Objective. To compare the clinical value of serum microRNA21 (miR21) and other tumor markers in early diagnosis of non-small cell lung cancer (NSCLC). Methods. Serums carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), neuron-specific enolase (NSE), and miR21 were detected in 50 NSCLC cases and 60 healthy control individuals. Results. Average serums miR21, CEA, NSE, and CYFRA21-1 levels were significantly higher in the case group than in control group (P < 0.01). Analysis of areas under the receiver operating characteristic (ROC) curve (AUC) revealed that CEA had the highest diagnostic efficiency for NSCLC. Serums miR21 and CYFRA21-1 levels were significantly lower at TNM stages I-II than stages III-IV (P < 0.05). Further, logistic multivariate regression analysis showed that the incidence of early NSCLC (TNM stages I-II) was correlated with serums CYFRA21-1 (OR = 1.076) and miR21 (OR = 2.473) levels (P < 0.05). By AUC analysis, miR21 had the highest diagnostic efficiency for early NSCLC, and single or combined detection of serums CYFRA21-1 and miR21 levels showed improved diagnostic efficiency for joint detection of both markers. Conclusions. Serum miR21 could serve as an important marker for auxiliary diagnosis of early NSCLC, while joint detection of serums miR21 and CYFRA21-1 levels could improve diagnostic efficiency.  The annual morbidity rate of non-small cell lung cancer (NSCLC) has been increasing in recent years. Both in China and worldwide, NSCLC has become one of the most lethal tumor types . With clBiopsy by bronchoscope, mediastinoscope, or thoracentesis is the most reliable method to diagnose NSCLC. However, these techniques have many contraindications in application and thus are not practical for early screening and continuous monitoring of the disease. Serum marker detection—with advantages including easy operation, low price, noninvasiveness, accessibility of samples, and ability for continuous monitoring—is a high-profile topic for auxiliary diagnosis of early NSCLC . ClinicaDuring the initiation and development of NSCLC, driver genes that induce and maintain molecular changes of malignant tumors, such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), fibroblast growth factor receptor 1 (FGFR1), and phosphoinositide 3-kinase catalytic subunit A (PIK3CA), play an important role . PreviouRecent studies have indicated that miRs participate in the occurrence, development, and prognosis of pulmonary cancer and have similar effects as protooncogenes or tumor-suppressing genes. In pulmonary cancer tissues, miRs have unique expression profiles and participate in multiple processes, such as regulating tumor angiogenesis , 12. TheThe study included a case group of 50 NSCLC patients admitted to Affiliated Yancheng Hospital, School of Medicine, Southeast University  from January 2013 to January 2014. Patients underwent pulmonary tumor resection, and NSCLC was confirmed by postoperative histopathology. Patients did not receive radiotherapy or chemotherapy before surgery. The case group included 38 men and 12 women, 45–81 years old with a mean age of 66.9 ± 8.7 years. Of the 50 cases, 29 were squamous cell carcinomas and 21 were adenocarcinomas. Analysis of TNM staging indicated that 7 tumors were stage I, 15 tumors were stage II, 19 tumors were stage III, and 9 tumors were stage IV.P > 0.05). All subjects included in the study provided written informed consent. The study protocol was approved by the Medical Ethics Committee of Affiliated Yancheng Hospital, School of Medicine, Southeast University .The study also included a control group of 60 individuals verified to be healthy volunteers by physical examination in Affiliated Yancheng Hospital, School of Medicine, Southeast University  during the same period. The control group included 47 men and 13 women, 42–78 years old with a mean age of 62.4 ± 7.9 years. Age and gender significantly differed between the two groups  automatic electrochemical luminescent immunoassay analyzer per manufacturer's instructions.μL total volumes, containing 0.15 μL of 100 mmol/L dNTPs; 1 μL of MultiScribe reverse transcriptase; 1.5 μL of 10X reverse transcription buffer; 0.19 μL of RNase inhibitor; 4.16 μL of sterile water; 5 μL of total RNA diluted 1 : 1 with sterile water; and 3 μL of primer (20 μM). The primer sequence, obtained from the Sanger miR sequence database, was 5′-GTGCAGGGTCCGAGGT-3′. Samples were reverse transcribed at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min and held at 4°C in the PCR Thermal Cycler Dice .Real-time fluorescent quantitative PCR (RT-PCR) was used to detect and compare expression of miR21 in serum specimens. RNA was extracted with an RNA extraction kit  at short notice, and RNA was immediately stored at −80°C. To detect miR expression, samples were reverse transcribed using a reverse transcription kit . Reactions were performed in 15 μL reactions, containing 10 μL of TaqMan Universal Master Mix; 4 μL of sterile water; 1 μL of TaqMan MicroRNA Assay reagent; and 5 μL of reverse transcription product diluted 1 : 1 with sterile water. Reactions were amplified on a Roche Cobas z 480 RT-PCR system, using the following conditions: 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. PCR system software was used to produce baseline and threshold values, with miR16 as the internal reference [−ΔΔCt refers to the expression level of miR21.Quantitative detection was performed on reverse-transcribed products in 20 eference –21. Cyclt-test was used for comparisons between case and control groups. Enumeration data were expressed as percentages. Area under receiver operating characteristic (ROC) curve (AUC) was used to compare serum marker diagnostic efficiency. P < 0.05 was considered statistically significant.The SPSS 13.0 statistical package  was utilized to establish a database of all study data. Measurement data were expressed as mean ± standard deviation. Independent sample Serum miR21, CEA, NSE, and CYFRA21-1 levels were, on average, significantly higher in patients with NSCLC than in control individuals .t = 2.575, −2.301, resp.; P < 0.05).Serum miR21, CEA, NSE, and CYFRA21-1 levels did not significantly differ with gender or age of NSCLC patients or with tumor pathology . In addiAUC of using serum miR21, CEA, NSE, and CYFRA21-1 levels to diagnose NSCLC was 0.918, 0.826, 0.853, and 0.866, respectively. CEA had the highest diagnostic efficiency for NSCLC , Table 3χ2 = 4.025) and miR21  levels (P < 0.05)  was 0.752, 0.806, 0.843, and 0.882, respectively. miR21 had the highest diagnostic efficiency for early NSCLC , Table 4 < 0.05) .Comparison of joint detection of serum CYFRA21-1 and miR21 levels with single detection of either marker indicated that AUC of single detection for CYFRA21-1, single detection for miR21, and joint detection of both markers were 0.843, 0.872, and 0.909, respectively , Table 6This study indicates that, on average, serum miR21 and CYFRA21-1 levels were lower in patients with NSCLC at TNM stages I-II than NSCLC at TNM stages III-IV. However, logistic multiple regression analysis indicated that early NSCLC (TNM stages I-II) was associated with serum CYFRA21-1 and miR21 levels. AUC analysis showed that miR21 had the highest diagnostic efficiency for early NSCLC. Analysis of the utility of single or joint detection of CYFRA21-2 and miR21 indicated that miR21 and CYFRA21-1 expression were somewhat related and played an auxiliary role in diagnosis of early NSCLC. In single marker detection, the efficiency of serum miR21 levels to diagnose early NSCLC was higher than other examined markers. Therefore, miR21 could serve as an important serum marker for auxiliary diagnosis of early NSCLC, while joint detection of serum miR21 and CYFRA21-1 levels could improve diagnostic efficiency. EGFR mutation rate and can serve as an indicator to guide targeted therapy [CEA is a polysaccharide protein complex antigen produced during embryonic development. CEA is a broad-spectrum tumor marker because it is expressed in multiple tumors and is closely associated with malignant tumors. The marker is mainly used in clinical practice to assist diagnosis, assess tumor staging and lesion degree, monitor treatment, and predict recurrence of malignant tumors. Recent studies have verified that serum CEA expression in NSCLC patients is related to therapy . Serum C therapy . HoweverConsidering a main driving force of miR transcription, miR21 plays an important regulatory role in cell proliferation, differentiation, and apoptosis and has a close relationship with tumor biological behaviors, such as growth, invasion, and migration . PreviouThe results of this study also indicate that CYFRA21-1 is valuable for diagnosis of early NSCLC. CYFRA21-1 is an epithelial tumor marker with abnormal expression associated with occurrence and development of multiple common malignant epithelial carcinomas, such as pulmonary carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, laryngeal carcinoma, urinary bladder carcinoma, mammary adenocarcinoma, oophoroma, and colorectal cancer . StudiesIn this study, NSE showed no diagnostic value for NSCLC, especially early NSCLC. NSE is a marker present in neurons and neurosecretory cells and can sensitively reflect central nervous system injuries, such as acute cerebrovascular disease, epilepsy, acute brain trauma, and neonate hypoxic-ischemic encephalopathy. As a tumor marker, NSE is mainly associated with specificity of neuroblastoma and small cell lung cancer (SCLC), and its ability to diagnose NSCLC is inferior to other tumor markers . NSE theA limitation of this study is its small sample size. Larger populations should be investigated to confirm these findings and further establish the utility of miR21 expression as a clinical tool. Work that demonstrates the action of miR21 on targets involved in apoptosis (reviewed by ) suggestAmong abnormally expressed tumor markers in NSCLC patients, miR21 has high efficiency in diagnosing early NSCLC and therefore can serve as an important serum marker in auxiliary diagnosis. Joint detection of miR21 with serum CYFRA21-1 can further improve diagnostic efficiency for early NSCLC.


