Screening of lymph nodes metastasis associated lncRNAs in colorectal cancer patients

AIM: To screen lymph nodes metastasis associated long noncoding RNAs (lncRNAs) in colorectal cancer through microarray analysis.METHODS: Metastatic lymph node (MLN), normal lymph node (NLN) and tumor tissues of 3 colorectal cancer (CRC) patients were collected during the operation and validated by pathological examinations. RNAs were extracted from MLN, NLN, and cancer tissues separately. RNA quantity and quality were measured with a NanoDrop ND-1000 spectrophotometer and RNA integrity was assessed by standard denaturing agarose electrophoresis. Agilent Feature Extraction Software (Version 11.0.1.1) was used to analyze acquired array images. Four differently expressed lncRNAs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) in 26 subsets of MLN, NLN, and tumor tissues.RESULTS: Of 33045 lncRNAs, 1133 were differentially expressed in MLN compared with NLN, of which 260 were up-regulated and 873 down-regulated (≥ 2 fold-change). Five hundred and forty-five lncRNAs were differentially expressed in MLN compared with tumor tissues, of which 460 were up-regulated and 85 down-regulated (≥ 2 fold-change). Compared with NLN and cancer tissues, 14 lncRNAs were specifically up-regulated and 5 specifically down-regulated in MLN. {"type":"entrez-nucleotide","attrs":{"text":"AK307796","term_id":"164693359","term_text":"AK307796"}}AK307796, ENST00000425785, and {"type":"entrez-nucleotide","attrs":{"text":"AK021444","term_id":"10432630","term_text":"AK021444"}}AK021444 were confirmed to be specifically up-regulated in MLN and ENST00000465846 specifically down-regulated in MLN by qRT-PCR in 26 CRC patients.CONCLUSION: The specifically expressed lncRNAs in MLN may exert a partial or key role in the progress of lymph nodes metastasis of CRC.     

LY294002 potentiates the anti-cancer effect of oxaliplatin for gastric cancer via death receptor pathway

AIM:To examine the effects of combined treatment of oxaliplatin and phosphatidylinositol 3’-kinase inhibitor,2-(4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one(LY294002) for gastric cancer. METHODS:Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay.Apoptotic cells were detected by flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay.Western blotting and immuno-precipitation were used to examine protein expres...     

CP100356 Hydrochloride,a P-Glycoprotein Inhibitor,Inhibits Lassa Virus Entry: Implication of a Candidate Pan-Mammarenavirus Entry Inhibitor

Lassa virus (LASV)—a member of the family Arenaviridae—causes Lassa fever in humans and is endemic in West Africa. Currently, no approved drugs are available. We screened 2480 small compounds for their potential antiviral activity using pseudotyped vesicular stomatitis virus harboring the LASV glycoprotein (VSV-LASVGP) and a related prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). Follow-up studies confirmed that {"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356 hydrochloride ({"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356), a specific P-glycoprotein (P-gp) inhibitor, suppressed VSV-LASVGP, LCMV, and LASV infection with half maximal inhibitory concentrations of 0.52, 0.54, and 0.062 μM, respectively, without significant cytotoxicity. Although {"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356 did not block receptor binding at the cell surface, it inhibited low-pH-dependent membrane fusion mediated by arenavirus glycoproteins. P-gp downregulation did not cause a significant reduction in either VSV-LASVGP or LCMV infection, suggesting that P-gp itself is unlikely to be involved in arenavirus entry. Finally, our data also indicate that {"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356 inhibits the infection by other mammarenaviruses. Thus, our findings suggest that {"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356 can be considered as an effective virus entry inhibitor for LASV and other highly pathogenic mammarenaviruses.     

Signature of prognostic epithelial–mesenchymal transition related long noncoding RNAs (ERLs) in hepatocellular carcinoma

Reliable biomarkers are of great significance for the treatment and diagnosis of hepatocellular carcinoma (HCC). This study identified potential prognostic epithelial–mesenchymal transition related lncRNAs (ERLs) by the cancer genome atlas (TCGA) database and bioinformatics.The differential expression of long noncoding RNA (lncRNA) was obtained by analyzing the lncRNA data of 370 HCC samples in TCGA. Then, Pearson correlation analysis was carried out with EMT related genes (ERGs) from molecular signatures database. Combined with the univariate Cox expression analysis of the total survival rate of hepatocellular carcinoma (HCC) patients, the prognostic ERLs were obtained. Then use “step” function to select the optimal combination of constructing multivariate Cox expression model. The expression levels of ERLs in HCC samples were verified by real-time quantitative polymerase chain reaction.Finally, we identified 5 prognostic ERLs ({"type":"entrez-nucleotide","attrs":{"text":"AC023157.3","term_id":"6980118","term_text":"AC023157.3"}}AC023157.3, {"type":"entrez-nucleotide","attrs":{"text":"AC099850.3","term_id":"25046596","term_text":"AC099850.3"}}AC099850.3, AL031985.3, {"type":"entrez-nucleotide","attrs":{"text":"AL365203.2","term_id":"9743577","term_text":"AL365203.2"}}AL365203.2, CYTOR). The model showed that these prognostic markers were reliable independent predictors of risk factors (P value <.0001, hazard ratio [HR] = 2.400, 95% confidence interval [CI] = 1.667–3.454 for OS). In the time-dependent receiver operating characteristic analysis, this prognostic marker is a good predictor of HCC survival (area under the curve of 1 year, 2 years, 3 years, and 5 years are 0.754, 0.720, 0.704, and 0.662 respectively). We analyzed the correlation of clinical characteristics of these prognostic markers, and the results show that this prognostic marker is an independent factor that can predict the prognosis of HCC more accurately. In addition, by matching with the Molecular Signatures Database, we obtained 18 ERLs, and then constructed the HCC prognosis model and clinical feature correlation analysis using 5 prognostic ERLs. The results show that these prognostic markers have reliable independent predictive value. Bioinformatics analysis showed that these prognostic markers were involved in the regulation of EMT and related functions of tumor occurrence and migration.Five prognostic types of ERLs identified in this study can be used as potential biomarkers to predict the prognosis of HCC.     

