The worldwide emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis threatens to make this disease incurable. Drug resistance mechanisms are only partially understood, and whether the current understanding of the genetic basis of drug resistance in M. tuberculosis is sufficiently comprehensive remains unclear. Here we sequenced and analyzed 161 isolates with a range of drug resistance profiles, discovering 72 new genes, 28 intergenic regions (IGRs), 11 nonsynonymous SNPs and 10 IGR SNPs with strong, consistent associations with drug resistance. On the basis of our examination of the dN/dS ratios of nonsynonymous to synonymous SNPs among the isolates, we suggest that the drug resistance-associated genes identified here likely contain essentially all the nonsynonymous SNPs that have arisen as a result of drug pressure in these isolates and should thus represent a near-complete set of drug resistance-associated genes for these isolates and antibiotics. Our work indicates that the genetic basis of drug resistance is more complex than previously anticipated and provides a strong foundation for elucidating unknown drug resistance mechanisms.

Lysophosphatidylcholines (lysoPCs) are a class of compounds that have a constant polar head, and fatty acyls of different chain lengths, position, degrees of saturation, and double bond location in human plasma. LysoPCs levels can be a clinical diagnostic indicator that reveals pathophysiological changes. In this work, a method was developed to discriminate between different types of lysoPCs using reversed phase ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, using mass spectrometry MS E . Isomeric lysoPCs were distinguished based on retention time and the peak intensity ratio of product ions, and 14 pairs of lysoPCs regioisomers were identified in human plasma. The plasma samples of 12 lung cancer patients and 12 healthy persons were collected and analyzed by principal component analysis to generate metabolic profiles of the identified lysoPCs. Both electrospray ionization ESI? and ESIresults showed that all lung cancer patients had the same five lysoPC metabolic abnormalities, specifically in sn-1 lyso16:0, sn-2 lysoPC 16:0, sn-1 lysoPC 18:0, sn-1 lysoPC 18:1 and sn-1 lysoPC 18:2. Thus, the function of isomers with different fatty acyl positions may be related to lung cancer, and this may help elucidate the mechanism of the disease.

Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.

e To explore the phenotypic and genotypic characterization of pyrazinamide (PZA) resistance among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates in Zhejiang province, a total of 274 MDR-TB isolates were collected. Drug susceptibility testing and spoligotyping were performed on all clinical isolates. In addition, the mutated features of PZA-resistant loci, including pncA and rpsA, were also analyzed by DNA sequencing. Our results showed that the prevalence of PZA resistance among MDR-TB strains in Zhejiang province was 43.07% and that PZA resistance was associated with concomitant resistance to streptomycin. The majority of PZA-resistant MDR-TB isolates belonged to the Beijing family. Mutations within pncA, not rpsA, constituted the primary mechanism of PZA resistance. Among 118 PZA-resistant isolates, 53 different mutations were observed in pncA, and most of them were point mutations. Compared with the phenotypic data, DNA sequencing of pncA has sensitivity and specificity of 77.97% and 96.79%, respectively. Analysis of pncA provided a robust tool for rapid detection of PZA drug resistance.

e Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(؊16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306 -497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.M ultidrug-resistant tuberculosis (MDR-TB) is attributed to an estimated 3.7% new cases and 20.2% previously treated cases of TB annually worldwide and is becoming a major threat to global public health (1). In China, the significantly high prevalence (5.7% new cases and 25.6% previously treated cases) of MDR-TB makes TB control especially challenging (2). Ethambutol (EMB) is an important first-line anti-TB drug routinely recommended for therapy of drug-resistant TB, including MDR-TB. Disturbingly, in some regions of China, substantial proportions (51.3% to 66.7%) of MDR-TB isolates demonstrated EMB resistance (3-5). Development of new rapid and reliable molecular methods for detecting drug resistance is essential to optimize treatment regimens, prevent treatment failure, and thus reduce the further spread of drug-resistant isolates. However, these molecular assays require precise knowledge of the genetic mutations associated with drug resistance. Prior studies indicated that the characteristics of resistance-associated mutations vary in different regions (6, 7). EMB acts against TB by inhibiting membrane-associated arabinosyl transferases encoded by the embCAB operon (including embC, embA, and embB), which are involved in the synthesis of cell wall arabinogalactan (8,9). Approximately 50% to 70% of EMB-resistant TB isolates harbor mutations in a relatively short fragment (codons 306 -497) in embB genes, with mutations occurring most frequently at codon 306 in embB (embB306), embB406, and embB497 (5,8,(10)(11)(12)(13). Sequence analysis of this fragment has been a tool for the rapid detection of EMB resistance. However, approximately one third of EMB-resistant isolates do not carry changes in this region and therefore are not detectable by using DNA sequencing (12,14). Although other mutations in the embCAB...