Osteoarthritis (OA) is an degenerative disease characterized by chronic joint pain. Complementary and alternative treatment such as acupuncture have been utilized to alleviate pain. The objective of this study was to investigate the analgesic mechanisms of electroacupuncture (EA) in the collagenase-induced osteoarthritis (CIOA) rat model.Four weeks after inducing CIOA by injecting collagenase solution into the left knee of 5-week-old male Sprague-Dawley rats, 2 Hz and 100 Hz EA on Zusanli (ST 36) was performed. The analgesic effect of EA was evaluated by the tail flick latency (TFL) and paw pressure threshold (PPT) tests. To investigate the analgesic mechanism, serotonergic and muscarinic cholinergic receptor agonists and antagonists were injected 20 min prior to EA and the resultant changes were evaluated by the TFL and PPT tests.EA on Zusanli (ST 36) demonstrated an analgesic effect in the CIOA rat model. The 2 Hz EA treatment showed a significantly greater analgesic effect than the 100 Hz treatment. The analgesic effect of 2 Hz EA was not strengthened by 5-HT1, 5-HT2, 5-HT3, and muscarinic cholinergic receptor agonist pretreatment, was blocked by 5-HT1, 5-HT3, and muscarinic cholinergic receptor antagonist pretreatment, but not blocked by 5-HT2 receptor antagonist pretreatment.In the CIOA rat model, EA on Zusanli (ST 36) exhibited analgesic effects, and 2 Hz EA resulted in a significantly greater analgesic effect than 100 Hz EA. The analgesic effect of 2 Hz EA was reduced by pretreatment of 5-HT1 receptor, 5-HT3 receptor and muscarinic cholinergic receptor antagonists. Osteoarthritis (OA) is one of the most prevalent chronic joint disease at present. It is characterized by loss of articular cartilage, osteophyte formation, subchondral bone change, and synovitis . OA has Acupuncture has been widely used to alleviate many types of pain, particularly chronic pain . Human aThe analgesic effects of EA have also been established through many studies. Researchers have conducted clinical trials and animal model studies on neuropathic pain and collagen-induced arthritis and demonstrated the descending modulation of nociceptive processing –11. The Five-week-old male Sprague-Dawley rats weighing 200 mg were obtained from Samtaco  and housed under controlled temperature (22 ± 1°C), humidity (55 ± 5 %), and 1:1 light-dark cycle (light from 6 AM to 6 PM). All animals had free access to food and water. All experiments were approved and conducted under the guidelines of the International Association for the Study of Pain and the Institutional Animal Care and Use Committee of Kyung Hee University .After one week of adaptation to the laboratory conditions, intra-articular collagenase injection was performed; 0.05 ml of 4 mg/ml collagenase solution  was injected into the left knee of all rats. Four days after the first injection, a booster injection was administered. The gross articular manifestations were assessed and histopathological and serological analyses were performed according to previous CIOA studies , 13. BriAfter four weeks of induction of CIOA and adaptation in the laboratory room conditions, tail flick latency (TFL) and paw pressure threshold (PPT) tests were performed at baseline, 10, 20, 30, 45, 60, and 90 min after initiation of EA.To evaluate the analgesic effect on the thermal stimuli, the TFL test was performed using the tail flick unit  . The ratFor evaluation of the analgesic effect on the mechanical stimuli, a PPT test was performed . Rats weAfter four weeks of induction of CIOA and adaptation to the laboratory condition, EA was performed into Zusanli (ST36), located laterally from tibial tuberosity and caudally below knee joint on the anterior tibialis muscle. The Zusanli acupoint is generally used to alleviate pain in clinical trials and animal studies –13, 17. To investigate the analgesic mechanism, the 5-HT1 receptor agonist 8-OH-DPAT (8 ODT) and antagonist spiroxatrine (SPROX), the 5-HT2 receptor agonist DOI (DOI) and antagonist ketanserin (KTSRN), the 5-HT3 receptor agonist m-chlorophenyl-biguanide (mCLBG) and antagonist ondansetron (ODSTN), and the muscarinic cholinergic receptor agonist neostigmine (NSTM) and antagonist atropine (ATRP) were dissolved in sterile 10 % DMSO (dimethyl sulfoxide) and intraperitoneally injected 20 min before EA.p < 0.05) were determined by Friedman’s rank test followed by Dunnett’s post-hoc test within a group, Mann–Whitney U test between two groups, and Kruskal–Wallis ANOVA followed by Dunnett’s post-hoc test among groups.All results were expressed in mean ± standard error of mean. In nonparametric procedures, statistically significant differences (n = 10). The 2 Hz EA treatment group (n = 10) showed a significantly greater TFL change than the 100 Hz EA treatment group (n = 10) . Between the two EA treatment groups, the 2 Hz EA treatment group (n = 10) showed a significantly higher PPT than the 100 Hz EA treatment group (n = 10)  Fig. . PPT als10) Fig. .Fig. 1Thn = 10) and the EA + DMSO group (n = 10). However, TFL increases induced by ST36 EA were significantly suppressed by SPROX pretreatment (n=10) 10–90 min after initiation of EA  and the EA + DMSO group (n = 10). However, PPT increases induced by ST36 EA were significantly suppressed by SPROX pretreatment (n=10) 10–90 min after initiation of EA , the EA + KTSRN group, and the EA + DMSO group (n = 10) , the EA + KTSRN group, and the EA + DMSO group (n = 10)  Fig. . In the 10) Fig. .Fig. 3Thn = 10) and the EA + DMSO group (n = 10) except 30 min after initiation of EA. However, TFL increases induced by ST36 EA were significantly suppressed by ODSTN pretreatment (n=10) 20–90 min after initiation of EA  and the EA + DMSO group (n = 10) except 30 min after initiation of EA. However, PPT increases induced by ST36 EA were significantly suppressed by ODSTN pretreatment (n=10) 20–90 min after initiation of EA  and the EA + DMSO group (n = 10). However, TFL increases induced by ST36 EA were significantly suppressed by ATRP pretreatment (n = 10) 10–90 min after initiation of EA  and the EA + DMSO group (n=10). However, PPT increases induced by ST36 EA were suppressed by ATRP pretreatment (n=10) 10–90 min after initiation of EA  have been considered as the primary therapy for OA . DespiteEA has been used to treat a diverse range of painful conditions. Previous research has suggested a relationship between pain modulatory mechanisms and acupuncture analgesia, focusing on the role of transmitters and modulators . The EA Various methods, including surgical procedures and intra-articular injections, can be used to induce OA. Intra-articular injection with chemical substances can be more conveniently performed in many studies because surgical procedures are complicated and take a longer period of time to induce degeneration . When seIn order to determine the proper time to conduct experiments with the CIOA model, the gross articular manifestations and histopathological and serological features were evaluated. In accordance with the results of previous studies, most of the osteoarthritic clinical and histopathological features were observed from 4 weeks after first collagenase injection. These features included altered pain-related behaviors, gross articular manifestations, cartilage-destructive features and serological biomarker activities , 13.TFL and PPT tests are commonly used to evaluate the degree of nociception through the change of animal behavior. TFL is focused on the thermal stimuli and PPT is focused on the mechanical stimuli. Pain in OA is associated with thermal and mechanical hyperalgesia and affected by local mechanical and thermal factors . TFL andThe acupoint Zusanli (ST 36) is traditionally used to reduce pain. Previous studies showed that EA on Zusanli (ST 36) led to analgesia using the tail flick method and c-Fos expression in the brain , and itsOur results demonstrated that both 2 Hz and 100 Hz EA showed analgesic effect, evidenced in the TFL and PPT tests. The 2 Hz EA more effectively relieved thermal and mechanical hyperalgesia than the 100 Hz EA, in accordance with the results of previous studies. It has been shown that low-frequency EA is more effective for nociceptive pain whereas high-frequency EA is more effective for neurogenic pain . Recent In order to investigate the analgesic mechanism of EA, we conducted experiments with serotonergic and muscarinic cholinergic receptor agonists and antagonists. With respect to the serotonergic mechanism, our study showed that the analgesic effect of 2 Hz EA was not strengthened by the 5-HT1, 5-HT2, and 5-HT3 receptor agonists. In experiments with antagonists, the analgesic effect of 2 Hz EA was blocked by the 5-HT1 and 5-HT3 receptor antagonists, but not blocked by the 5-HT2 receptor antagonist. These results suggest that the 5-HT1 and 5-HT3 receptors partially mediate the analgesia induced by 2 Hz EA in the CIOA rat model. Ryu et al.  conducteWith respect to the cholinergic mechanism, our study showed that the analgesic effect of 2 Hz EA was not strengthened by the muscarinic cholinergic receptor agonist and blocked by muscarinic cholinergic receptor antagonist. These results suggest that the muscarinic cholinergic receptor partially mediates the analgesia induced by 2 Hz EA in the CIOA rat model. Baek et al.  also conConflicting study results have been reported in the role of the serotonergic agonists and antagonists themselves in pain modulatioin. McCleane et al.  suggesteThe present study demonstrated that 5-HT antagonists ODSN intraperitoneal pretreatment suppressed the ST-36 2Hz EA-induced analgesia but there was no significant TFL and PPT changes when EA was not administered. It appears that 5-HT3 agonist mCLBG intraperitoneal pretreatment increased pain thresholds assessed by TFL and PPT not only in absence of EA treatment and also in ST-36 2Hz EA. Ali Z et al.  demonstrIn summary of the results of our research and previous studies, we speculate that the pain threshold-modulatory effects of serotonergic agonists and antagonits could depend on differences among the agonists, antagonists, time of agonists and antagonits pretreatment, administrative maneuver , and the type of pain models.Considering limited knowledge on the therapeutical active components of EA and the physiological reactions followed by EA, restricted conclusive implication whether 2 Hz EA induced analgesia was mediated by serotonergic and cholinergic receptor mechanisms could be made. Because current study did not perform a comparison with the effect of non-acupoint EA, further studies are needed to investigate the acupoint specific effects of ST36 EA using various EA stimulation parameters including frequency.In summary, these observation suggest that 5-HT1, 5-HT3 and muscarinic cholinergic receptors partially mediate the analgesic effects of EA in CIOA. These results suggests that EA could be a potential option for the relieving osteoarthritic pain.8 ODT, 8-OH-DPAT; ATRP, atropine; CIA, collagen-induced arthritis; CIOA, collagenase-induced osteoarthritis; DMSO, dimethyl sulfoxide; DOI, 5-HT2 receptor agonist DOI; EA, electroacupuncture; KTSRN, ketanserin; mCLBG, m-chlorophenyl-biguanide; NSAIDs, Non-steroidal anti-inflammatory drugs; NSTM, neostigmine; OA, osteoarthritis; ODSTN, ondansetron; PPT, paw pressure threshold; SPROX, spiroxatrine; TFL, tail flick latency


Klebsiella pneumoniae CH1034, a sequence type 6 (ST6) strain isolated in 2012 from a central venous catheter of a hospitalized patient.We report here the genome sequence of  Klebsiella pneumoniae is an important Gram-negative opportunistic pathogen frequently associated with nosocomial infections, including urinary tract infections, pneumonia, infections of surgical wounds, bacteremia, and septicemia  with a K7 capsular serotype  sequence of the serotype , 6 and ide novo assembled using clc_assembler from the CLC Genomics Workbench analysis package with standard parameters. The 46 resulting contigs, ranging from 567 bp to 412,668 bp, were ordered by genome position and orientation using the previously published K. pneumoniae NTUH-K2044 genome as a reference with Mauve Contig Mover (http://www.genoscope.cns.fr/agc/microscope).The genomic DNA from CH1034 was sequenced by next-generation sequencing using an Illumina HiSeq 2000 instrument with 100-bp paired-end reads and 2,000× coverage. After trimming, a total of 109,160,225 reads with a mean read length of 93.42 bp were K. pneumoniae CH1034. Moreover, 83.77% of the CDSs are classified in at least one COG group , of which 8.75%  are classified in the unknown function group. Eleven putative transposase-encoding genes were detected in the genome.The assembled genome size of isolate CH1034 was 5,271,927 bp in length, with a G+C composition of 57.4%. Genome annotation resulted in 5,248 genomic objects, including 5,114 predicted coding sequences (CDSs), 15 pseudogenes, 5 rRNAs , 70 tRNAs, and 44 miscellaneous RNAs (miscRNAs). The annotation results showed that 1,245 CDSs were annotated with at least 1 EC number, corresponding to 138 KEGG maps, with at least one reaction catalyzed in CH1034_250036 to CH1034_250050) was found in only 3 other bacterial genomes: K. pneumoniae subsp. pneumoniae PittNDM01, Enterobacter hormaechei isolate 05545, and Escherichia coli ECOR31. Among the 15 CDSs that were annotated, 5 encoded potentially proteins with enzymatic functions , and 10 encoded potentially proteins with unknown function.A comparison with the genome sequences available in the MicroB  and nr databases showed that a region of 16.4 kb (CDSs PRJEB9899 and contig accession numbers CXPD01000001 to CXPD01000046.This whole-genome shotgun project has been deposited at ENA/EMBL-EBI under the accession no.