A Suggested New Bacteriophage Genus, “Kp34likevirus”, within the Autographivirinae Subfamily of Podoviridae

Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 ({"type":"entrez-nucleotide","attrs":{"text":"NC_013649","term_id":"294661411","term_text":"NC_013649"}}NC_013649), F19 ({"type":"entrez-nucleotide","attrs":{"text":"NC_023567","term_id":"612508640","term_text":"NC_023567"}}NC_023567) and NTUH-K2044-K1-1 ({"type":"entrez-nucleotide","attrs":{"text":"NC_025418","term_id":"725949105","term_text":"NC_025418"}}NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called “Kp34likevirus” after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.     

Phosphatidylethanolamine-binding protein 4 promotes lung cancer cells proliferation and invasion via PI3K/Akt/mTOR axis

Background

While phosphatidylethanolamine-binding protein 4 (PEBP4) is a key factor in the malignant proliferation and metastasis of tumor cells, the exact regulatory network governing its roles remains unclear. This study was designed to investigate the effect of PEBP4 on PI3K/Akt/mTOR pathway and explore its molecular network that governs the proliferation and metastasis of tumor cells.

Methods

After the recombinant plasmid pcDNA3.1-PEBP4 was constructed, the recombinant plasmid pcDNA3.1-PEBP4 and PEBP4-targeting siRNA were transfected into lung cancer HCC827 cell line. The expressions of PI3K/Akt/mTOR pathway components in HCC827 cells in each group were determined using Western blotting. In the HCC827 cells, the effect of PI3K pathway inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay. Furthermore, the effect of mTOR inhibitor rapamycin (RAPA) on the expressions of PI3K/Akt/mTOR pathway components under the effect of PEBP4 was determined using Western blotting, and the effects of RAPA on the cell viability, proliferation, and migration capabilities under the overexpression of PEBP4 were determined using MTT method, flow cytometry, and Transwell migration assay.

Results

As shown by Western blotting, the protein expressions of p-Akt and phosphorylated mTOR (p-mTOR) were significantly higher in the pcDNA3.1-PEBP4-transfected group than in the normal control group and PEBP4 siRNA group (P<0.05); furthermore, the protein expressions of p-Akt and p-mTOR significantly decreased in the PEBP4 targeting siRNA-transfected group (P<0.05). Treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 significantly inhibited the protein expressions of p-Akt and p-mTOR in HCC827 cells (P<0.05). In contrast, treatment with RAPA only significantly inhibited the protein expression of p-mTOR (P<0.05). As shown by MTT, flow cytometry, and Transwell migration assay, both {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and RAPA could significantly lower the viability of HCC827 cells and inhibit their proliferation and invasion (P<0.05); meanwhile, they could reverse the effect of PEBP4 in promoting the proliferation and migration of HCC827 cells (P<0.05).

Conclusions

The overexpression of PEBP4 increases the phosphorylation levels of Akt and mTOR in lung cancer cells. The PI3K/Akt/mTOR signaling axis may be a key molecular pathway via which PEBP4 promotes the proliferation and invasion of non-small cell lung cancer (NSCLC) cells; also, it may serve as a potential therapeutic target.     

Laboratory Diagnosis and Genotype Identification of Scrub Typhus from Pinggu District,Beijing, 2008 and 2010

This study was conducted to determine the diagnosis and genotype of Orientia tsutsugamushi in Pinggu district, Beijing. Indirect immunofluorescence assay (IFA) was performed to detect O. tsutsugamushi-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. Nested polymerase chain reaction (PCR) and DNA sequencing analysis targeting the O. tsutsugamushi-specific groEL gene and 56 kDa protein gene were performed on whole-blood samples from scrub typhus patients. We confirmed that 47 patients were infected with scrub typhus in Pinggu district, Beijing. Representative sequences amplified by primers according to the groEL gene (BJ-PG-2008; GenBank accession No. {"type":"entrez-nucleotide","attrs":{"text":"JQ894502","term_id":"388252860","term_text":"JQ894502"}}JQ894502) and the 56 kDa protein gene (PG-56kDa; GenBank accession No. {"type":"entrez-nucleotide","attrs":{"text":"JX843795","term_id":"410832938","term_text":"JX843795"}}JX843795) both clustered with Kawasaki. PG-56kDa had sequence homology of 100% with TADY12-0308, shandong-XDM2, Neimeng-90, and sdu-1 and sequence homology of 96% with Kawasaki, Taguchi, Oishi, and Kanda. We confirmed the genotype of O. tsutsugamushi in Pinggu district, Beijing, as Kawasaki, and the patient in 2008 confirmed in this study was the first patient with confirmed scrub typhus in Beijing.     

