75 men (59.1%) and 52 women (40.9%); mean age, 45.39 ± 21.18 years, with a range of 1–85 years] formed the study cohort. The most common cause of MSI was odontogenic infection (57.5%). The most common space involved was the submandibular space. All patients were treated by antibiotics as well as surgical incision and drainage. Sixteen patients developed life-threatening complications, and the dominant complication was respiratory obstruction. Multivariate logistic regression analysis revealed the percentage of neutrophils (NEUT%) upon hospital admission ≥85.0% to be associated with life-threatening complications (P < 0.05). Even though adequate antibiotic therapy and incision and drainage of abscess were given, MSI patients with NEUT% upon hospital admission ≥85.0% carry a higher risk of life-threatening complications. In these patients, an aggressive treatment strategy is mandatory.
PMCID: PMC4782818  PMID: 26967077
Complication; maxillofacial space infection; odontogenic infection; risk factor
16.  Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage K8 and Its Genomic Analysis 
Phage therapy requires the comprehensive understanding of the mechanisms underlying the host-phage interactions. In this work, to identify the genes related to Pseudomonas aeruginosa phage K8 receptor synthesis, 16 phage-resistant mutants were selected from a Tn5G transposon mutant library of strain PAK. The disrupted genetic loci were identified and they were related to O-specific antigen (OSA) synthesis, including gene wbpR, ssg, wbpV, wbpO, and Y880_RS05480, which encoded a putative O-antigen polymerase Wzy. The Lipopolysaccharide profile of the Y880_RS05480 mutant was analyzed and shown to lack the O-antigen. Therefore, the data from characterization of Y880_RS05480 by TMHMM and SDS-PAGE silver staining analysis suggest that this locus might encode Wzy. The complete phage K8 genome was characterized as 93879 bp in length and contained identical 1188-bp terminal direct repeats. Comparative genomic analysis showed that phage K8 was highly homologous to members of the genus PaP1-like phages. On the basis of our genetic findings, OSA of P. aeruginosa PAK is proven to be the receptor of phage K8. The highly conserved structural proteins among the genetic closely related phages suggest that they may recognize the same receptor.
PMCID: PMC4773840  PMID: 26973628
Pseudomonas phage K8; phage receptor; O-specific antigen (OSA); wzy gene; genome annotation
18.  Bilateral Neuropathy of Primary Sensory Neurons by the Chronic Compression of Multiple Unilateral DRGs 
Neural Plasticity  2015;2016:2130901.
To mimic multilevel nerve root compression and intervertebral foramina stenosis in human, we established a new animal model of the chronic compression of unilateral multiple lumbar DRGs (mCCD) in the rat. A higher occurrence of signs of spontaneous pain behaviors, such as wet-dog shaking and spontaneous hind paw shrinking behaviors, was firstly observed from day 1 onward. In the meantime, the unilateral mCCD rat exhibited significant bilateral hind paw mechanical and cold allodynia and hyperalgesia, as well as a thermal preference to 30°C plate between 30 and 35°C. The expression of activating transcription factor 3 (ATF3) was significantly increased in the ipsilateral and contralateral all-sized DRG neurons after the mCCD. And the expression of CGRP was significantly increased in the ipsilateral and contralateral large- and medium-sized DRG neurons. ATF3 and CGRP expressions correlated to evoked pain hypersensitivities such as mechanical and cold allodynia on postoperative day 1. The results suggested that bilateral neuropathy of primary sensory neurons might contribute to bilateral hypersensitivity in the mCCD rat.
