Molecular Characterization and Drug-resistance of Mycobacterium tuberculosis Strains in Xuzhou, China

To understand the genetic diversity and drug resistance status of Mycobocterium tuberculosis （M. tuberculosis） circulating in Xuzhou of China, the spacer-oligonucleotide typing （Spoligotyping） and multi-loci VNTRs （variable number tandem repeats） analysis （MLVA） were utilized for the genotyping of the isolates. Drug susceptibility test （DST） was performed by the proportion method on the Lowenstein-Jensen （L-J） medium using isoniazid, rifampicin, ethambutol, and streptomycin. By Spoligotyping, 287 M. tuberculosis isolates were differentiated into 14 clusters. Then with 15-1oci MLVA, these strains could be divided into 32 clusters, 228 genotypes. Of 15 VNTRs, 6 loci had the highly discriminatory powers, 6 loci presented moderate discrimination and 3 loci demonstrated less polymorphism. The DST results showed that 46 strains were resistant to at least one first-line anti-tuberculosis agent. There was a difference in the isoniazid resistance between Beijing and non-Beijing genotype strains. We concluded that the combination of Spoligotyping and 15 VNTR loci as the genotyping in our study was applicable for this region, the drug resistant isolates were identified, and the Beijing family was the most prevalent genotype in the rural counties of Xuzhou.

科学术语的翻译实践及其概念的语境重置——从“bacterium”到“细菌”

19世纪末20世纪初,“bacterium”概念由西方传播至我国,并最终以“细菌”二字成为汉语词。这是一则外来概念在华传播的成功案例。“细菌”的入华“旅行”先后经历了术语“bacterium”的翻译实践、概念“bacterium”的在华接受及译词“细菌”的最终确立。“微虫”“微生物”“微菌”“霉菌”“微生毒”“微生虫”“璧他利亚”等译词的涌现、共存与淘汰体现了概念跨文化传播在语言层面的复杂表征,语境重置则是此番过程的本质。汉语语境“虫”概念和本土传统的病因学体系为“旅行”概念的接受创造条件。“细菌”二字被确立为术语“bacterium”的译词则是社会权力制约下的结果。深入挖掘这则成功案例,厘清概念跨文化传输的特点,或能为提升概念跨文化传播效率提供些许启示。

Molecular Characterization of Drug-Resistant Beijing Family Isolates of Mycobacterium Tuberculosis from Tianjin,China

Objective Tuberculosis remains a severe public health issue, and the Beijing family of mycobacterium tuberculosis （M. tuberculosis） is widespread in East Asia, especially in some areas in China, like Beijing and Tianjin. This study aimed at determining the mutation patterns of drug-resistant Beijing strains of M. tuberculosis isolated from Tianjin, China. Methods A total of 822 M. tuberculosis isolates were screened for drug resistance by an absolute concentration method and the genotype was identified by PCR. 169 drug-resistant isolates of the Beijing family were analyzed for the potential mutations in the rpoB, katG, inhA promoter region and in rpsL, rrs and embB genes, which are associated with resistance to rifampin （RFP）, isoniazid （INH）, streptomycin （SM） and ethambutol （EMB） respectively by PCR and DNA sequencing. Results Fifty-eight out of 63 RFP-resistant isolates were found to carry the mutations within the 81-bp RFP resistance determining region （RRDR） of the rpoB gene and the most frequent mutations occurred at codon 531 （44.4%）, 526 （28.6%）, and 516 （7.9%） respectively. 16 mutation pattems affecting 12 different codons around the RRDR of rpoB were found. Of 116 INH-resistant isolates, 56 （48.3%） had the mutation of katG 315 （AGC→ACC） （Ser→Thr）, 3 （2.6%） carried S315N （AGC→AAC） and 27 （16.0%） had the mutation of inhA-15A→T. 84 out of 122 SM-resistant isolates （68.9%） displayed mutations at the codons 43 or 88 with AAG→AGG （Lys→Arg） of the rpsL gene and 22 （18.0%） with the mutations at positions 513A→C, 516C→T or 905 A→G in the rrs gene. Of 34 EMB-resistant isolates, 6 had mutation with M306V （ATG→GTG）, 3 with M306I （ATG→ATT）, 1 with M306I （ATG→ATA）, 1 with D328Y （GAT→TAT）, 1 with V348L （GTC→CTC）, and 1 with G406S （GGC→AGC） in the embB gene. Conelusion These novel findings extended our understanding of resistance-related mutations in the Beijing strains of M. tuberculosis and may provide a scientific basis for development of new strategies for diagnosis and control of tuberculosis in China and other countries where Beijing strains are prevalent.