Phylogenetic Characteristics of Canine Parvovirus Type 2c Variant Endemic in Shanghai,China

Canine parvovirus type 2 (CPV-2) has spread and mutated globally over the past 40 years. In the present study, 206 samples from dogs suspected of CPV-2 infection were collected from five veterinary clinics in Shanghai city, China. The average positive rate for CPV-2 was detected to be 40.78% using the PCR method. Using an F81 cell (feline kidney cell) culture, the isolates of three CPV-2c strains were obtained. The near full-length genome sequences of the isolates were determined and submitted to GenBank: CPV-SH2001 ({"type":"entrez-nucleotide","attrs":{"text":"MW650830","term_id":"2075754664"}}MW650830), CPV-SH2002 ({"type":"entrez-nucleotide","attrs":{"text":"MW811188","term_id":"2078827845"}}MW811188), and CPV-SH2003 ({"type":"entrez-nucleotide","attrs":{"text":"MW811189","term_id":"2078827850"}}MW811189). By comparing the amino acid sequences of 12 CPV strains with those of 48 related strains retrieved from GenBank, all of the CPV strains from Shanghai were typed as belonging to a relatively new CPV-2c variant spreading in Asia, with typical amino acid residues (5Gly, 267Tyr, 324Ile, and 370Arg) in the VP2 protein. The divergence time of this new CPV-2c clade was estimated by the phylogenetic tree using the maximum likelihood and RelTime with Dated Tips (RTDT) approaches. Our results indicate that the 426 and 324 VP2 amino acid residues are under strong selection pressure with a posterior probability of 0.966 and 0.943, respectively. Therefore, this study provides insight into the phylogenetic characteristics of the current CPV-2c variant in Shanghai city, China.     

Infection with Leptospira kirschneri Serovar Mozdok: First Report from the Southern Hemisphere

Leptospirosis is a global zoonosis caused by pathogenic Leptospira spp. In this study, we characterized two Leptospira kirschneri serogroup Pomona serovar Mozdok isolates, one obtained from a dog and the other from a patient with severe leptospirosis, 4 years later. Histopathological analysis showed that both isolates caused severe tissue damage when used to infect hamsters. While L. kirschneri serogroup Pomona serovar Mozdok is endemic in animals in Europe, there is only one report of human leptospirosis in the literature. Although strains belonging to L. kirschneri serogroup Pomona have been identified in cases of human leptospirosis in Europe, serovar Mozdok has not yet been implicated. The 4-year interval between isolations and the fact that this is the first report of serovar Mozdok as the causative agent of human leptospirosis in the southern hemisphere, demonstrates its epidemiological importance to public health. Moreover, the presence of serovar Mozdok in Brazil has the potential to affect vaccine and diagnostic test development.Leptospirosis is a reemerging zoonotic disease, and the global burden is showing an upward trend. The original estimates in 1999¹ predicted some 500,000 annual cases compared with the latest prediction of 873,000 cases and 49,000 mortalities per year, a 74.6% increase over 15 years.² Accurate laboratory diagnosis continues to be a limiting factor, meaning that the true global burden of leptospirosis is likely to be much higher.³ In Latin America, the prevalence of severe leptospirosis is high (10,000 cases a year) due to the tropical climate and lack of appropriate sanitation.³ Although the city of Pelotas has a subtropical climate, > 50 cases of human leptospirosis per 100,000 inhabitants are reported each year, one of the highest rates in southern Brazil.⁴ The infection rate in Pelotas is higher than the Brazilian average for the same period (3.5/100,000) and other regions with similar climatic conditions (> 10/100,000).⁵At present, there are 10 pathogenic Leptospira spp. classified into > 260 serovars⁶ and Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri are most commonly associated with human leptospirosis.⁷ In Brazil, L. interrogans serogroup Icterohaemorrhagiae serovars Icterohaemorrhagiae and Copenhageni are the main cause of urban leptospirosis and have been widely studied,³ whereas rural leptospirosis and the associated serovars have been largely neglected. To the best of our knowledge, L. kirschneri serogroup Pomona serovar Mozdok has only been implicated in a case of human leptospirosis in Cuba.⁸ Serovar Mozdok is endemic to Croatia where it is prevalent in wild rodents. Human leptospirosis caused by serogroup Pomona is common in that region and while serovar Mozdok has not been implicated in any human cases,⁹ it is a causative agent of canine leptospirosis in Europe.¹⁰We report the isolation and characterization of two isolates of serovar Mozdok recovered from cases of canine and human leptospirosis in Pelotas, southern Brazil. The canine strain was isolated in 2009 during a municipal dog neutering campaign. Urine samples were aseptically collected from the bladder during ovarian hysterectomy, via aspiration using an insulin 30-G needle and syringe (BD Biosciences, Franklin Lakes, NJ). The urine was immediately inoculated into unsupplemented Ellinghausen-McCullough-Johnson-Harris (EMJH; Difco, Sparks, MD) medium (100 μL urine/5 mL EMJH), incubated for 1 hour and then subcultured into EMJH containing 10% of a commercial supplement (Difco). The dog from which the strain was isolated was asymptomatic and was released after the surgical procedure. The second isolate was obtained from the blood culture of a 56-year-old female patient from a rural area of the city. The patient presented with headache, myalgia, fever, vomiting, fatigue, sleepiness, and arthralgia and reported contact with dogs, rats, pigs, cattle, and flood water. The isolate was cultured in EMJH medium as described for the canine isolate. Both isolates were identified as L. kirschneri by means of secY gene sequencing.¹¹ Multilocus sequence typing (MLST)⁷ further characterized the isolates as L. kirschneri serogroup Pomona serovar Mozdok (ST 117). All sequencing procedures were performed using the paired-end technology on an Illumina Solexa platform (Illumina, San Diego, CA; GenBank accession numbers for sequences are shown in

Molecular Detection and Identification of Bartonella Species in Rat Fleas from Northeastern Thailand

The presence of Bartonella species in Xenopsylla cheopis fleas collected from Rattus spp. (R. exulans, R. norvegicus, and R. rattus) in Khon Kaen Province, Thailand was investigated. One hundred ninety-three fleas obtained from 62 rats, were screened by polymerase chain reaction using primers specific for the 16S–23S intergenic spacer region, and the presence of Bartonella DNA was confirmed by using the citrate synthase gene. Bartonella DNA was detected in 59.1% (114 of 193) of fleas examined. Sequencing demonstrated the presence of Bartonella spp. similar to B. elizabethae, B. rattimassiliensis, B. rochalimae, and B. tribocorum in the samples tested with a cutoff for sequence similarity ≥ 96% and 4 clustered together with the closest match with B. grahamii (95.5% identity). If X. cheopis proves to be a competent vector of these species, our results suggest that humans and animals residing in this area may be at risk for infection by several zoonotic Bartonella species.Bartonella species are small, pleomorphic, gram-negative bacteria that infect a variety of mammalian hosts, including cats, dogs, rodents, ruminants, and humans. Clinical symptoms associated with Bartonella range from mild, influenza-like symptoms to more severe manifestations such as endocarditis, myocarditis, uveitis, bacillary angiomatosis, and peliosis hepatis.¹ Approximately half of the 20 Bartonella species or subspecies identified to date are known or suspected human pathogens,² and most are believed to be transmitted by arthropod vectors (fleas, lice, sandflies, and ticks).³Xenopsylla cheopis, the Oriental rat flea, is a suspected vector of several Bartonella species (B. tribocorum, B. elizabethae, B. queenslandensis, B. rochalimae, and novel Bartonella genotypes), and Bartonella DNA has been detected in these fleas from various locations worldwide.^3–8 Although generally found on rodents, X. cheopis have been found to parasitize humans and are known vectors of the zoonotic agents Yersinia pestis (plague) and Rickettsia typhi (murine typhus).⁹Numerous surveys have been performed to identify the presence of Bartonella species affecting humans and domestic and peri-domestic animals in Thailand.^10–17 Bartonella henselae, (the agent of cat scratch disease),¹⁴ B. tamiae,¹⁰ B. elizabethae, B. rattimassiliensis, and B. tribocorum have been isolated from febrile patients,¹⁵ B. henselae and B. clarridgeiae have been reported in cats,¹¹ and B. clarridgeiae, B. vinsonii subsp. arupensis, B. elizabethae, B. grahamii, B. quintana, B. taylorii, and novel Bartonella genotypes have been found in dogs.^11,16 In rodent species, B. grahamii, B. elizabethae, Candidatus Bartonella thailandensis, B. coopersplainensis, B. phoceensis, B. rattimassiliensis, B. tribocorum, and novel Bartonella genotypes have been detected by culture and polymerase chain reaction (PCR) analysis.^12,13,17 However, little information has been obtained to identify potential arthropod vectors of Bartonella species in Thailand. Bartonella henselae, B. clarridgeiae and B. koehlerae were detected in Ctenocephalides felis fleas removed from cats^18–20 and B. henselae was identified in two C. canis¹⁹ also collected from cats. Furthermore, a Bartonella sp., similar to B. grahamii, was found in a rodent flea, Nosopsyllus fasciatus, obtained from Rattus surifer.¹⁸ Bartonella tamiae DNA has also been found in chigger mites (genera Leptotrombidium, Schoengastia, and Blankarrtia) and in a tick (genus Haemaphysalis) collected from rodents in Thailand, suggesting a potential role for these arthropods in the transmission of B. tamiae.²¹The aim of the current study was to investigate the prevalence of Bartonella species in rodent-associated fleas collected in Khon Kaen Province, Thailand, and to determine what potential role, if any, these fleas may play in the transmission of Bartonella species to individuals residing in this area.For this study, 62 rats (10 R. norvegicus, 9 R. rattus, and 43 R. exulans) were trapped in and around homes in 4 villages, 1 market, and on farm land (a pig farm and 2 rice fields) in Khon Kaen Province, Thailand during May–June 2011 (22 Work involving rodents was conducted as outlined in our approved animal use protocol (#11-003), under the supervision of the Institutional Animal Care and Use Committee of the Division of Vector Borne Diseases.