We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes  whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se Escherichia coli genome, there are three formate dehydrogenases FdhN, FdhO, and FdhH with each consisting of one selenopolypeptide requiring SeCys [0) [Selenium (Se) is a nonmetal element with atomic number 34, which is chemically related to sulfur (S) and tellurium but rarely found in its elemental form in nature. Se is an essential micronutrient for mammals and some bacteria and a component of selenocysteine (SeCys), an amino acid used by a group of proteins , 2. In Eng SeCys  for the ng SeCys , 8. Highng SeCys . PrevioueCys [0) –13. In neCys [0) . Hence,  E. coli have the capacity to convert selenite to Se0 [ E. coli strains can grow in the presence of 9.2 mM selenite and efficiently metabolize selenite into Se0 [ cysA, cysU, and cysW [0 [Several bacterial species includinge to Se0 –27. E. cinto Se0 . Howeverinto Se0 , 28 althinto Se0 , 15, 26.and cysW . Selenitand cysW . After eand cysW , followe cysW [0 . Further E. coli is limited to four genes that have been identified to respond to sodium selenite (Na2SeO3) treatment. Three of them are oxidative stress stimulons sodA, gor, and trxB, encoding the manganese superoxide dismutase, glutathione reductase, and thioredoxin reductase, respectively, which are upregulated ≥5-fold by 2 mM Na2SeO3 treatment [ gutS  that encodes the GutS polypeptide, a homolog of membrane transport proteins, which can be induced by 0.03–0.06 mM Na2SeO3 and 0.002 mM sodium tellurite [ Caulobacter crescentus cells treated with 0.3 mM Na2SeO3; only 12 genes were found to be upregulated about 2- to 5-fold [2SeO3 in C. crescentus [Currently, the knowledge concerning the molecular mechanism involved in selenite reduction inreatment . The fouellurite . Microaro 5-fold . Howevero 5-fold , suggesto 5-fold  and membo 5-fold  were fouescentus . Therefo E. coli, there are only three proteins FdhN, FdhO, and FdhH that each contains one polypeptide requiring SeCys residue for their activities [ fdnG encodes a 110 kD selenopolypeptide, α subunit of FdhN. The fdoG encodes another 110 kD selenopolypeptide for FdhO while the fdhF encodes a 80 kD selenopolypeptide of FdhH. In addition, there is a group of SeCys biosynthesis and incorporation machinery genes for the biosynthesis of SeCys and selenopolypeptides such as selA (selenocysteine synthase), selB (selenocysteinyl-tRNA-specific translation factor), selC (tRNA specific for selenocysteine), selD (selenophosphate synthase) [ ybbB (selenophosphate-dependent tRNA 2-selenouridine synthase) [ sufS (PLP-dependent selenocysteine lyase) [2SeO3 concentrations that are 30% stimulatory and 50% inhibitory on bacterial cell growth were determined and used to study their expressions by quantitative real-time PCR (qRT-PCR). In addition, microarray analysis was also used to screen selenite-responsive genes besides above selected genes.Intivities –6. The gynthase) , ybbB (synthase) , and sufe lyase) . We quesE. coli strain K12 was used in this study. Bacterial cells were grown in Luria-Bertani (LB) medium in 14-mL polystyrene round-bottom tubes. A final volume of 3 mL was used for cultures and incubated at 37°C in a rotary shaker at 225 rpm for different periods of time depending on the experiment.2SeO3 inhibited and stimulated bacterial growth, an overnight culture of K12 adjusted to the optical density at 600 nm (OD600) of 1 was used as bacterial stock solution for testing. Na2SeO3 (Sigma-Aldrich) was dissolved in ddH2O to prepare 1 M selenite stock solution. Initially, each 20 μL of bacterial stock solution was inoculated into 3 mL LB medium containing Na2SeO3 with a final concentration of 0, 25, 50, 75, 100, or 125 mM. The experiment was performed on three biological replicates in triplicate for each concentration tested. After 14 h of growth, the extent of selenite to Se0 reduction as indicated by the degree of red color in each culture was determined qualitatively and photographs were compared. Since reduced red Se0 can interfere with optical density measurements, bacterial growth was determined by counting viable cell numbers. Cells were diluted using LB medium and then 20 μL of diluted culture was spread onto a LB agar and incubated at 37°C overnight. The serial dilutions were done in triplicate for each culture. Thus, the colony-forming units (CFUs) were calculated from nine plates for each treatment.To determine which concentrations of Na2SeO3 were used to determine the concentration that would inhibit the bacterial growth. Another experiment using lower concentrations  was performed to determine which concentration stimulated bacterial growth. Both experiments were performed on three biological replicates in triplicate for each concentration tested.After the initial test, 0, 5, 10, 15, 20, 25, and 30 mM Na2SeO3 to determine when cells should be harvested for RNA extraction at which significant difference in bacterial growth between treated and untreated control could be observed. The same culture conditions were used as previously mentioned; treated and untreated cultures were incubated at 37°C for 0, 2, 4, 6, 8, 10, 12, or 14 h. Colony forming units were recorded as previously mentioned for each time point. Three biological replicates done in triplicate were done for each culture.After screening the inhibitory selenite concentration, cultures were treated with selected 5 mM Na600 of 0.2, 0.4, and 0.8; then, these were served as initial inoculant concentrations for culturing for 0.5, 1, 1.5, 2, 2.5, and 3 h. The cell density of each culture was recorded by measuring OD600 value. Three biological replicates with triplicate assay for each time point were done for the entire experiment.To study the effect of culture duration on induced selenite-responsive genes, two previously reported growth times, 0.5 h , 32 and Total RNA was isolated using the RiboPure-Bacteria RNA Isolation Kit  and treated with DNase I  to remove any DNA contamination. RNA concentration was quantified using a NanoDrop ND 1000 spectrophotometer. The quality of the RNA was visualized on a 1.2% agarose gel. For microarray analysis, RNA quality was further checked using a dual beam spectrophotometer and an Agilent Bioanalyzer 2100 Lab-on-a-Chip system.fdnG, fdoG, and fdhF) and five machinery genes  for SeCys biosynthesis and insertion were selected to quantify their expression levels in response to selenite treatments. The selC was not selected because its sequence (95 bp) is too short to meet the requirement of primer design software Primer Express 3.0  for designing a pair of primers. Previously reported four selenite-induced genes  [sbp, sulfate transporter subunit, thiP, thiamine transporter membrane protein, and iscS, cysteine desulfurase) [2SeO3 were used for RNA isolation. Equal amounts of RNA for each sample were used to synthesize first-strand of cDNA with a High Capacity cDNA Reverse Transcription kit and random primers . PCR was carried out with the Power SYBR Green mix . Primer design and ΔΔCt calculation were carried out as described previously [http://www.ncbi.nlm.nih.gov/) for designing primers. The 16S rRNA was used as an endogenous control. 16S rRNA primers were designed based on 719 bp conserved region of seven members  also retrieved from strain K12 genome. Each sample was assayed in triplicate and the experiment was repeated with three biological replicates. Detailed primer information for each gene is listed in Three selenopolypeptide genes (nd gutS) , 31 and lfurase) –38 were eviously . Sequenc2SeO3 for 6 h were used for microarray analysis for screening additional selenite-responsive genes which were not in the previous qRT-PCR analysis. Three replicates for treated and untreated samples were used. The microarray chip used in this experiment was the Affymetrix GeneChip E. coli genome 2.0 arrays  containing 10,208 probe sets for detecting the entire 20,366 genes in the K12 strain and three other E. coli strains. The RNA quality control analysis, standardization, cRNA labeling, and array hybridization were processed at Genome Explorations Inc. . Raw signals (CEL files) were normalized and transformed into log2 values by MAS 5 . For statistical analysis, the significance analysis tool set in gene traffic was employed to perform multiclass ANOVA. Pairwise comparisons were made between untreated control and 0.01 or 5 mM treatment. All probe sets having comparisons reaching absolute fold change ≥1.5 and t-test P values ≤0.05 were selected for further qRT-PCR confirmation. Aforementioned qRT-PCR conditions and endogenous control were used. Sequences of related genes were retrieved from strain K12 genome. Detailed primer information for each gene is listed in Bacterial cells untreated and treated with 0, 0.01, or 5 mM Na2SeO3 treatments, Student's t-test was used.For the comparison of cell numbers between untreated control and Na2SeO3 inhibitory effects on bacterial growth, E. coli K12 was cultivated in the presence of high concentration of Na2SeO3 at 0, 25, 50, 75, 100, or 125 mM. Results showed that all these selenite treatments caused more than 50% inhibition of E. coli growth  was used to determine a concentration of Na2SeO3 that would inhibit bacterial growth by 50%. We found that 5 mM Na2SeO3 treatment reduced cell numbers by 53% and turned cultures red  is inoculated into 3 mL LB medium. To determine which initial culture concentration would allow the culture to reach log phase after culturing for 0.5 to 3 h, a pretest culture experiment was performed with initial OD600 of 0.2, 0.4, and 0.8. The results showed that OD600 values increased from initial 0.2, 0.4, and 0.8 to 0.4, 0.6, and 0.9 after culturing for 0.5 h and to 0.9, 1.1, and 1.2 after culturing for 2 h, respectively  were induced 2.6- and 3.8-fold in 5 mM treatment for 2 and 6 h but not in the other treatments (gor and trxB) did not meet the 2-fold cutoff under either 0.01 or 5 mM treatment, which is not in agreement with previous report by Bébien et al. [QRT-PCR results showed that two selenopolypeptide genesat 0.5 h . The thieatments . These r and 6 h . The remeatments . The expn et al. . yegB (probe set ID 1764732) were induced 2.2- and 1.8-fold by 0.01 and 5 mM treatments, respectively , selenite is readily incorporated into SeCys for the synthesis of selenoproteins, such as formate dehydrogenases [ via the mechanism of oxidative stress [Previous studies have shown thatogenases . At modeogenases , 42. At e stress . In the e stress , 41, 42  fdnG, fdoG, and fdhF encoding selenopolypeptides for FdhN, FdhO, and FdhH were induced more than 2-fold by both 0.01 and 5 mM Na2SeO3 ( E. coli has only these three proteins with each containing one selenopolypeptide requiring SeCys for its activity [ fdnG, fdoG, and fdhF are only induced by 5 mM Na2SeO3 treatment. Since they were induced more than 2-fold by 0.01 mM Na2SeO3 as well, whether these proteins containing SeCys have an additional function other than antioxidation needs to be further investigated. In the presence of 0.01 mM Na2SeO3, a small part of selenite may be used to synthesize selenopolypeptides while large part may be metabolized with the S as suggested by previous studies [Using qRT-PCR analysis, we found that all three selenopolypeptide genes Na2SeO3 , which hactivity . A possiactivity . FdhN anactivity , 45. The studies , 42. Nev iscS and two previously reported selenite-induced genes sodA and gutS were induced only by 5 mM (2SeO3 treatment, bacterial cells were already under toxic condition with more than 50% growth inhibition even though the selenite reduction was still active as judged from the accumulation of Se0 (Figures  sodA encoding the antioxidant enzyme [ gor and trxB, encoding antioxidant proteins glutathione reductase and thioredoxin reductase, respectively [ gutS by selenite was similar to the previous report [ gutS gene product may allow Se to permeate into cells because it shares homology with membrane transport proteins. Induction of iscS under the selenite toxic conditions ( iscS is also required for biosynthesis of 2-selenouridine in tRNA and FdhH [Moreover, we also found that one sulfur metabolic gene by 5 mM , which i Figures  and 2. It enzyme  is inducectively , could ns report . The gutnditions  may be dand FdhH .2SeO3 concentration. Our results of microarray experiment are similar to those reported by Hu and coworkers [In the present study, we also made an attempt to identify selenite-responsive genes genome-wide by microarray screening. Surprisingly, this screening did not yield any selenite-responsive genes in cells treated with 50% inhibitory or 30% stimulatory Naoworkers , in whicoworkers , Hu and oworkers  could stoworkers  from sel2SeO3 and determined that 0.001 to 0.01 mM promoted bacterial growth approximately by 30%, whereas 5 mM treatment inhibited cell growth by more than 50%. Although microarray analysis was not sensitive enough to identify these selenite-responsive genes in bacteria, we were able to determine that genes encoding selenopolypeptides and some antioxidant proteins were involved in selenite reduction. Our findings will help to further elucidate the mechanism responsible for selenite reduction.We have comprehensively studied the bacterial growth under various concentrations of Na