A STUDY OF DECREASING DRUG-RESISTANCE OF HUMAN OVARIAN CANCER CELLS IN VITRO

A chemosensitivity test for ovarian cancer using tritiated thymidine incorporation assay was carried out. A dose-response relationship for cisplatin and potentiation of verapamil in increasing vincristine inhibition to ovarian cancer were investigated. A 5- fold increase of cisplatin density converted the tumors which were initially resistance to standard-dose cisplatin Into drug-sensitive ones. Vera-pamil was found to be able to overcome vincristine-resistance of some tumors in vitro. These results suggest that using high dose cisplatin therapy or increasing local drug concentration by using other administration way, we could expect some ovarian cancers that had failed to standard dose cisplatin therapy to be effective. Combination of vincristine with verapamll may be helpful in treating some vincristineresistant cases.

Bio-inhibitive effect of an algal symbiotic bacterium on corrosion of magnesium in marine environment

It is a longstanding and challenging task to develop sustainable environment-friendly and cost-effective corrosion-protection technologies for Mg alloys, especially under marine conditions in which corrosion can normally be significantly accelerated by bacterial activity. However,this paper reports on the corrosion of highly active Mg interestingly inhibited by an algal-symbiotic bacterium Bacillus altitudinis. The corrosion of Mg in the presence of the bacterium drastically reduced by one order of magnitude after 14 days of immersion. This means that the algal-symbiotic bacterium widely available in natural ocean environments may be employed as a green and sustainable inhibitor in the marine industry. Based on electrochemical measurements, surface analyses and microbe experiments, a combined inhibition mechanism is proposed in the paper to interpret the interesting corrosion behavior of Mg.

Establishment and evaluation of an overlap extension polymerase chain reaction technique for rapid and efficient detection of drug-resistance in Mycobacterium tuberculosis

Background:Rapid and accurate detection of drug resistance inMycobacterium tuberculosis is critical for effective control of tuberculosis(TB).Herein,we established a novel,low cost strategy having high accuracy and speed for the detection ofM.tuberculosis drug resistance,using gene splicing by overlap extension PCR(SOE PCR).Methods:The SOE PCR assay and Sanger sequencing are designed and constructed to detect mutations of rpoB,embB,katG,andinhA promoter,which have been considered as the major contributors to rifampicin(RFP),isoniazid(INH),and ethambutol(EMB)resistance inM.tuberculosis.One hundred and eightM.tuberculosis isolates came from mycobacterial cultures of TB cases at Chongqing Public Health Medical Center in China from December 2018 to April 2019,of which 56 isolates were tested with the GeneXpert MTB/RIF assay.Performance evaluation of the SOE PCR technique was compared with traditional mycobacterial culture and drug susceptibility testing(DST)or GeneXpert MTB/RIF among these isolates.Kappa identity test was used to analyze the consistency of the different diagnostic methods.Results:We found that the mutations of S531L,S315T and M306V were most prevalent for RFP,INH and EMB resistance,respectively,in the 108 M.tuberculosis isolates.Compared with phenotypic DST,the sensitivity and specificity of the SOE PCR assay for resistance detection were 100.00% and 88.00% for RFP,94.64% and 94.23% for INH,and 68.97% and 79.75% for EMB,respectively.Compared with the GeneXpert MTB/RIF,the SOE PCR method was completely consistent with results of the GeneXpert MTB/RIF,with a concordance of 100% for resistance to RFP.Conclusions:In present study,a novel SOE PCR diagnostic method was successfully developed for the accurate detection ofM.tuberculosis drug resistance.Our results using this method have a high consistency with that of traditional phenotypic DST or GeneXpert MTB/RIF,and SOE PCR testing in clinical isolates can also be conducted rapidly and simultaneously for detection of drug resistance to RFP,EMB,and INH.

Reverse effect of curcumin on CDDP-induced drug-resistance via Keap1/p62-Nrf2 signaling in A549/CDDP cell

Objective: To assess the effect of curcumin on CDDP-induced drug resistance and explore the underlying molecular mechanism through Nrf2 system and autophagy pathway.Methods: A drug-resistant cell model was established by exposing A549/CDDP cell to2 μg/mL CDDP. A549/CDDP cell was treated with 20 μg/mL CDDP and 10 μM curcumin. The cell viability and apoptosis level, the signals of Keap1/P62-Nrf2 and autophagy pathway were analyzed.Results: CDDP induction promoted drug-resistant phenotype in A549/CDDP cell and activated autophagy as well as Nrf2 signals in A549/CDDP cell. Meanwhile, curcumin combination attenuated autophagy and Nrf2 activation induced by CDDP, and reversed the drug-resistant phenotype. Notably, curcumin combination augmented Keap1 transcription. Furthermore, Keap1 ablation with short hairpin RNAs hampered the efficacy of curcumin, suggesting Keap1 played a crucial role on reversal effect of curcumin.Conclusions: The present findings demonstrate that CDDP promotes abnormal activation of Nrf2 pathway and autophagy, leading to drug resistance of A549/CDDP cell.Curcumin attenuates this process and combat drug-resistance through its potent activation on Keap1 transcription, which is essential for interplay between oxidative stress induced Nrf2 activation and autophagy/apoptosis switch.