Table 1

Number of rats trapped per site by species and total number of fleas examined per site, northeastern Thailand

Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation

Rhizobia are Gram-negative soil bacteria able to establish nitrogen-fixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithine-containing lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse–chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase {"type":"entrez-protein","attrs":{"text":"SMc00171","term_id":"1174089800","term_text":"SMC00171"}}SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which {"type":"entrez-protein","attrs":{"text":"SMc00171","term_id":"1174089800","term_text":"SMC00171"}}SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that {"type":"entrez-protein","attrs":{"text":"SMc00171","term_id":"1174089800","term_text":"SMC00171"}}SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria.     

Detection of Entebbe Bat Virus after 54 Years

Entebbe bat virus (ENTV; Flaviviridae: Flavivirus), closely related to yellow fever virus, was first isolated from a little free-tailed bat (Chaerephon pumilus) in Uganda in 1957, but was not detected after that initial isolation. In 2011, we isolated ENTV from a little free-tailed bat captured from the attic of a house near where it had originally been found. Infectious virus was recovered from the spleen and lung, and the viral RNA was sequenced and compared with that of the original isolate. Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Further study of this virus would provide valuable insights into the ecological and genetic factors governing the evolution and transmission of bat- and mosquito-borne flaviviruses.Bat virus investigations in Uganda were initially inspired in the mid-1950s by the isolations of rabies virus and a novel flavivirus, later to become known as Rio Bravo virus (Flaviviridae: Flavivirus), from the salivary glands of insectivorous bats in the United States.^1–3 Subsequently, investigators at the Uganda Virus Research Institute (UVRI) collected bats from the attic of UVRI, dissected salivary glands, and isolated viruses by intracerebral inoculation of triturated salivary gland extracts into neonatal mice.^4,5 This first effort resulted in the isolation of Entebbe bat salivary gland virus, strain IL-30 (Flaviviridae: Flavivirus) from a little free-tailed bat (Chaerephon pumilus) (Cretzchmar).^4,5 The taxon was then recognized by one of its synonyms: Tadarida (Chaerephon) limbata (Peters). Lumsden and others^4,5 determined that this isolate was a novel flavivirus through a series of neutralization tests using immune sera against 20 arboviruses as well as against Rio Bravo virus. By 1964, bat virus research had expanded at UVRI, and sampling of 1,022 additional bats yielded 14 strains of viruses including Dakar bat virus (Flaviviridae: Flavivirus) also from little free-tailed bats, and Bukalasa bat virus (Flaviviridae: Flavivirus) from Angolan free-tailed bats (Mops condylurus) (A. Smith).⁶ Isolation of Mount Elgon bat virus (Rhabdoviridae: Ledantevirus) followed shortly thereafter from an eloquent horseshoe bat (Rhinolophus hildebrandtii eloquens) (K. Anderson) captured in Kenya, with the virus being isolated and characterized at UVRI.^7,8 Additional bat virus discoveries in Uganda later included Kasokero virus (Bunyaviridae, unassigned) from an Egyptian fruit bat (Rousettus aegyptiacus) (E. Geoffroy).⁹ Entebbe bat virus (ENTV, renamed), Bukalasa bat virus, and Dakar bat virus were all isolated from insectivorous bats captured in the attic of UVRI.¹⁰ Serological surveys suggested that the infection prevalence of ENTV in wild populations of little free-tailed bats was high, but ENTV was never isolated again after the original discovery.^10,11To follow up on these early studies, arbovirus surveillance of bats was conducted in Uganda from 2011 to 2013. Bats were captured from attics in several locations around Entebbe and Kampala as part of a larger, country-wide sampling effort to be reported elsewhere. All bat captures were conducted under the approval of CDC/DVBD IACUC protocol 010-015. Bats were captured in attics using mist nets, taking appropriate biosafety precautions. On capture, bats were placed individually in cloth holding bags. Bats were anesthetized with halothane, exsanguinated by cardiac puncture, and euthanized by halothane overdose and cervical dislocation. Blood from bats was collected directly into serum separator tubes, centrifuged in the field, and placed immediately in a nitrogen dry vapor shipper. Liver, spleen, heart, lung, and kidney were all collected directly into cryovials and stored immediately on dry ice. In total, 95 little free-tailed bats and 34 Angolan free-tailed bats were captured in 2011, 15 little free-tailed bats in 2012, and 70 little free-tailed bats in 2013.Virus isolation was attempted first from spleens, and on a positive result, virus isolation was also performed from the remaining tissues harvested from the infected bat. A single virus was isolated from 1 of 180 (0.5%) little free-tailed bats. The infected bat was an adult male, captured in Banga, Nakiwogo (0°04.884′ N, 32°27.030′ E) on June 23, 2011, near UVRI. Infectious virus was isolated from the spleen and lung; no infectious virus was recovered from the heart, liver, or kidney. No other tissues, oral or fecal swabs were collected from the insectivorous bats captured in 2011. The isolate was initially identified as ENTV by sequencing of the flavivirus NS5 amplicon using primers FU2/CFD3,¹² followed by next-generation sequencing (NGS). Virus isolations, nucleic acid preparations, and NGS were performed as described previously.