We aime3-) for circuit priming solution. Arterial blood gases and laboratory parameters were collected at 0, 5, 30, 60, 90, and 120 minutes after pump start. SID, base excess (BE) and total weak acids (Atot) were calculated.We randomly assigned patients with acute respiratory failure in need of ECMO to receive either NaCl 0.9% , Ringer lactate , or a novel balanced solution . ECMO was initiated for ARDS (45%), bridge to lung transplant (25%), acute graft failure after transplant (15%), and acute on chronic respiratory failure (15%). Average priming volume was 10 ± 5 ml/kg; patients' baseline HCO3 was 28 ± 6 mEq/l. During the first 2 hours after ECMO initiation, arterial pH raised similarly in all groups (P = 0.39) due to CO2 removal. In contrast, BE decreased starting after 5 minutes in both the NS and RL groups . No BE changes were observed in the Solution × group (0.3 ± 0.8 mEq/l). In the NS group, BE reduction was associated with a reduction in SID , entirely due to an increase in Cl . In the RL group, BE and SID reductions  were associated with an increase in both Cl  and lactate . No changes were observed in other electrolyte concentrations. Dilution did not differ between groups (P = 0.25 for Atot variation). The acidifying effect of NS and RL was amplified in patients with higher baseline HCO3-.We enrolled 20 patients (23 priming procedures - RL, 3- level for ECMO priming uniquely avoids the addition of metabolic acidosis to patients with uncompensated hypercapnia.As compared with NS and RL, the use of a novel balanced solution with a SID equal to the patient HCO


Vibrio alginolyticus 63. Remarkably, Amy63 possesses amylase, agarase and carrageenase activities. Amy63 is a substrate promiscuous α-amylase, with the substrate priority order of starch, carrageenan and agar. Amy63 maintains considerable amylase, carrageenase and agarase activities and stabilities at wide temperature and pH ranges, and optimum activities are detected at temperature of 60 °C and pH of 6.0, respectively. Moreover, the heteroexpression of Amy63 dramatically enhances the ability of E. coli to degrade starch, carrageenan and agar. Motif searching shows three continuous glycosyl hydrolase 70 (GH70) family homologs existed in Amy63 encoding sequence. Combining serial deletions and phylogenetic analysis of Amy63, the GH70 homologs are proposed as the determinants of enzyme promiscuity. Notably, such enzymes exist in all kingdoms of life, thus providing an expanded perspective on studies of multifunctional enzymes. To our knowledge, this is the first report of an amylase having additional agarase and carrageenase activities.A multifunctional enzyme is one that performs multiple physiological functions, thus benefiting the organism. Characterization of multifunctional enzymes is important for researchers to understand how organisms adapt to different environmental challenges. In the present study, we report the discovery of a novel multifunctional enzyme Amy63 produced by marine bacterium  Multifunctional enzymes (MFEs) are enzymes that are considered to perform multiple independent functions, and are often also moonlighting or promiscuous enzymes1234Since the first multifunctional enzyme, crystallins, was reported by Piatigorsky and Wistowα-amylase represents the most intensively studied amylolytic enzyme, which degrades starch substrates and is applied widely in various branches of the food, pharmaceutical, and chemical industries10115Vibrio alginolyticus 63, which possesses agarase and carrageenase activities in addition to its main amylase activity towards soluble starch, amylose, amylopectin and glycogen. The heteroexpression of Amy63 dramatically enhances the degradation abilities of E. coli towards starch, carrageenan and agar. Serial deletions together with sequence analysis of Amy63, lead us to propose that the GH70 homologs determine multifunctionality. Phylogenetic analysis of Amy63 indicates that this kind of multifunctional amylases is widely spread in the all kingdoms of life, which profoundly broaden our knowledge about the α-amylase.In this study, we report a novel multifunctional α-amylase Amy63, isolated from Vibrio alginolyticus by the 16S rRNA gene sequencing (Accession no. KT224384), the Amy63-producing strain was designated as Vibrio alginolyticus 63.The initial purpose of this study was to identify robust marine bacterial agarases. By screening crude extracts, we discovered that the agarase produced by marine bacterium strain 63 exhibited the highest activity and stability in various temperatures and pHs, even in presence of surfactants or chelating agents. Therefore, we purified the native agarase, hereafter named Amy63, from the producing strain and confirmed it was functional by zymogram analysis  and a plVibrio alginolyticus (Accession no. gi|491538798). To check whether Amy63 had amylase activity, zymogram analysis and plate-based activity assays were performed. Remarkably, the results showed that purified Amy63 had strong amylase activity which effectively degraded starch in a PAGE gel  is an ideal system for testing this because it provides a background which does not express any agarases, amylases or carrageenases. To heterologously express Amy63 in E. coli BL21(DE3), we first cloned the coding sequence of Amy63 (Accession no. KT224383) based on the mass spectrometry results  typical for α-amylase were identified in Amy63  and a Weet al.kcat/KM values of the promiscuous sugar kinase YajF is approximately 102 M−1s−1 and thus ~104 lower than that of the primary E. coli glucokinase (Glk)To measure the activities of Amy63 on its substrates, the changes in the average molecular weights of starch, agar and carrageenan in a hydrolysis reaction mixture were measured using gel permeation chromatography (GPC) as described by Suzuki Marinobacter sp. EMB8Because the amylase activity is the primary function of Amy63, we next sought to identify the substrate specificity of amylase activity of Amy63. We tested the enzyme against different glucose polymers and found that Amy63 has wide substrate specificity, with soluble starch being the best substrate . Amy63 eBecause temperature and pH are decisive parameters for enzyme activity18+ and K+  system because it has no any amylase, agarase and carrageenase activities. In view of Amy63’s multifunctionality, we hypothesized that the expression of Amy63 could enhance the degradation abilities of E. coli cells towards starch, agar or carrageenan, thus increasing the carbon source. If this were the case, then we would expect the host cells to grow faster in the basic medium supplemented with starch, agar or carrageenan as the sole-carbon source upon expression of Amy63. As expected, the growth rates of E. coli cells in the different mediums were very similar before the expression of Amy63; however, the growth rates changed dramatically once Amy63 was expressed , and three conserved GH70 homologs and a DUF1939 domain were identified in the polypeptide chain of Amy63 , which has a very similar GH70 homologs distribution pattern with Amy63  containing α-amylases ubiquitously exist in all kingdoms of life  Both native and recombinant Amy63 have strong hydrolytic activities on agar and carrageenan in addition to starch by zymogram anyalyses and plate-based activity assays; (ii) Amy63 shares seven typical conserved sequence regions (CSRs) of α-amylase and phylogenetically clusters with the typical GH13 family α-amylases; (iii) Gel permeation chromatography results further confirm Amy63 could degrade starch, agar and carrageenan. Moreover, gel permeation chromatography results indicate that starch is the best substrate of Amy63, and the degradation capabilities of agar and carrageenan by Amy63 are weaker than that of starch. Therefore, we conclude that Amy63 is a substrate promiscuous enzyme. The priority order for substrates of Amy63 is starch, carrageenan and agar. To our knowledge, there are no other enzymes with such activities reported thus far.By characterizing Amy63, we found that the optimal pH and temperature of Amy63 is around 6.0 and 60 °C, respectively. Moreover, the enzymatic activity is stable within the pH range from 5.0 to 11.0 and the temperature range between 0 and 70 °C. In addition to starch, hydrolysis product analysis showed that carrageenan and agar could be degraded into glycans with a lower degree of polymerization by Amy63. This suggests Amy63 could have potential applications in many industrial processes such as starch or other complex substrate saccharification containing agar or carrageenan in sugar and textile industriesThe effects of metal ions and other reagents on activity of Amy63 were also checked. It is noteworthy that DTT dramatically enhanced Amy63’s activity, especially for its agarase and carrageenase activities, which is similar to ScAmy43http://www.cazy.org) family enzymes displayed a variety of catalytic activities, including dextransucrase (EC 2.4.1.5), alternansucrase (EC 2.4.1.140), reuteransucrase (EC 2.4.1.-), α-4,6-glucanotransferase (EC 2.4.1.-) and α-1,2-branched dextransucrase (EC 2.4.1.-). The enzymes from glycoside hydrolase families GH13, GH70, and GH77 together form clan GH-H. GH13 family amylosucrase enzymes have the GH13 type of domain architecture and the GH70 type of glucansucrase activityThe gene sharing model, where a gene with a given function is recruited for a different, moonlighting function without any changes in the coding regionMFEs are able to employ alternative approaches to coordinate multiple activities and regulate their own expression which could benefit the living systems by providing competitive survival capabilitiesMultifunctional α-amylases, like Amy63, may generally exist in the biological world, since GH70 homolog containing α-amylases are ubiquitous in all kingdoms of life. For Amy63, it will be important to investigate the detailed mechanisms of catalytic promiscuity and the evolvability of promiscuous functions in the future.2 and 10% KI in distilled water). Colonies with a clear zone formed by the hydrolysis of agar were evaluated as agarase producers. Marine bacterium strain 63 was isolated from algae of South China sea, inoculated in marine broth 2216E, and grown at 28 °C. Genomic DNA was extracted from the isolate, and PCR (polymerase chain reaction)http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the phylogenetic position of the bacterium strain 63.Marine bacterial strains were isolated from South China Sea and screened for their abilities to hydrolyze agar by streaking strain in the 2216E agar plate containing 5 g/l tryptone, 1 g/l yeast extract, and 20 g/l agar, one liter filtered seawater, pH adjusted to 7.4–7.6. The plate was incubated at 28 °C for 48 h and stained with Lugol’s iodine solution  pre-equilibrated with buffer A. Bound proteins were eluted with 150 ml linear gradient of 0.1–1.0 M of NaCl in 10 mM Tris-HCl (pH 8.0) at a flow rate of 5 ml/min. Active fractions were collected and concentrated by ultrafiltration , and loaded onto a HiloadTM 16/600 superdexTM 200 column  pre-equilibrated with 10 mM Tris-HCl (pH 8.0) containing 0.15 M NaCl. Bound proteins were eluted with the equivalent buffer at a flow rate of 1 ml/min (2 ml per tube). Active fractions were collected for further study. All purifications were carried out at 4 °C using an AKTA purifier system .Amy63 was purified as described previously32For plate-based activity assays of Amy63, the same amount of purified Amy63 was put in the center hole of plates containing 2% agar, 5% carrageenan, or 1% soluble starch with 1.5% agar plus 0.01% Trypan Blue in 20 mM Tris–HCl buffer (pH 8.0), respectively. The plates were kept in 50 °C for 2 h. Plates containing agar and carrageenan for checking agarase and carrageenase activities were stained with Lugol’s iodine solution and then visualized the clear zone around the hole. Plates containing soluble starch and Trypan Blue were checked directly for the amylase activity by visualization of clear zone around the hole.For zymogram analyses of Amy63, the same amount of purified Amy63 was performed with SDS-PAGE on a 10% gel as described by Laemmliin situ with trypsin using established methodsThe procedure of MALDI-TOF⁄TOF mass spectrometry was carried out as described previouslyVibrio alginolyticus (Accession no. gi|491538798). Therefore, a pair of degenerate primers P2F and P2R based on the gi491538798 sequence were designed to amplify the full length Amy63 encoding sequence. The amplified DNA was ligated to the pGEM-T vector  for sequencing.Based on the first peptide RVDWNNR and last peptide QHSYLDHWDVIGWTR identified from Mass spectrometry, a pair of degenerate primers P1F and P1R were designed to amplify the corresponding DNA. The amplified PCR products were sequenced and blasted the homology in NCBI. The sequence alignments result showed that it had high homology (99%) with cytoplasmic α-amylase of For sequence analysis of Amy63, sequences of ten typical α-amylases from GH13 subfamilies 1, 5, 6, 7, 15, 24, 27, 28, 36, 37 together with Amy63 were aligned using the program Clustal-W2amy63 DNA fragment with artificial EcoRI and HindIII sites was amplified by PCR using primers 63F and 63R. The PCR products were gel-purified and cloned into pET-30a(+) vector. The positive recombinant was confirmed by sequencing and transformed into E. coli BL21 (DE3) cells, and induced with isopropyl-β-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mM at 16 °C for 16 h. The recombinant Amy63 was purified as described beforeTM HP column , HiTrapTM Q HP column  and HiloadTM 16/600 superdexTM 200 column . Further detailed purification was taken as that of native Amy63.The full-length 3 at a flow rate of 0.6 ml/min. The columns in the GPC apparatus were thermostatted at 40 °C.Amy63 (1 mg/ml) was incubated separately with 0.25% agar, 0.25% carrageenan or 1% soluble starch in 20 mM Tris buffer (pH 8.0) at 37 °C for 24 h and the product mixtures were measured by gel permeation chromatography (GPC). G-5000, G-3000 and KS-802 gel exclusion columns were used and eluted with 0.2 M NaNO540) was then recordedTo determine the substrate specificity of amylase, the enzyme activity was assessed against 1% (w/v) of soluble starch, dextrin, amylose, amylopectin, α-cyclodextrin, β-cyclodextrin, glycogen and pullulan in acetic acid buffer (pH 6.0) at 50 °C using 3,5-dinitrosalicylic acid (DNS) methodFor maltohexaose degradation assay, Amy63 (1 mg/ml) was incubated with 25 mM maltohexaose in 20 mM Tris buffer (pH 8.0) at 37 °C for 12 and 24 h, respectively, and the saccharide product mixtures were analyzed by hydrophilic interaction chromatography.et al.The effect of pH on Amy63 amylase, agarase and carrageenase activities were performed at 50 °C in 0.2 M Glycine-HCl , sodium acetate buffer , Tris-HCl buffer , Glycine-NaOH buffer  for 30 min with the method described by Suzuki To evaluate the optimal temperature of Amy63, the DNS assay which uses soluble starch, agar or carrageenan as substrates, respectively, was conducted at various temperatures ranging from 0 °C to 100 °C in acetic acid buffer (pH 6.0). The thermostability of Amy63 was determined by measuring the residual activity after pre-incubating them for 1 h at various temperatures ranging from 0 °C to 100 °C and the relative activity was defined as the percentage of activity against the highest activity2, CaCl2, MgCl2, MnCl2, FeSO4), surfactant (SDS) and chelating agent (EDTA) on Amy63 amylase, agarase and carrageenase activities were evaluated by measuring the residual enzyme activity with DNS method described above. And the relative activity in the absence of any additives was taken as 100% as described by Mohammad et al.Effects of different concentrations  of metal ions  cells containing pET30a or pET30a/Amy63 (overexpress Amy63) were cultured in LB medium supplemented with 50 mg/l kanamycin until OD600 reached 0.8. Thereafter, the same amount of E. coli BL21 (DE3) cells containing pET30a or pET30a/Amy63 were taken out from the flasks and centrifuged in 6,000 rpm for 3 min, and the pellets were washed with basic medium  three times. After the washes, the pellets were resuspended with the same amount of basic medium, and 1 ml of resuspension cells containing pET30a or pET30a/Amy63 was added into 50 ml basic medium supplemented with 50 mg/l kanamycin and 0.5% starch, 0.1% agar or 0.1% carrageenan, respectively. The cells were inoculated in 37 °C at 150 rpm. When the OD600 reached around 0.8, the cells were induced with IPTG at a final concentration of 0.2 mM and cultured at 25 °C with shaking for another 20 h. The OD600 values were checked every several hours after the IPTG was added. Each treatment was done with three independent replicates.In order to analyze the physiological function of Amy63, the http://www.genome.jp/tools/motif/). The UniProt accession numbers of α-amylases from different kingdoms of life used for GH70-1, GH70-2 and GH70-3 Weblogo analyses are as following: AGV20183.1, ACY52744.1, AEM05860.1, AIA26430.1, ACQ73554.1, AJH89352.1, AJH21062.1, AAB86961.1, BAA32431.1, AAW44866.1, YP_007313109.1, BAP33443.1, AIN23306.1, AGJ85226.1, AHB43819.1, AKF38621.1, AJJ12514.1, BAB71820.1, AKG37901.1, AKG73076.1, AEP00550.1.The motifs of Amy63 were analyzed by using the MOTIF Search program (E. coli BL21 (DE3) cells, those four mutants were purified using the procedures described above and used for further plate-based activity assays and zymogram analyses.Four truncates of Amy63 which lack the first GH70 homolog, the first two GH70 homologs, the third GH70 homolog or the DUF1939 motifs, respectively, were constructed using primers F1, F2, R2, R3, R4. After over-expression in http://www.genome.jp/tools/motif/).The program MEGA version 6.0 was used to construct phylogenetic treesHow to cite this article: Liu, G. et al. Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities. Sci. Rep.6, 18726; doi: 10.1038/srep18726 (2016).


The repetitive unit was likely composed of 3 As, 3 Cs, 13 Bs and 1 D. Together with the average molecular weight, it was predictable that HB-1 consisted of about seven of the repetitive unit. The inhibition activity of HB-1 on human glioma cell line SF188 was also measured, only to find it inactive.A water soluble polysaccharide, HB-1, with a molecular weight of 23,930, was isolated from radix Ranunculi ternati. by hot water extraction, ethanol precipitation, deproteination，ultrafiltration and gel-filtration column chromatography. Its sugar composition was determined by GLC as Glc, Ara, and Gal in a molar ration of 16.071: 2.722: 1. And the absolute configuration of Glc was identified as D. Smith degradation and methylation reaction showed the proportion of — Many of these bioactive polysaccharides are relatively non-toxic with no significant side effects  was isolated and structurally elucidated. The isolation, purification, identification and its biological activities are reported in this paper.During our investigation on the extracts from GeneralXL (7.8×30 cm) column, and HT-230A column oven made by Tianmeida Scientific Instrument Factory in Shenyang, China. Microplate reader was μQuant and was produced by Bio-tek Instruments Inc. ZDUF ultrafiltration machine was made by Zhongda Membrane Separation Instrument Factory in Hangzhou, China. L-cysteine methyl ester hydrochloride, N-Trimethysiylimidzole (TMSI), D-Glc and L-Glc were purchased from Sigma-aldrich company . All other chemicals and reagents were analytical grade.GCMS spectra were taken on a Shimadzu GCMS-QP5050A spectrometer. GC analysis was carried out with GC9790 produced by Fuli company of China on columns of SE-30 (30 m × 0.32 mm × 0.5 μm) and OV-17 (30 m × 0.32 mm × 0.5 μm). HPLC was carried out using successive UV  and evaporative light scattering (ELSD) (softa corporation model 200es ELSD) detector, series Ⅲ pump controlled by Ezchrom Elite Client/server(version 3.1.6), WYK-16b4 air compressor made by Dayin Instrument Factory in Tianjin, China, TOSOH TSK gel G4000PWPlant materialRadix Ranunculi ternati was purchased from Bozhou county, Anhui province, China in 2004, and was identified by Qishi Sun, professor of Shenyang Pharmaceutical School, China. Voucher specimens were deposited at the Faculty of Pharmacy, Medical College, Xiamen University, Xiamen, China.Extraction and fractionationRanunculus ternatus Thunb (2 Kg) was extracted with hot water for three times. The extraction was put together and concentrated in vacuum to 1000 mL, then precipitated with 70% ethanol. The precipitation was deproteinated by Sevage method  monitored by ELSD, eluting with water at 0.5 mL/min, using purified dextran fractions with definite molecular weights as standards (The average molecular weight of HB-1 was determined by high - performance liquid chromatography (HPLC) on a column of TSK gel G4000PWtandards . Sugar analysis2, one part of the hydrolysate was analyzed by PC. The other part (2 mg) was derivatized with 0.4 mL hexamethyldisilazane (HMDS) and 0.2 mL trimethylchlorosilane (TMCS). The supernatant was subjected to GLC, which was performed with a SE-30 column (100 m*0.25 mm*0.25 μm) and an H2 flame ionization detector at a column temperature of 200 ºC, vaporizer temperature of 280 ºC, and detector temperature of 280 ºC (2) was 2 mL/min.HB-1 was hydrolyzed with 2 mol/L trifluoroacetic acid (TFA) at 110 ºC in a sealed tube for 2 h (Absolute configuration determination2. Pyridine solution (1 mL) of the sugar (2 mg) was mixed with L-cysteine methyl ester hydrochloride (2 mg), and kept at 60 ºC for 2 h. After drying the mixtures with N2, N-Trimethysiylimidzole  was added, and the warming at 60 ºC was continued for another hour. The reaction was ended by adding water (1 mL) into the container, and then the solution was extracted with cyclohexane (3 × 1 mL). Finally, the cyclohexane layer was collected and concentrated to 1 mL for GLC analysis, which was performed with an OV-17 column and an H2 flame ionization detector at a column temperature of 220 ºC, vaporizer temperature of 280 ºC , and detector temperature of 280 ºC  with column temperature program: 50 °C (5 min) to 235 °C (5 min.) at 10 °C/min , vaporizer temperature: 280 °C, and detector temperature:280 °C (HB-1(25.4 mg) was oxidized by 25 mL 15 mM sodium metaperiodate at 4 ºC in the dark. The production of formic acid and consumption of periodate were periodically determined by titration with 10 mM sodium hydroxide and colorimetry. After completion of the oxidation, the reaction was terminated by addition of ethylene glycol. Then KBHe:280 °C .Methylation2O5) in methyl sulfoxide (3 mL) was added powdered NaOH  and methyl iodide . The mixture was stirred away from light at room tempreture for 1 h. The methylated polysaccharide was extracted with chloroform–water (1:1) for three times and the organic phase was dried with N2 stream. The methylated polysaccharide was hydrolyzed with 90% formic acid and 2 M TFA (1 mL), successively, for 6 h at 100 °C. Then the solution was dried with an N2 stream. The products were dissolved in water (1 mL) and reduced with 30 mg KBH4 (2 h at rt). Excess KBH4 was destroyed by drop wise addition of acetic acid (HAc) in ice. Then the mixture was treated with 3 mL of a solution of acetic acid: methanol (1:9) and dried with an N2 stream. This procedure was repeated four times. Successively, the mixture was repeatedly treated three times with methanol. The resulting alditols were acetylated with acetic anhydride (1 mL)–pyridine (1 mL) at 100 °C for 1 h, and then cooled and dried with an N2 stream. The alditol acetates were partitioned between chloroform and water (three times). The chloroform fractions were analyzed by GC–MS on a Shimadzu QP2010 GC–MS, equipped with a DB1capillary column  using a temperature programming of 150 °C (5 min) to 280 °C (10 min) at 10 °C/min. The carrier gas was Helium at a flow rate 1 mL/min  -2, 5-diphenyltetrazolium bromide (MTT) assay. Briefly, exponentially growing cells were plated into 96-well plates . After 24 h, serial dilutions of the drugs were added to the cells, which were incubated for 24 h. Then the cells were incubated with 10 μL of MTT (5 mg/mL) at 37 °C for 5 h. One hundred microliters of DMSO was added to solubilize the formazan crystals formed, and the optical densities at 570 nm were measured using a microplate reader.The human glioma cell line SF188 was grown as a monolayer in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were maintained in a humidified atmosphere of 5% CORanunculi ternate. The crude polysaccharides HA and HB were prepared from this herb by hot water extraction, EtOH precipitation, deproteinization, and ultrafiltration. HB-1 was obtained from HB by DEAE Sephadex A-50  was about 16%, —1Glc4— (B) about 62%, (C) about 14%, and —1Gal6— (D) about 8%. The proportion of A was almost the same as that of C, so came the presumption that there were 3 fragments of a attached to C, then C linked together with 13 Bs and 1 D, which was the unit that repeated to form the polysaccharide. The possible structure of the repetitive unit was shown in The lack of absorbance at 260 nm and 280 nm indicated that this polysaccharide contained no nucleic acid or protein. The sugar composition of HB-1 was determined by GC-MS as glucose (Glc), arabinose (Ara), and galactose  in a molar ratio of 16.071: 2.722: 1.000 and the absolute configuration of Glc were identified as D. The result of periodate oxidation and Smith degradation showed that the reaction products were mainly phycitol derived from (1→4) Glc and a few glycerine from (1→) Glc or Ara, (1→4) Ara, or (1→2) Ara. Methylation reaction showed that the proportion of —Lanoderma lucidum polysaccharides and lentinan with (1→3)-D-glucan structure have anticancer actions (Ranunculi ternate polysaccharides. Since HB-1 consists of (1→2)- glucans, (1→4)- glucans and (1→6)- glucans instead of (1→3)- glucans, it is, in this way, explicable that it did not inhibit human glioma cell line SF188. Unfortunately, HB-1 did not possess the inhibition activity on human glioma cell line SF188. Studies show that, generally, both  actions . Maybe tRanunculi ternate has been used as hospital preparation to treat breast cancer and proves to be very effective, there must be a substance, or some substances working together to support this therapeutic effect, which needs further investigation. But since the water extraction, which mostly consists of polysaccharides, of radix