Three-dimensional collagen-based scaffold model to study the microenvironment and drug-resistance mechanisms of oropharyngeal squamous cell carcinomas

Objective:Squamous cell carcinoma(SCC)represents the most common histotype of all head and neck malignancies and includes oropharyngeal squamous cell carcinoma(OSCC),a tumor associated with different clinical outcomes and linked to human papilloma virus(HPV)status.Translational research has few available in vitro models with which to study the different pathophysiological behavior of OSCCs.The present study proposes a 3-dimensional(3 D)biomimetic collagen-based scaffold to mimic the tumor microenvironment and the crosstalk between the extracellular matrix(ECM)and cancer cells.Methods:We compared the phenotypic and genetic features of HPV-positive and HPV-negative OSCC cell lines cultured on common monolayer supports and on scaffolds.We also explored cancer cell adaptation to the 3 D microenvironment and its impact on the efficacy of drugs tested on cell lines and primary cultures.Results:HPV-positive and HPV-negative cell lines were successfully grown in the 3 D model and displayed different collagen fiber organization.The 3 D cultures induced an increased expression of markers related to epithelial–mesenchymal transition(EMT)and to matrix interactions and showed different migration behavior,as confirmed by zebrafish embryo xenografts.The expression of hypoxia-inducible factor 1α(1α)and glycolysis markers were indicative of the development of a hypoxic microenvironment inside the scaffold area.Furthermore,the 3 D cultures activated drug-resistance signaling pathways in both cell lines and primary cultures.Conclusions:Our results suggest that collagen-based scaffolds could be a suitable model for the reproduction of the pathophysiological features of OSCCs.Moreover,3 D architecture appears capable of inducing drug-resistance processes that can be studied to better our understanding of the different clinical outcomes of HPV-positive and HPV-negative patients with OSCCs.

Investigation of immune escape-associated mutations of hepatitis B virus in patients harboring hepatitis B virus drug-resistance mutations

It is unclear whether immune escape-associated mutations in the major hydrophilic region of hepatitis B virus surface antigen(HBsAg)are associated with nucleoside/nucleotide analog resistance.AIM To evaluate the association between immune escape-associated mutations and nucleoside/nucleotide analog resistance mutations.METHODS In total,19440 patients with chronic hepatitis B virus infection,who underwent resistance testing at the Fifth Medical Center of Chinese PLA General Hospital between July 2007 and December 2017,were enrolled.As determined by sequence analysis,6982 patients harbored a virus with resistance mutations and 12458 harbored a virus lacking resistance mutations.Phenotypic analyses were performed to evaluate HBsAg production,replication capacity,and drug-induced viral inhibition of patient-derived drug-resistant mutants with or without the coexistence of sA159V.RESULTS The rate of immune escape-associated mutation was significantly higher in 9 of the 39 analyzed mutation sites in patients with resistance mutations than in patients without resistance mutations.In particular,these mutations were sQ101H/K/R,sS114A/L/T,sT118A/K/M/R/S/V,sP120A/L/Q/S/T,sT/I126A/N/P/S,sM133I/L/T,sC137W/Y,sG145A/R,and sA159G/V.Among these,sA159V was detected in 1.95%(136/6982)of patients with resistance mutations and 1.08%(134/12,458)of patients lacking resistance mutations(P<0.05).The coexistence of sA159V with lamivudine(LAM)and entecavir(ETV)-resistance mutations in the same viral genome was identified during follow-up in some patients with drug resistance.HBsAg production was significantly lower and the replication capacity was significantly higher,without a significant difference in LAM/ETV susceptibility,in sA159V-containing LAM/ETV-resistant mutants than in their sA159V-lacking counterparts.CONCLUSION In summary,we observed a close link between the increase in certain immune escape-associated mutations and the development of resistance mutations.sA159V might increase the fitness of LAM/ETV-resistant mutants under environmental pressure in some cases.