¹³ Novel primers were designed from contigs generated from the NGS data using Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA)¹⁴ (Supplemental Table 1), and the sequence for the entire genome was confirmed by direct sequencing. End terminal sequences were obtained using 5′/3′ RACE (FirstChoice^® RLM-RACE, Life Technologies, Carlsbad, CA) according to the manufacturer''s instructions. Virus-specific RACE primers are provided in Supplemental Table 1. The 5′ outer reaction used primer 514R in combination with the 5′ RACE outer primer. The 5′ RACE inner reactions used primer 388R together with the 5′ RACE inner primer; amplification was also achieved with primers 15F + 388R. The 3′ outer reaction used either primer 9882F or 10328F in combination with the 3′ RACE outer primer. The 3′ inner RACE was not successful. A maximum likelihood phylogenetic tree was generated in MEGA version 6.0¹⁵ (Center for Evolutionary Medicine and Informatics, Biodesign Institute, Tempe, AZ) using 10,493 nucleotides spanning the open reading frame sequence of select flavivirus genomes available in GenBank (Figure 1 ). The analysis was performed using a complete deletion substitution model and 1,000 bootstrap replicates.Open in a separate windowFigure 1.Maximum likelihood tree based on open reading frame sequence (complete gap deletion). Scale bar indicates nucleotide substitutions per site. Bootstrap values > 50% are shown (1,000 replicates). CFAV = cell fusing agent virus (NC001564); DENV1 = dengue type 1 ({"type":"entrez-nucleotide","attrs":{"text":"M87512","term_id":"323660","term_text":"M87512"}}M87512); DENV2 = dengue type 2 ({"type":"entrez-nucleotide","attrs":{"text":"M20558","term_id":"323449","term_text":"M20558"}}M20558); DENV3 = dengue type 3 ({"type":"entrez-nucleotide","attrs":{"text":"M93130","term_id":"323468","term_text":"M93130"}}M93130); DENV4 = dengue type 4 ({"type":"entrez-nucleotide","attrs":{"text":"AF326573","term_id":"12659201","term_text":"AF326573"}}AF326573); EHV = Edge Hill virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ859060","term_id":"146411782","term_text":"DQ859060"}}DQ859060); ENTV = Entebbe bat virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641); RBV = Rio Bravo virus ({"type":"entrez-nucleotide","attrs":{"text":"JQ582840","term_id":"383215103","term_text":"JQ582840"}}JQ582840); SEPV = Sepik virus (NC008719); SLEV = St. Louis encephalitis virus (NC007580); SOKV = Sokoluk virus ({"type":"entrez-nucleotide","attrs":{"text":"NC_026624","term_id":"765702601","term_text":"NC_026624"}}NC_026624); TBEV = tick-borne encephalitis virus (NC001672); WNV = West Nile virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ211652","term_id":"77166600","term_text":"DQ211652"}}DQ211652); WSLV = Wesselsbron virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ859058","term_id":"146411778","term_text":"DQ859058"}}DQ859058); YFV = yellow fever virus ({"type":"entrez-nucleotide","attrs":{"text":"K02749","term_id":"336192","term_text":"K02749"}}K02749); YOKV = Yokose virus (NC005039); ZIKV = Zika virus ({"type":"entrez-nucleotide","attrs":{"text":"AF013415","term_id":"2731554","term_text":"AF013415"}}AF013415).In total, 10,663 nucleotides of the ENTV genome were obtained (GenBank Accession number {"type":"entrez-nucleotide","attrs":{"text":"KP233893","term_id":"873167403","term_text":"KP233893"}}KP233893), of which the polypeptide is encoded between nucleotides 120 and 10,355. Kuno and Chang¹⁶ published the only other existing ENTV genome ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641) sequenced from the original IL-30 strain. Compared with that original sequence, we report an additional 153 nucleotides in the 3′ noncoding region, but also experienced difficulty in obtaining the complete 3′ terminal sequence.¹⁶ Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Before sequencing, the 1957 isolate had been passaged in suckling mouse brain, but the earlier passage history is unclear; the 2011 isolate was passaged twice in Vero cells. This 2.2% amino acid divergence is within the range that has been observed for other flaviviruses with isolations ranging between 1 and 49 years apart,^17–19 indicating relatively little change in the ENTV genome in 54 years since the first isolation.Every gene had at least one amino acid change except for PrM and M (20 It has also been shown to associate with host cellular vimentin during dengue virus replication to stabilize the replication complex.²¹ The functional significance of this high percentage of amino acid divergence has yet to be determined, but Hahn and others²² surmised that a large number of amino acid substitutions could be accommodated among the small, hydrophobic polypeptides NS2A, NS2B, NS4A, and NS4B so long as the hydrophobicity profile remained unchanged. In this case, changes to the NS4A hydrophobicity profile between these two isolates were inconsequential. Two of the 76 amino acid changes occurred at the junctions of NS2A/NS2B, and at NS4A/2K. The lengths of all of the genes were identical to those previously reported.¹⁶