In mice MEOX2/TCF15 heterodimers are highly expressed in heart endothelial cells and are involved in the transcriptional regulation of lipid transport. In a general population, we investigated whether genetic variation in these genes predicted coronary heart disease (CHD).MEOX2 and four in TCF15. Over 15.2 years (median), CHD, myocardial infarction, coronary revascularisation and ischaemic cardiomyopathy occurred in 106, 53, 78 and 22 participants. For SNPs, we contrasted CHD risk in minor-allele heterozygotes and homozygotes (variant) vs. major-allele homozygotes (reference) and for haplotypes carriers (variant) vs. non-carriers. In multivariable-adjusted analyses with correction for multiple testing, CHD risk was associated with MEOX2 SNPs (P ≤ 0.049), but not with TCF15 SNPs (P ≥ 0.29). The MEOX2 GTCCGC haplotype (frequency 16.5 %) was associated with the sex- and age-standardised CHD incidence ; the multivariable-adjusted hazard ratio [HR] of CHD was 1.78 . For myocardial infarction, coronary revascularisation, and ischaemic cardiomyopathy, the corresponding HRs were 1.96 (1.16–3.31), 1.87 (1.20–2.91) and 3.16 (1.41–7.09), respectively. The MEOX2 GTCCGC haplotype significantly improved the prediction of CHD over and beyond traditional risk factors and was associated with similar population-attributable risk as smoking (18.7 % vs. 16.2 %).In 2027 participants randomly recruited from a Flemish population , we genotyped six SNPs in MEOX2, but not TCF15, is a strong predictor of CHD. Further experimental studies should elucidate the underlying molecular mechanisms.Genetic variation in The online version of this article (doi:10.1186/s12863-015-0272-2) contains supplementary material, which is available to authorized users. Endothelial cells lining the microvasculature constitute the interface between the circulating blood and tissues . They diLPL is expressed at the luminal endothelial surface of arteries and capillaries and hydrolyses circulating lipoprotein triglycerides into free fatty acids and glycerol . Local  and systhttp://www.privacycommission.be). The Ethics Committee of the University of Leuven approved the study. Recruitment for the FLEMENGHO study started in 1985  × [R −1] + 1), where R is the multivariable-adjusted hazard ratio, D is the incidence of CHD in the whole population, and G is the prevalence of the GTCCGC haplotype [MEOX2 GTCCGC haplotype to predict CHD over and beyond classical risk factors, using the integrated discrimination improvement (IDI) and the net reclassification improvement (NRI), as described by Pencina and colleagues for survival data [We computed the positive predictive value of the risk carrying aplotype . The attaplotype . Finallyval data .2, and total cholesterol 5.49 ± 1.15 mmol/L. Among all participants, 486 (24.0 %) had hypertension, of whom 214 (44.0 %) were on antihypertensive drug treatment, 33 (1.6 %) had diabetes mellitus, and 41 (2.0 %) reported a history of CHD. Previous coronary complications included angiographically proven coronary stenosis, myocardial infarction, and coronary revascularisation in 8 (0.4 %), 11 (0.5 %) and 22 (1.1 %) patients, respectively. Of 1034 women and 993 men, 277 (26.8 %) women and 328 (33.0 %) men were smokers, and 168 (16.3 %) women and 418 (42.1 %) men reported intake of alcohol. In smokers, median tobacco use was 15 cigarettes per day . In drinkers, the median alcohol consumption was 14 g per day (8 to 26 g per day).All 2027 participants were White Europeans, of whom 1034 (51.0 %) were women. The study population consisted of 332 singletons and 1695 related subjects, belonging to 49 single-generation families and 191 multi-generation pedigrees. Age averaged (±SD) 43.6 ± 14.3 years, blood pressure 125.0 ± 15.4 mm Hg systolic and 76.2 ± 9.5 mm Hg diastolic, body mass index 25.7 ± 4.3 kg/mvs. 36.8 %; P = 0.13), while the prevalence of drinking was lower among cases . Heart rate at baseline was similar in participants without and with incident CHD .Table n = 29) or percutaneous (n = 56) coronary revascularisation. Coronary events also included 5 fatal and 17 non-fatal cases of ischaemic cardiomyopathy.Over a median follow-up of 15.2 years , 106 new coronary events occurred, 24 fatal and 82 non-fatal. Coronary events comprised 12 fatal and 34 non-fatal myocardial infarcts and 7 sudden deaths. There were 78 patients who underwent surgical (MEOX2 and the four SNPs in TCF15 complied with Hardy-Weinberg equilibrium (0.30 ≤ P ≤ 0.80). In the whole study population  with the MEOX2 SNPs except for rs1050290 (P = 0.058). There were no differences in these estimates between homozygous and heterozygous minor allele carriers (0.23 ≤ P ≤ 0.98) except for rs12056299 (P = 0.014). For all coronary events combined, the sex- and age-standardised incidence rates (0.11 ≤ P ≤ 0.39) and the sex- and age-adjusted cumulative incidence (0.11 ≤ P ≤ 0.71) did not differ among minor allele carriers and major allele homozygotes of the four TCF15 SNPs.The sex- and age-standardised incidence rates of coronary events associated with the Next, we accounted for family clusters and adjusted the hazard ratios for baseline characteristics, including sex, age, body mass index, systolic pressure, the total-to-HDL cholesterol ratio, smoking and drinking, and antihypertensive drug treatment. Compared with homozygotes of the major allele, for rs10777, rs12056299, rs7787043, rs4532497, and rs1050290, CHD risk was higher in minor allele carriers, whereas the opposite was the case for rs6959056 . However, Additional file P = 0.011) between rs12624577 in TCF15 and rs4532497 in MEOX2. Among C allele carriers of MEOX2 rs4532497, the hazard ratio expressing the risk of the C allele relative to the TT genotype in TCF15 was 2.44 , whereas among MEOX2 TT homozygotes the corresponding hazard ratio was 0.75 .For the four MEOX2 SNPs had a frequency of over 10 % and were carried through in the analysis. With letters referring to the alleles in rs10777, rs12056299, rs7787043, rs4532497, rs6959056, and rs1050290 in MEOX2, these haplotypes were TCTTAT (27.5 %), TCTTGT (26.4 %), and GTCCGC (16.5 %). For all coronary events, rates standardised for sex and age  and cumulative incidence estimates adjusted for sex and age  and after adjustment for multiple testing (P ≤ 0.036), showed significant association with GTCCGC. In multivariable-adjusted analyses  were 7.2, 39.3 and 16.2 %, respectively. Table GTCCGC haplotype to the basic model including traditional risk factors improved (0.016 ≤ P ≤ 0.056) IDI and NRI.With adjustments applied as before, the positive predictive value and the attributable and population-attributable CHD risks associated with MEOX2 and TCF15, two transcription factors that are highly expressed by cardiac endothelium and that in a heterodimeric fashion interfere with cardiac energy metabolism by driving endothelial CD36 and LPL expression, thereby facilitating fatty acid transport across the cardiac endothelium [MEOX2, as captured by six tagging SNPs. On the other hand, genetic variation in TCF15, coding for the heterodimeric partner of MEOX2, was not associated with the incidence of coronary events. Nonetheless, the CHD risk associated with MEOX2 rs4532497 was confined to TCF15 rs12624577 variant allele carriers, which might reflect the known heterodimeric action picked up in our experimental studies [MEOX2, the molecular mechanisms underlying this relation need further clarification in experimental studies back translating our epidemiological findings. For now, working hypotheses might be developed along two lines respectively involving disturbed lipid handling [To our knowledge, our study is the first to relate in a general population CHD incidence to genetic variation in handling –19, a kehandling –24 or inhandling , 26.MEOX2 gene was not among these SNPs, but LPL (rs264) was [LPL is associated with the levels of circulating LPL activity [MEOX2, a gene that facilitates fatty acid transport into and through heart endothelial cells. As for genetic variation in LPL [LPL [MEOX2 variants (data not shown), pointing to a local coronary rather than a systemic underlying mechanism.A large-scale genome-wide association study identified 46 significant lead SNPs associated with CHD. Twelve showed a significant association with a lipid trait. Variation in the 264) was . LPL cat264) was . Geneticactivity , the plaactivity , 18 and activity , 18 and activity , albeit activity . Cardiacn in LPL , 18, we LPL [LPL , 18, we MEOX2 as a potentially important regulatory gene inhibiting not only the angiogenic response of endothelial cells to pro-angiogenic factors, but also their response to chronic inflammatory stimulation that normally activates NF-κB [MEOX2. Chen and Gorski did an in silico search for micro-RNA binding sites in the GAX 5’UTR and identified consensus sites for multiple candidate micro-RNAs, of which only miR-130a was expressed in proliferating endothelial cells [Moving to the second hypothetical pathophysiological pathway, MEOX2 is also known as growth arrest-specific homeobox (GAX) , During es NF-κB . Inflammes NF-κB . These oes NF-κB –24, 27 mal cells . miR-130al cells .MEOX2. However, these GWAS studies relied on comparing cases and controls drawn from heterogeneous sources [-6 to 10-8. In contrast, our study was prospective and population-based and tested a prior hypothesis involving only six SNPs in MEOX2 and four in TCF15. We did therefore not rely on such extreme P-values, but applied the Benjamin-Hochberg approach for multiple testing. Admittedly, our sample size was smaller than in the GWAS studies. This is particularly relevant for the interaction between MEOX2 rs4532497 and TCF15 rs12624577 . As shown in Additional file MEOX2. Moreover, according to Ensemble 75 annotation (http://www.ensembl.org/Homo_sapiens/Info/Annotation) and GENCODE 22 (http://www.gencodegenes.org/releases/22.html), rs4532497 and rs6959056 also map into the ENSG00000237070 (AC005550.3) antisense non-protein coding gene. In particular, rs4532497 maps into intron 1, whereas rs6959056 maps into exon 4 of ENSG00000237070. rs6959056 and rs1050290 fall into a promoter regulatory region (ENST00000622287) both in human umbilical vein endothelial cells and in human dermal fibroblasts, where MEOX2 is expressed. Further functional studies are needed to investigate the possible modulation of MEOX2 expression. Moreover, rs6959056 in exon 4 of the non-coding transcript ENST00000451240 could affect gene function, although no information on the ENSG00000237070 gene is currently available.We performed the annotation of the genomic context surrounding the SNPs retained for Xenopus laevis, TCF15 directly activates the expression of MEOX2 [MEOX2 and TCF15 genes. Several reasons may explain why such associations were not detected. Indeed, heart failure is a heterogeneous disease caused by a multitude of instigators, including ischaemic or valvular heart disease, comorbidities, or risk factors such as hypertension. Moreover, the diagnosis of heart failure depends on the clinical interpretation of a combination of signs and symptoms, that are difficult to recognise [TCF15, also known as Paraxis, is a member