PMCID: PMC4706945  PMID: 26819761
19.  Adjuvant Immune Enhancement of Subunit Vaccine Encoding pSCPI of Streptococcus iniae in Channel Catfish (Ictalurus punctatus) 
Channel catfish (Ictalurus punctatus) is an important agricultural fish that has been plagued by Streptococcus iniae (S. iniae) infections in recent years, some of them severe. C5a peptidase is an important virulent factor of S. iniae. In this study, the subunit vaccine containing the truncated part of C5a peptidase (pSCPI) was mixed with aluminum hydroxide gel (AH), propolis adjuvant (PA), and Freund’s Incomplete Adjuvant (FIA). The immunogenicity of the pSCPI was detected by Western-blot in vitro. The relative percent survival (RPS), lysozyme activity, antibody titers, and the expression of the related immune genes were monitored in vivo to evaluate the immune effects of the three different adjuvants. The results showed that pSCPI exerted moderate immune protection (RPS = 46.43%), whereas each of the three adjuvants improved the immune protection of pSCPI. The immunoprotection of pSCPI + AH, pSCPI + PA, and pSCPI + FIA was characterized by RPS values of 67.86%, 75.00% and, 85.71%, respectively. Further, each of the three different adjuvanted pSCPIs stimulated higher levels of lysozyme activity and antibody titers than the unadjuvanted pSCPI and/or PBS buffer. In addition, pSCPI + FIA and pSCPI + PA induced expression of the related immune genes under investigation, which was substantially higher than the levels stimulated by PBS. pSCPI + AH significantly stimulated the induction of MHC II β, CD4-L2, and IFN-γ, while it induced slightly higher production of TNF-α and even led to a decrease in the levels of IL-1β, MHC I α, and CD8 α. Therefore, we conclude that compared with the other two adjuvants, FIA combined with pSCPI is a more promising candidate adjuvant against S. iniae in channel catfish.
PMCID: PMC4691029  PMID: 26602918
Channel catfish; Streptococcus iniae; subunit vaccine; pSCPI; adjuvant; immune effect
20.  The Significance of Myriophyllum elatinoides for Swine Wastewater Treatment: Abundance and Community Structure of Ammonia-Oxidizing Microorganisms in Sediments 
PLoS ONE  2015;10(10):e0139778.
Myriophyllum elatinoides was reported to effectively treat wastewater by removing nitrogen (N) and phosphorus (P). However, little is known about the abundance and community structure of ammonia-oxidizing microorganisms associated with M. elatinoides purification systems. The objective of this research was to characterize the abundance and community structure of ammonia-oxidizing microorganisms in swine wastewater and determine the main nitrogen removal pathways. In this study, five different waters were treated by M. elatinoides in microcosms for one month. The five waters included tap water (Control), swine wastewater (SW), 50% diluted swine wastewater (50% SW), and two synthetic wastewaters: 200 mg NH4+-N L−1 (200 NH4+-N) and 400 mg NH4+-N L−1 (400 NH4+-N). The most dramatic changes were in NH4+-N and total N (TN) concentrations, with average removal rates of 84% and 90%, respectively, in the treatments containing swine wastewater. On days 7, 14, and 28, the dissolved oxygen (DO) increased by 81.8%, 210.4% and 136.5%, respectively, compared with on day 0, in the swine wastewater. The results also showed that the bacterial amoA (AOB) copy numbers in the sediments of the treatments were significantly higher than those of archaeal amoA (AOA) copy numbers (p = 0.015). In addition, the high DO concentrations in swine wastewater responded well to the high abundance of AOB. The AOA and AOB community distributions were positively related with NO3-N and were negatively related with DO in swine wastewater treatments. In summary, our experimental results suggested that the M. elatinoides purification system could improve the activity of ammonia-oxidizing microorganisms and consequently might contribute to the significant N removal from the swine wastewater.
PMCID: PMC4596523  PMID: 26444015
21.  Structural basis of Ornithine Decarboxylase inactivation and accelerated degradation by polyamine sensor Antizyme1 
Scientific Reports  2015;5:14738.
Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis in humans. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Excessive accumulation of polyamines has a cytotoxic effect on cells and elevated level of ODC activity is associated with cancer development. To maintain normal cellular proliferation, regulation of polyamine synthesis is imposed by Antizyme1 (AZ1). The expression of AZ1 is induced by a ribosomal frameshifting mechanism in response to increased intracellular polyamines. AZ1 regulates polyamine homeostasis by inactivating ODC activity and enhancing its degradation. Here, we report the structure of human ODC in complex with N-terminally truncated AZ1 (cAZ1). The structure shows cAZ1 binding to ODC, which occludes the binding of a second molecule of ODC to form the active homodimer. Consequently, the substrate binding site is disrupted and ODC is inactivated. Structural comparison shows that the binding of cAZ1 to ODC causes a global conformational change of ODC and renders its C-terminal region flexible, therefore exposing this region for degradation by the 26S proteasome. Our structure provides the molecular basis for the inactivation of ODC by AZ1 and sheds light on how AZ1 promotes its degradation.