Table 1

Length of each gene region in the Entebbe bat virus genome and the number (%) of amino acid (aa) substitutions in the 2011 isolate ({"type":"entrez-nucleotide","attrs":{"text":"KP233893","term_id":"873167403","term_text":"KP233893"}}KP233893) relative to the 1957 isolate ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641)

Gain in sensitivity and loss in temporal contrast of STDP by dopaminergic modulation at hippocampal synapses

Spike-timing-dependent plasticity (STDP) is considered a physiologically relevant form of Hebbian learning. However, behavioral learning often involves action of reinforcement or reward signals such as dopamine. Here, we examined how dopamine influences the quantitative rule of STDP at glutamatergic synapses of hippocampal neurons. The presence of 20 μM dopamine during paired pre- and postsynaptic spiking activity expanded the effective time window for timing-dependent long-term potentiation (t-LTP) to at least −45 ms, and allowed normally ineffective weak stimuli with fewer spike pairs to induce significant t-LTP. Meanwhile, dopamine did not affect the degree of t-LTP induced by normal strong stimuli with spike timing (ST) of +10 ms. Such dopamine-dependent enhancement in the sensitivity of t-LTP was completely blocked by the D1-like dopamine receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, but not by the D2-like dopamine receptor antagonist sulpiride. Surprisingly, timing-dependent long-term depression (t-LTD) at negative ST was converted into t-LTP by dopamine treatment; this conversion was also blocked by {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390. In addition, t-LTP in the presence of dopamine was completely blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid, indicating that D1-like receptor-mediated modulation appears to act through the classical NMDA receptor-mediated signaling pathway that underlies STDP. These results provide a quantitative and mechanistic basis for a previously undescribed learning rule that depends on pre- and postsynaptic ST, as well as the global reward signal.     

Mitochondrial ATP 6 and 8 polymorphisms in irritable bowel syndrome with diarrhea

AIM: To investigate mitochondrial ATP 6 and 8 poly-morphisms in the colon and ileum of patients with ir-ritable bowel syndrome with diarrhea （IBS-D）. METHODS: Twenty-eight patients fulfilling the Rome Ⅲ criteria for IBS-D and 28 healthy subjects were in-vestigated. All study participants underwent screening colonoscopy and mucosal biopsies were obtained from the colon and/or terminal ileum. Genomic DNA was ex-tracted from specimens based on standard protocols. Mitochondrial ATP （MT-ATP） 6 and 8 genes in speci-mens were polymerase chain reaction amplified and sequenced. Sequencing data were analyzed via Variant Reporter Software and compared with the reference sequence from Genbank （accession No. NC₀12920） to indicate possible polymorphisms. The protocol was registered at www.clinicaltrials.gov as NCT01028898. RESULTS: Twenty-five polymorphic sites of MT-ATP 6 and 8 genes were detected and 12 of them were missense mutations. A median of two polymorphic sites in MT-ATP genes was found in colon specimens of controls while a median of three polymorphic sites was noted in patients with IBS-D （Mann-Whitney test, P=0.012）. The variants of the colon and ileum speci-mens from the same subjects were identical in all but one case. Symptom duration in IBS was not found to be a significant factor associated with the mtDNA polymorphism （Spearman correlation, P=0.592）. The mitochondrial DNA change at 8860 was present in all cases of both groups. The frequency of the 8701 poly-morphism was found to be the second most frequent; however, no statistical difference was noted between the groups （χ2 test, P=0.584）. CONCLUSION: Patients with IBS-D have a higher inci-dence of MT-ATP 6 and 8 polymorphisms than healthy subjects, implying that the mtDNA polymorphism may play a role in IBS-D.     

Bovine NK-lysin: Copy number variation and functional diversification

NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ∼30–35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer’s patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants.Antimicrobial peptides (AMPs) are effector molecules in the innate immune system and are widespread in all kingdoms of life (1, 2). Human granulysin (GNLY) and pig NK-lysin are orthologs and belong to the same group of AMPs (3, 4). They are secreted from the granules of cytotoxic T lymphocytes and natural killer (NK) cells and are active against a wide spectrum of microorganisms including Gram-positive and Gram-negative bacteria, fungi, protozoa, viruses, and even tumor cells (5–11). NK-lysin orthologs have been identified and characterized in many species, including human, pig, cattle, horse, water buffalo, and several species of birds (12–15). Bovine NK-lysin was first reported a decade ago (16), when two bovine cDNA fragments were obtained from each of four different cows. It was unclear whether the detected sequences, Bo-lysin 89 and Bo-lysin 62, were from two different NK-lysin genes or were alleles of a single gene. Also, multiple variants of NK-lysin sequences exist in the bovine nucleotide database, suggesting the existence of more than one copy of NK-lysin in the cattle genome (Fig. S1 and Open in a separate windowFig. S1.Phylogenetic analysis of seven different bovine NK-lysin–related mRNA sequences (mRNA-1–7) from the NCBI nucleotide database. Four clades were formed and annotated as NK1 and NK2A, 2B, and 2C. Bootstrap values are shown at branch points.