of the Twist subfamily of basic helix-loop-helix transcription factors that regulate specification of mesodermal derivatives during vertebrate embryogenesis . TCF15 pof MEOX2 . Our expof MEOX2 . In our ecognise , 34.P = 0.031), rs45324977 (P = 0.019) and haplotype GTCCGC (P = 0.0054) in relation to CHD. On the other hand, we did not consider applying a correction for multiple testing based on the four coronary endpoints. Indeed, such events are highly correlated. Multiple testing is therefore not indicated, because each new test does not provide an independent opportunity for a type-I error [MEOX2 might be causal, taking into account (i) the strength and consistency of the association across different SNPs; (ii) temporality, genetic variability preceding the event; (iii) plausibility based on the experimental studies [LPL [MEOX2 and TCF15 are currently known. In this regard, it is important to note that we noticed that of the genes overexpressed in cardiac endothelial cells, those most upregulated included genes involved in lipid homeostasis, including LPL [vs. 43.6 years) and had a higher systolic blood pressure (129.5 vs. 125.0 mm Hg), resulting in a higher prevalence of hypertension (40.0 vs. 24.0 %). We cannot ascertain whether these factors might have biased our analyses.The present study must be interpreted within the context of some potential limitations. First, even though our analysis was hypothesis-driven based on published evidence from experimental studies , we adju-I error . Second,-I error  suggest ies [LPL –19. ThirMEOX2 GTCCGC carrying state and smoking. Several investigators proposed the use of genetic risk scores based on genome-wide association studies to stratify for the probability of CHD [MEOX2 improved IDI and NRI over and beyond the basic model including traditional CHD risk factors.The clinical implications of our current findings can be gauged by the observation that the attributable and population-attributable CHD risks were similar for the y of CHD , 38. In y of CHD , a scoreMEOX2 gene as a novel risk factor for CHD in a white population. However, further experimental studies are required to back-translate our epidemiological observations into underlying molecular mechanisms. Elucidation of these pathways might reveal new targets for the prevention and treatment of CHD.Our current study based on a predefined hypothesis generated by data from our experimental studies , identif


Pasteurella multocida sequence type 128, which was isolated from the infected finger bone of an adult female who was bitten by a domestic dog. The draft genome will be a valuable addition to the scarce genomic resources available for P. multocida.We report here the first high-quality draft genome sequence of  Pasteurella multocida is a Gram-negative coccobacillus and the most common causative microbe isolated from >50% of dog bites and 70% of cat bites  based on biochemical tests. Genomic DNA was extracted from the culture and prepared with Nextera XT . The library was quantified with Qubit and sequenced on the MiSeq desktop sequencer (2 × 250-bp run configuration) located at the Monash University Malaysia Genomics Facility, Selangor, Malaysia. Raw reads generated from the MiSeq were subsequently adapter trimmed and assembled using Trimmomatic 0.33 and SPAdes 3.5.0, respectively  multilocus sequencing type (MLST) database (http://pubmlst.org/pmultocida_rirdc/) hosted at the PubMLST website (http://www.pubmlst.org)  .De novo genome assembly produced 23 contigs with total genome length, N50, G+C content, and coverage of 2,276,171 bp, 202,696 bp, 40.27%, and 70×, respectively. Annotation was done with NCBI Prokaryotic Genome Annotation Pipeline, leading to the prediction of 2,045 protein-coding genes, 10 rRNAs, and 50 tRNAs. The 16S rRNA and whole-genome sequences of strain SMC1 showed 100% similarity and 98.54% average nucleotide identity, respectively, to those of P. multocida ATCC 43137T, thus confirming its identity as P. multocida instead of P. canis, which was previously determined based on biochemical tests. Based on in silico MLST analysis, the sequence type (ST) of strain SMC1 was found to be ST128. The only other strain of P. multocida ST128 that has been reported to date is P. multocida strain M25/017, which was isolated from a sheep in New Zealand in 1995 (A total of 844,167 paired-end reads were generated for strain SMC1. P. multocida strain SMC1 has been deposited at DDBJ/EMBL/GenBank under the accession no. LNCO00000000. The version described in this paper is version LNCO01000000.The whole-genome shotgun project for


All near-equilibrium systems under linear regime evolve to stationary states in which there is constant entropy production rate. In an open chemical system that exchanges matter and energy with the exterior, we can identify both the energy and entropy flows associated with the exchange of matter and energy. This can be achieved by applying statistical mechanics (SM), which links the microscopic properties of a system to its bulk properties. In the case of contractile tissues such as human placenta, Huxley’s equations offer a phenomenological formalism for applying SM. SM was investigated in human placental stem villi (PSV) (n = 40). PSV were stimulated by means of KCl exposure (n = 20) and tetanic electrical stimulation (n = 20). This made it possible to determine statistical entropy (S), internal energy (E), affinity (A), thermodynamic force (A / T) (T: temperature), thermodynamic flow (v) and entropy production rate (A / T x v). We found that PSV operated near equilibrium, i.e., A ≺≺ 2500 J/mol and in a stationary linear regime, i.e., (A / T) varied linearly with v. As v was dramatically low, entropy production rate which quantified irreversibility of chemical processes appeared to be the lowest ever observed in any contractile system. Human placental stem villi (PSV) have been shown to possess contractile properties ,2 and thth percentile [Placenta (n = 40) were obtained from pregnant women undergoing delivery at the maternity of Meaux hospital, Meaux. Patients gave informed written consent. The study was approved by the « Comité Consultatif sur le Traitement de l’Information en matière de Recherche dans le domaine de la Santé » 2012.181, the « Local Ethical Committee », 2011, 206, the « Direction Générale pour la Recherche et l’Innovation » and the Direction of the Meaux Hospital, Meaux. All pregnancies were normal without complications. Several exclusion criteria were retained: delivery before 37 weeks of gestational age, newborn with birth weight below the 10rcentile , pre-ecl-2 was imposed to the PSV in such a way it induced neither shortening nor lengthening of the PSV. The bath chamber contained a Krebs-Henseleit solution (in mM): 118 NaCl, 4.7 KCl, 1.2 MgSO4.7 H2O, 1.1 KH2PO4, 24 NaHCO3, 2.5 CaCl2.6H2O and 4.5 glucose, maintained at 22°C, pH 7.4, and bubbled with 95% O2-5% CO2. Stimulation of human PSV was induced by either electrical tetanus   or after exposure to KCl  at 0.05 M concentration.Human PSV were carefully cut from the middle part of a cotyledon (resting length Lo was 10.2±2.6 mm and diameter was 1.5±0.3 mm) belonging to the type 1 category of the Demir classification . PSV werThe electromagnetic device was previously described in details . A preci2 and in Lo/s, respectively. G is dimensionless.The load level was progressively decremented by successive steps of 0.1mN from isometric tension until zero-load. At each afterload level, PSV shortened at isotonic tension level (T) and constant velocity (V) . The T-V1 in s -1) and CB detachment (g1 and g2 in s -1), catalytic constant (kcat in s -1), myosin content (mole number per liter in nM/L), and thermodynamic flow (v), i.e., maximum myosin ATPase activity under isometric conditions where v = kcat x mole number per liter. Moreover, w is the maximum mechanical work of a single CB (w / e = 0.75) and e is the free energy required to split one ATP molecule [−19 J. The molecular step size h is defined by the translocation distance of the actin filament per ATP hydrolysis. In vivo double-head myosin displacement during a unitary interaction with actin was 10 nm [1, g1, g2 and kcat were given by the following equations [The CB cycle was described in molecule . Accordias 10 nm . The paras 10 nm , the valS) was in a container (C), both exchanging energy and matter. S was composed of all the cycling myosin CBs that could individually be found in one of the seven distinct states, either attached to or detached from actin, and bound or not to a nucleotide  or to Pi. C was composed of all the non-cycling myosin CBs, all the non-cycling actin molecules, and all the ATP and ADP and Pi which were not attached to the cycling myosin CBs. Actin, myosin CBs and small soluble molecules  can be exchanged between S and C, so that the number of cycling CBs may slightly fluctuate with the number of non cycling CBs becoming cycling CBs and vice versa. In the grand canonical ensemble, the average number of independent, non interacting cycling CBs within S was determined from A. Huxley’s equations [S. The method for determining the probability (Pr) of the seven  conformational states of the CB cycle has been previously described [r was individualized. The energy level Er increased from E0 to E6. By convention, the lowest level (E0), which coincided with the ground state (gs), was equal to zero; thus Eo = Egs = 0. The most probable detached state was D3; this implies that Po = PD3 and consequently Eo = ED3 = 0. The Boltzmann distribution is given by the equation:The grand canonical ensemble is a useful tool for applying SM to complex open systems such as living contractile tissues, and has been previously used ,10. The quations  and was escribed  Fig 1)..S) was ir = exp (−βEr)/∑ exp (−βEr), and by definition the molecular partition function is equal to z = ∑exp(−βEr), thus Pr = exp (−βEr)/z, where β = 1 / kT. As Eo = 0 consequently, Po = PD3 = 1/z.PMoreover, we have the thermodynamic equation: E−TS = −RT lnzS tended towards zero. Under these conditions, A also tended towards zero. Thus E−TS tended towards Ψ. The extrapolation at v = 0 of the E−TS versus v relationship  was equal to Ψ. Affinity A was calculated by the equation: A = Ψ−RT lnPD 3 and thermodynamic force was equal to A/T.Thus E−TS = Ψ−A = −RT lnz = RT lnPmax = RT lnPD 3. Consequently E = RTlnPmax+TS. The CB cycle stopped when the thermodynamic flow ϖ of the system If A ≺≺ 2500 J/mol, a chemical system operates near-equilibrium. A near-equilibrium chemical system under linear regime evolves to a stationary state. This is achieved if the thermodynamic force A/T varies linearly with the thermodynamic flow v ,20. In oAffinity (A) of PSV was 573±562 J/mol (mean ± SD) in KCl and 549±441 J/mol in tetanus ; NS. ThuThe purpose of our study was to apply the formalism of statistical mechanics (SM) to human placental stem villi (PSV). SM made it possible to determine thermodynamic parameters such as statistical entropy, internal energy, chemical affinity, thermodynamic force, thermodynamic flow, entropy production rate of a given chemical system . We founNumerous experimental studies have shown that human PSV behaved as a contractile tissue. Contractile properties of the human placenta have long been recognized. Early in the twentieth century, several authors have described smooth-like muscle cells in human PSV –27. Then-1 [+—K+ ATPase pump [In human PSV, near-equilibrium thermodynamic behavior was validated because the chemical affinity (A) of the global CB cycle was A ≺≺ 2500 J.mol-1 ,20,39  was particularly low because of the low values of both kcat and myosin content in PSV , which refers to pathological states in which a fetus is unable to achieve its genetically determined potential size. IUGR which represents 3 to 5% of births, causes numerous perinatal complications, including fetal morbidity and mortality, iatrogenic prematurity, fetal compromise in labor, etc. Numerous placental abnormalities due to hypertension, pre-eclampsia or diabetes may cause IUGR. From a clinical point of view, it would be interesting to study thermodynamic properties of placenta in pathological states such as IUGR.Stem villi of human placenta were a contractile tissue containing myosin molecular motors which cycle dramatically slowly. This confers a particular thermodynamic profile, with the lowest entropy production rate ever observed in any contractile system. Moreover, human placenta behaved as a near-equilibrium system under linear regime evolving to a stationary state. The low values of both thermodynamic flow and myosin content conferred to human PSV a dramatically low rate of entropy production compared to other contractile tissues.