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatumZ7产氢效率的因素,采用生理学方法对其进行了研究。结果表明:乙醇型发酵菌A.elongatumZ7的最适产氢温度为37℃,最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度>60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。

高效产氢细菌新种——Ethanologenbacterium sp.X-1的分离鉴定及其产氢效能

为获得高效产氢发酵细菌 ,采用改进的厌氧Hungate培养技术 ,从生物制氢反应器CSTR中分离一株产氢细菌X 1。对该株细菌进行了形态学特征、生理生化指标、16SrDNA和 16S 2 3SrDNA间隔区序列分析等研究。结果表明与最相近的种属Clostridiumcellulosi和Acetanaerobacteriumelongatum等的 16SrRNA基因序列同源性为 94 %以下。16S 2 3SrRNA间隔区基因序列比对分析显示保守区域仅为tRNAAla和tRNAIle序列 ,其它可变部位没有同源性区域 ,鉴定为新属Ethanologenbacteriumsp .。该株细菌为专性厌氧杆菌 ,代谢特征为乙醇发酵 ,葡萄糖发酵产物主要为乙醇、乙酸、H2 和CO2 。在pH4 0和 36℃条件下最大产氢速率是 2 8 3mmolH2 (gdrycell·h)。

Isolation of a Bacterium Strain Degraded Agar

One in 58 strains of bacteria isolated from the compost showed clear colonies after a few days of growth on the plates containing medium made of only agar and water.Water suspension contained only agar (2 and 8g·L -1 ) with two controls (normal saline,LB medium) was inoculated with the bacterium BR5-1 to see whether there was an increasement of the alive bacteria concentration after 48 h of the growth.The results showed that there was a significant rising of the alive bacteria concentration in the agar susp...

来源于P.bacterium 1109的甘露醇脱氢酶的重组纯化及酶学性质研究

本文将来自P.bacterium 1109的甘露糖醇脱氢酶(MDH)表达并提取纯化,研究了该重组酶的酶学性质及其在甘露醇生产中的工艺条件。结果显示重组MDH是一个相对分子量为37 kDa的四聚体。氨基酸序列比对发现其与大多数MDHs的同源性小于40%。该酶的最适pH和温度分别为8.5和80℃,且当金属离子Zn^(2+)存在时,重组MDH的活力提高到对照组的260%。此外,重组MDH在75℃孵育6 h后仍可保留超过85%的残留活性,热稳定性较高,比大多数MDHs的活性高。底物特异性研究表明其对D-果糖具有较高的专一性。重组MDH催化D-果糖的米氏常数(K_m)和催化效率(k_(cat)/K_m)分别为20 mmol/L和7.5 L/(mmol·min)。重组MDH在以400 mmol/L的D-果糖为底物的反应系统中,可将80%以上的D-果糖转化为甘露糖醇。通过对反应条件的优化,为后续工业化生产制备甘露醇奠定了基础。

Effect of Sulfate Reduced Bacterium on Corrosion Behavior of 10CrMoAl Steel

The effects of sulfate reduced bacterium （SRB） on the corrosion behavior of 10CrMoAl steel in seawater were studied by chemical immersion, potentiodynamic polarization, electrochemical impedance spectroscopy measurement, and scanning electron microscope techniques. The results show that the content of element sulfur in the corrosion product of 10CrMoAl steel in seawater with SRB is up to 9. 23 %, which is higher than that of the same in sterile seawater. X-ray diffraction demonstrates that the main corrosion product is FeS. SRB increases the corrosion rate by anodic depolarization of the metabolized sulfide product. SEM observation indicates that the corrosion product is not distributed continuously; in addition, bacilliform sulfate-reduced bacterium accumulates on the local surface of 10CrMoAl steel. Hence, SRB enhances sensitivity to the localized corrosion of 10CrMoAl steel in seawater.

Purification and Characterization of 2-Haloacid Dehalogenase from Marine Bacterium Paracoccus sp. DEH99, Isolated from Marine Sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.

Isolation and characteristics of one marine psychrotrophic cellulase-generating bacterium Ar/w/b/75°/10/5 from Chuckchi Sea,Arctic

Microorganisms living in polar zones play an important part as the potential source of organic activity materials with low temperature characteristics in the bio-technological applications. A psychrotrophic bacterium (strain Ar/w/b/75°/10/5) , producing cellulose at low temperatures during late-exponential and early-stationary phases of cell growth, was isolated from sea ice-covered surface water in Chuckchi Sea, Arctic. This bacterium, with rod cells, was Gram-negative, slightly halophilic. Colony growing on agar plate was in black. Optimum growth temperature was 15℃. No cell growth was observed at 351 or above. Optimum salt concentration for cell growth was between 2 and 3 % of sodium chloride in media. Maximal cellulase activity was detected at a temperature of 35℃ and pH8. Cellulase was irreversibly inactivated when incubated at 55℃ within 30 min. Enzyme can be kept stable at the temperature no higher than 25℃. Of special interest was that this bacterium produced various extracellular enzymes including cellulase, amylase, agar hydrolase and protease, at low or moderate temperature conditions, which is certainly of it potential value for applications.