Table S1.

NK-lysin–related sequences from the NCBI bovine nucleotide database

Exploring new targets for the treatment of hepatitis-B virus and hepatitis-B virus-associated hepatocellular carcinoma: A new perspective in bioinformatics

Background:Hepatitis B Virus (HBV) infection is a global public health problem. After infection, patients experience a natural course from chronic hepatitis to cirrhosis and even Hepatitis B associated Hepatocellular Carcinoma (HBV-HCC). With the multi-omics research, many differentially expressed genes from chronic hepatitis to HCC stages have been discovered. All these provide important clues for new biomarkers and therapeutic targets. The purpose of this study is to explore the differential gene expression of HBV and HBV-related liver cancer, and analyze their enrichments and significance of related pathways.Methods:In this study, we downloaded four microarray datasets {"type":"entrez-geo","attrs":{"text":"GSE121248","term_id":"121248"}}GSE121248, {"type":"entrez-geo","attrs":{"text":"GSE67764","term_id":"67764"}}GSE67764, {"type":"entrez-geo","attrs":{"text":"GSE55092","term_id":"55092"}}GSE55092, {"type":"entrez-geo","attrs":{"text":"GSE55092","term_id":"55092"}}GSE55092 and {"type":"entrez-geo","attrs":{"text":"GSE83148","term_id":"83148"}}GSE83148 from the Gene Expression Omnibus (GEO) database. Using these four datasets, patients with chronic hepatitis B (CHB) differentially expressed genes (CHB DEGs) and patients with HBV-related HCC differentially expressed genes (HBV-HCC DEGs) were identified. Then Protein–protein Interaction (PPI) network analysis, Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to excavate the functional interaction of these two groups of DEGs and the common DEGs. Finally, the Kaplan website was used to analyze the role of these genes in HCC prognostic.Results:A total of 241 CHB DEGs, 276 HBV-HCC DEGs, and 4 common DEGs (cytochrome P450 family 26 subfamily A member 1 (CYP26A1), family with sequence similarity 110 member C(FAM110C), SET and MYND domain containing 3(SMYD3) and zymogen granule protein 16(ZG16)) were identified. CYP26A1, FAM110C, SMYD3 and ZG16 exist in 4 models and interact with 33 genes in the PPI network of CHB and HBV-HCC DEGs,. GO function analysis showed that: CYP26A1, FAM110C, SMYD3, ZG16, and the 33 genes in their models mainly affect the regulation of synaptic vesicle transport, tangential migration from the subventricular zone to the olfactory bulb, cellular response to manganese ion, protein localization to mitochondrion, cellular response to dopamine, negative regulation of neuron death in the biological process of CHB. In the biological process of HBV-HCC, they mainly affect tryptophan catabolic process, ethanol oxidation, drug metabolic process, tryptophan catabolic process to kynurenine, xenobiotic metabolic process, retinoic acid metabolic process, steroid metabolic process, retinoid metabolic process, steroid catabolic process, retinal metabolic process, and rogen metabolic process. The analysis of the 4 common DEGs related to the prognosis of liver cancer showed that: CYP26A1, FAM110C, SMYD3 and ZG16 are closely related to the development of liver cancer and patient survival. Besides, further investigation of the research status of the four genes showed that CYP26A1 and SMYD3 could also affect HBV replication and the prognosis of liver cancer.Conclusion:CYP26A1, FAM110C, SMYD3 and ZG16 are unique genes to differentiate HBV infection and HBV-related HCC, and expected to be novel targets for HBV-related HCC occurrence and prognostic judgement.     

Overcoming cancer cell resistance to Smac mimetic induced apoptosis by modulating cIAP-2 expression

Smac mimetics target cancer cells in a TNFα-dependent manner, partly via proteasome degradation of cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2. Degradation of cIAPs triggers the release of receptor interacting protein kinase (RIPK1) from TNF receptor I (TNFR1) to form a caspase-8 activating complex together with the adaptor protein Fas-associated death domain (FADD). We report here a means through which cancer cells mediate resistance to Smac mimetic/TNFα-induced apoptosis and corresponding strategies to overcome such resistance. These human cancer cell lines evades Smac mimetic-induced apoptosis by up-regulation of cIAP2, which although initially degraded, rebounds and is refractory to subsequent degradation. cIAP2 is induced by TNFα via NF-κB and modulation of the NF-κB signal renders otherwise resistant cells sensitive to Smac mimetics. In addition, other signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), have the potential to concurrently regulate cIAP2. Using the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, cIAP2 up-regulation was suppressed and resistance to Smac mimetics-induced apoptosis was also overcome.