TEM and EDX analysis of the process of transformation reveals that aminoclay coats negatively-charged DNA biomolecules and forms a self-assembled hybrid nanostructure. Subsequently, when this nanostructure is mixed with microalgal cells and plated onto selective agar plates with high friction force, cell wall is disrupted facilitating delivery of plasmid DNA into the cell and ultimately to the nucleus. This method is not only simple, inexpensive, and non-toxic to cells but also provides efficient transformation (5.03×102 transformants/µg DNA), second only to electroporation which needs advanced instrumentation. We present optimized parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates. It is also confirmed the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii through molecular methods.Genetic engineering in microalgae is gaining attraction but nuclear transformation methods available so far are either inefficient or require special equipment. In this study, we employ positively charged nanoparticles, 3-aminopropyl-functionalized magnesium phyllosilicate (aminoclay, approximate unit cell composition of [H Genetic engineering in prokaryotic unicellular organisms has reached newer heights with metabolic and pathway engineering, sometimes leading to drastic change in the identity of the parent strain Agrobacterium tumefaciens-mediated gene transfer is inefficient Among the various methods established, agitation with glass beads needs protoplast generation Since its synthesis Chlamydomonas reinhardtii cells. This method provides a simple, powerful, and non-invasive tool to transform cell wall-intact algal cells, without delicate pre-treatment such as enzymatic digestion and overcoming efficiency bottleneck that has limited genetic engineering of algal cells through nuclear transformation.Because of the attractive characteristics of aminoclay, such as its low toxicity profile with regards to environment C. reinhardtii culture, C. reinhardtii transformation, DNA extraction, PCR analysis, TEM examination, spreading friction apparatus, and commercially available kits used in this study are displayed in The detailed materials and equipments of aminoclay synthesis, The detailed Tris-Acetate-Phosphate (TAP) salts solution, liquid and solid TAP medium, and medium A (4% agar with Hygromycin B) and medium B (1.5% agar with Hygromycin B) are described in Secure the experimental stand, ∼1 meter height, on the desk with a clamp. Under the clamp, place a rotor with control buttons (speed and timer) and ensure it is level. Hang a triangle shaped polystyrene stir stick to a force control gauge that is perpendicularly tightened to the experimental stand .2•6H2O into 200 ml bulk ethanol solution in 500-ml beaker for 20 min. Add 13 ml 3-aminopropyl triethoxysilane to this bulk ethanolic solution and stir for 12 hours. Collect white-colored products by centrifugation at 6000 g for 10 min. Rinse the precipitate products two times with 200 ml bulk ethanol. Dry the harvested aminoclay on the oven at 50°C for 24 hours in order to let evaporation of the residual ethanol solvent. Grind dried aminoclay lumps using a mortar and pestle.According to literature 5 cells ml−1 in 1 liter of TAP liquid medium and grow at 25°C, under constant agitation at 10 g force continuous white light (100 µE m−2s−1) for 3–5 days. Harvest the cells (1×108 cells ml−1) by centrifugation .Based on the literature Option A) or tip ultrasonication for 5, 10, 20, and 30 s (Option B). Test mechanical properties  of 1.5, 3.0, and 4.0% agar plates. Pipet 100, 200, or 300 µl aliquots of cell and DNA-aminoclay mixture solution, generated by vortexing (Option A) or tip ultrasonication (Option B) on 4.0% agar plates containing hygromycin B (15 µg/ml). Rub them using spreading friction equipment for 4 min at a constant speed of 0.22 g force.Re-suspend 500 µl cell pellet to 500 µl mixture of aminoclay-DNA solution in a 1.5 ml-Eppendorf tube. Pre-treat the samples by vortexing for 10, 30, 60, 120, and 180 s , DNA (150 ng/µL), and DNA and aminoclay mixture (150 ng/µL in 100 mg/mL of aminoclay solution) were measured by dynamic light scattering (DLS) methods  in triplicate.in vitro, mix 500 µl hydrated aminoclay with DNA solution (at least 30 ng) in a 1.5 ml-Eppendorf tube. Drop 10 µl aliquot of the mixture of DNA-aminoclay solution onto a carbon mesh film copper grid. Dry the sample on the copper grid overnight at room temperature. Inspect it by TEM. To inspect the ultrastructural studyfor cross-sectioned cells To examine DNA and aminoclay hybrids −2s−1) for 5–7 d. Pick the colonies and plate each one into a well of a 96-well plate containing medium A supplemented with Hygromycin B (10 µg µl−1). Transfer the transformant colonies and plate each one into a well of a 24-well plate containing medium B supplemented with Hygromycin B (20 µg µl−1). Every data point should be based on at least three independent experiments, and expressed as an average of the individual values with standard deviation.Incubate cells at room temperature under static conditions and continuous light (60 µE mwww.appliedbiosystems.com). Using a DNA extraction kit (i-genomic DNA Extraction Mini Kit for Plant-iNtRON), it is confirmed the colony identity by checking the insertion of plasmid DNA by PCR. It is noted that when considering the high GC contents of Chlamydomonas genome, PCR efficiency can be improved by adding DMSO and 5 M of Betaine or by using high GC buffer to the PCR mix.Denature at 94°C for 5 min, followed by 35 cycles of 94°C for 45 sec, 59°C for 45 sec, 72°C for 1 min, ending with a final extension at 72°C for 7 min. PCR was performed in a thermal cycler . According to manufacturer’s guideline, the hybridization was conducted. Resultant signals were detected by the Bio-Imaging Analyzer BAS-1800 Π .Southern blotting assay was followed Microalgal transformation is difficult to achieve because of the complex cell wall structures. Although DNA adsorbed nanofibers have been developed for bacterial transformation, popularly referred to as Yoshida effect, they proved insufficient for microalgal transformation in our earlier experiments . Our ear). Single molecules of plasmid DNA were condensed by coating with aminoclay, forming nanostructures of 10.5–11.5 nm in diameter and ∼2 µm in length , corresponding to size reduction of hydrodynamic diameter in DNA and aminoclay mixture by DLS method. These observations were confirmed by elemental analysis  in which P indicates the presence of DNA molecules and Mg, Si, and Cl indicates elements derived from aminoclay, confirmation by comparison of only aminoclay , DNA (150 ng/µL), and DNA and aminoclay mixture (150 ng/µL in 100 mg/mL of aminoclay solution) showed +35.0±3.5 mV, −24.6 mV±5.2, and +26.2±5.1 mV, resulting in 49.87±5.1 nm, 466.53±20.34 nm, and 550.33±7.8 nm of hydrodynamic diameter size in aqueous solution, respectively. TEM analyses were performed Figure 2minoclay . AminoclAs ascertained in earlier experiments, friction force seemed to be an important parameter in facilitating cell rupture, however, further increase in friction force resulted in damage of agar surface and resultant false colonies or smear. In order to apply the maximum friction force to the agar plates, a 4% concentration of agar was chosen based on mechanical strength of the agar medium as well as high transformation efficiency  in whichOption A we obtained ∼25 colonies (corresponding to frequency of 4.0×10−6/cell), by vortexing for 60 seconds, adding 100 µl mixture of plasmid DNA-aminoclay solution and cells under 0.7 kgf force   .Using PCR, we confirmed the genetic identity of ten transformed colonies obtained by optimal vortexing pre-treatment Figure 5Kpn I and probed with a radiolabeled DNA fragment. Mutant T1 and T2 show a single band, corresponding to a unique integration site of the vector.For Soutrhern blot anlaysis , Figure Although confirmation of transformation and transformants were obtained using aminoclay, the mechanism of transformation and formation of a hybrid nanostructure involving DNA and aminoclay was not established.As mentioned before, TEM analysis confirmed that both an effective coating of aminoclay and cell wall rupture is important for efficient transformation of microalgal cells 2. The hInThe aminoclay method is one of the most efficient methods for transformation and is non-sophisticated Table 1.Neochloris and Nannochloropsis. or Chlorella sp. In précis, the aminoclay-based nuclear transformation protocol for intact microalgal cells that we present here is a very simple, efficient, safe, and reproducible. Microalgal transformation with this fast and effective method could hasten both basic and applied research in microalgal biotechnology. Further studies are being performed to demonstrate the applicability of the method into other important strains such as File S1Supporting Information. Method S1, List of materials and equipments. Text S1, Description of reagent setup. Table S1, Summary of the optimal conditions established for nuclear transformation of C. reinhardtii. Table S2, Limitations and its solution of transformation by aminoclay. Figure S1, Schematic representation of spreading friction equipment. Figure S2, Digital camera images of resultant transformant colonies. Figure S3, Schematic representation of the approximate unit structure of aminoclay ([H2N(CH2)3]8Si8Mg6O12(OH)4)). Figure S4, TEM images and energy dispersive X-ray (EDX) analysis of aminoclay. Figure S5, Mechanical properties of plates with increasing concentration of agar . Figure S6, Schematic diagram of an expression construct and Southern blot analysis.(DOC)Click here for additional data file.