Lignocellulosic biomass-based ethanol is categorized as 2 nd generation bioethanol in the advanced biofuel portfolio. To make sound incentive policy proposals for the Chinese government and to develop guidance for research and development and industrialization of the technology, the paper reports careful techno-economic and sensitivity analyses performed to estimate the current competitiveness of the bioethanol and identify key components which have the greatest impact on its plant-gate price (PGP). Two models were developed for the research, including the Bioethanol PGP Assessment Model (BPAM) and the Feedstock Cost Estimation Model (FCEM). Results show that the PGP of the bioethanol ranges $4.68-$6.05/gal (9,550-12,356 yuan/t). The key components that contribute most to bioethanol PGP include the conversion rate of cellulose to glucose, the ratio of five-carbon sugars converted to ethanol, feedstock cost, and enzyme loading, etc. Lignocellulosic ethanol is currently unable to compete with fossil gasoline, therefore incentive policies are necessary to promote its development. It is suggested that the consumption tax be exempted, the value added tax (VAT) be refunded upon collection, and feed-in tariff for excess electricity (byproduct) be implemented to facilitate the OPEN ACCESSEnergies 2015, 8 4097 industrialization of the technology. A minimum direct subsidy of $1.20/gal EtOH (2,500 yuan/t EtOH) is also proposed for consideration.

Although the ecological function of dark septate endophytes (DSEs) is well studied, little is known about the responses of the host plant to DSEs obtained from other plants, especially under conditions of heavy metal stress. This study aimed to investigate how DSEs from a heavy-metal habitat affect non-host plants in cadmium (Cd) stress soils, which then provides a basis for the application of DSEs in the cultivation of different plant and soil remediation strategies for polluted ecosystems. We isolated and identified two species of DSE (Acrocalymma vagum and Scytalidium lignicola) inhabiting the roots of Ilex chinensis (host plant) which are grown in metal-polluted habitats. Then, the Cd stress tolerance of the DSEs was tested using a pure culture of which the Cd concentration has been adjusted. Subsequently, we examined the performance of non-host plants (Medicago sativa and Ammopiptanthus mongolicus) which were inoculated with DSEs under Cd stress in a growth chamber. The results indicated that the two DSEs could grow under Cd stress in vitro, even when not exhibiting high levels of tolerance to Cd. The superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), soluble protein, and melanin of the DSE fungi reached maximal levels at concentrations of 30-60 mg Cd/L, indicating the important preventive strategies adopted by the DSE fungi in environments contaminated by Cd. Despite a decreased biomass of DSE hyphae with enhanced Cd concentrations, the accumulation of Cd in the DSE hyphae tended to show an increasing trend. Both DSEs were effective colonizers of the non-host plants. A. vagum and S. lignicola inoculation significantly promoted the biomass and the root architecture of the two non-host plants under Cd stress. A. vagum inoculation increased the total nitrogen (TN) of A. mongolicus, whereas inoculation with S. lignicola significantly increased the organic carbon (OC) of M. sativa. In particular, the DSE inoculation significantly improved the accumulation of Cd in plant tissues under Cd stress, demonstrating a potential application in the bio-remediation of heavy-metalpollution areas. Our findings suggest that the DSE inoculation improved the root growth and nutrient absorption of non-host plants, altered the soil Cd concentration, and facilitated plant growth and survival under Cd stress. These results contribute to a better understanding of DSE-plant interactions in habitats contaminated by heavy metals.

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