Extraction and physicochemical characteristics of a red pigment produced by marine bacterium strain S - 9801

A red pigment that has better biological properties is produced by marine bacterium strainS- 9801. The extraction methods, physicochemical and toxicity of the pigment have been studied. Dissolubility of pigment in the five organic solvent has been tested, and ethanol is optimally chosen for extraction. Physicochemical characteristics of this pigment was stable. The absorbance of the pigment solution was no losing when put under natural light for 10 days or treated by UV for 30 minutes, color of the pigment unchanged after 100℃ hythere for 1 h or 80 ℃ xerother for 2 h. The median lethal dose (LD_50) of the rat by celiac injection was 670.04 mg/kg and minimum lethal dose of oral was greater than 2 000 mg/kg.

Degradation of 2,6-di-tert-butylphenol by an isolated high-efficiency bacterium strain

An aerobic bacterium strain, F-3-4, capable of effectively degrading 2,6-di-tert-butylphenol(2,6-DTBP), was isolated and screened out from an acrylic fiber wastewater and the biofilm in the wastewater treatment facilities. This strain was identified as Alcaligenes sp. through morphological, physiological and biochemical examinations. After cultivation, the strain was enhanced by 26.3% in its degradation capacity for 2,6-DTBP. Results indicated that the strain was able to utilize 2,6-DTBP, lysine, lactamine, citrate, n-utenedioic acid and malic acid as the sole carbon and energy source, alkalinize acetamide, asparagine, L-histidine, acetate, citrate and propionate, but failed to utilize glucose, D-fructose, D-seminose, D-xylose, serine and phenylalanine as the sole carbon and energy source. The optimal growth conditions were determined to be: temperature 37℃, pH 7.0, inoculum size 0.1% and shaker rotary speed 250 r/min. Under the optimal conditions, the degradation kinetics of 2,6-DTBP with an initial concentration of 100 mg/L was studied. Results indicated that 62.4% of 2,6-DTBP was removed after 11 d. The degradation kinetics could be expressed by Eckenfelder equation with a half life of 9.38 d. In addition, the initial concentration of 2,6-DTBP played an important role on the degradation ability of the strain. The maximum initial concentration of 2,6-DTBP was determined to be 200 mg/L. Above this level, the strain was overloaded and exhibited significant inhibition.

Inhibition of spore germination of Ulva pertusa by the marine bacterium Pseudoalteromonas haloplanktis CI4

The effect of the bacterial strain C14 on the germination of spores from the green alga Ulva pertusa was assayed and it was found that the bacterial biofilm and cell-free supernatant strongly inhibited spore germination. In attempts to define the chemical nature of she antifouling substance in the supernatant of C14, the culture supernatants were tested for activity after heat treatment, enzymatic treatments, size fractionation, and separation into aqueous and organic fractions. Results suggest that this bacterium produces an extracellular component with specific activity toward algal spores that was heat-sensitive and between 3 and 10 kDa in molecular size. The exposure of the organic phase fraction to spores showed inhibitive effect on spore germination. Pronase and carboxypeptidase y did not significantly affect the activity of inhibitory component, suggesting that the component was not a protein or a peptide. The bacterium C14 was identified as Pseudoalteromonas haloplanktis based on the phenotypic characters and 16S rRNA gene analvsis.

Isolation and characterization of a marine bacterium producing protease from Chukchi Sea, Arctic

A Gram negative bacterium Ar/W/b/75°25′N/1 producing extracellular alkaline protease was isolated from surface water of latitude 75°25′N, and longitude 162°25′W in Chukchi sea, Arctic. The strain can grow at the temperature range from 7℃ to 30℃, and grow better at 30℃. It can not grow at 40℃. Keeping certain salinity concentration in medium is necessary for cell growth. It grows well in medium containing salinity concentration from 0.5% to 10% sodium chloride. Glucose, sucrose and soluble starch can be utilized by the strain, among which glucose is the optimal carbon source. Peptone is the optimal organic nitrogen source for cell growth and protease producing, and ammonium nitrate is the optimal inorganic nitrogen source. About 75 7% of total protease of the strain are extracellular enzyme. Optimal temperature for proteolytic activity is at 40℃. Protease of the strain keeps stable below 40℃, and shows high proteolytic activity within the pH range from 7 to 11.