PMCID: PMC4595762  PMID: 26443277
22.  MicroRNA 192 regulates chemo-resistance of lung adenocarcinoma for gemcitabine and cisplatin combined therapy by targeting Bcl-2 
Lung cancer is the most leading cause of cancer-related death worldwide, with non-small-cell lung cancer (NSCLC) accounting for over 80% of all lung cancer cases. Patients with NSCLC are mostly treated with platinum-based chemotherapy. Chemoresistance is a leading cause of chemo-therapy failure in NSCLC treatment. Recent studies have shown that dysregulation of microRNAs might modulate the resistance of cancer cells to anti-cancer drugs, yet the modulation mechanism is not fully understood. In this paper, we try to test whether miR-192 regulates chemo-resistance in human carcinoma A549 mice model by targeting Bcl-2. Mice model of human lung adenocarcinoma was built up, and was used for gemcitabine and cisplatin combined chemotherapy. MTT assay, real-time RT-PCR, western blotting assay were used to investigate miR-192 expression levels, cell viability ratio and Bcl-2 protein expression levels. MiR-192 expression level in A549 cells is significantly higher than in normal human bronchial epithelial cells. MiR-192 inhibitor treated tumor exhibits sensitivity to cisplatin and gemcitabine therapy. Bcl-2 mRNA and protein expression levels up-regulated in miR-192 inhibitor treated tumor. Bcl-2 is a key regulator for miR-192 related chemotherapy resistance. In this study, we demonstrate that miR-192 regulates chemoresistance for gemcitabine and cisplatin combined chemotherapy in human adenocarcinoma lung cancer A549 cells, and Bcl-2 is the target of miR-192.
PMCID: PMC4612835  PMID: 26550150
MicroRNA; chemoresistance; Bcl-2; pathways
23.  An Uncoordinated-5 Homolog B Receptor Monoclonal Antibody Regulates A375 Melanoma Cell Migration 
Uncoordinated-5 homolog B receptor (UNC5B) was first found to mediate neural chemorepulsive effects by binding to its ligand netrin-1 in the nervous system. Newer evidence indicated that UNC5B also has functions outside the nervous system. In this study, we report on the generation of a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of UNC5B using the hybridoma technique. Western blot, immunofluorescence, and flow cytometry analyses showed that the antibody specifically bound to UNC5B protein. Interestingly, the antibody blocked the Netrin-1-induced inhibitory effect on the mobility of melanoma A375 cells by wound healing assay and transwell migration assay, whereas it had no effects on cell proliferation measured by CCK-8 assay. Thus, the functional antibody may provide a useful tool for the study of UNC5B expression profiles and functions outside the nervous system.
PMCID: PMC4151061  PMID: 25171009
24.  Cloning and Characterization of Surface-Localized α-Enolase of Streptococcus iniae, an Effective Protective Antigen in Mice 
Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the α-enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant α-enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that α-enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant α-enolase can confer effective protection against S. iniae infection in mice, which suggests that α-enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae α-enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.
PMCID: PMC4519854  PMID: 26121302
Streptococcus iniae; α-enolase; characterization; functions; surface location; protective antigen; mice
25.  MicroRNA-21 regulates non-small cell lung cancer cell proliferation by affecting cell apoptosis via COX-19 
Aims: This study is to investigate the regulatory effect of microRNA-21 (miR-21) on bone metastasis of non-small cell lung cancer (NSCLC). Methods: In this study, 18 patients were diagnosed with vertebral column metastasis of NSCLC. MiR-21 or small interfering RNAs were transfected into H2170 cells using Lipofectamine 2000. Real-time PCR was performed to detect miR-21 expression. Western blotting was used to measure the expression of COX-19 protein. Enzymatic activity tests were performed to measure the activity of cytochrome C oxidase. Flow cytometry was used to monitor changes in cell apoptotic rate. MTT assay was used to determine the capability of cell proliferation. Results: Bone metastasis of NSCLC enhanced the levels of miR-21 in NSCLC patients. Proliferation capability of cells with high expression of miR-21 was greater than that of cells with the inhibition of miR-21 expression. High expression of miR-21 promoted cell proliferation by inhibiting cell apoptosis. COX-19 was a key factor in the inhibition of apoptosis by miR-21. Inhibition of COX-19 expression reduced cell proliferation by enhancing cell apoptosis. Conclusions: This study demonstrates that inhibition of miRNA-21 suppresses NSCLC cell proliferation by promoting cell apoptosis via the decrease of COX-19 expression.
PMCID: PMC4538100  PMID: 26309536
Bone metastasis; non-small cell lung cancer; microRNA-21; cytochrome C oxidase; COX-19; apoptosis

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