Manihot esculenta Crantz) is unclear. We measured soluble sugar concentrations of nine plant species with differing levels of drought tolerance and 41 cassava varieties using high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD). Significantly high amounts of trehalose were identified in drought-tolerant crops cassava, Jatropha curcas, and castor bean (Ricinus communis). All cassava varieties tested contained high amounts of trehalose, although their concentrations varied from 0.23 to 1.29 mg·g−1 fresh weight (FW), and the trehalose level was highly correlated with dehydration stress tolerance of detached leaves of the varieties. Moreover, the trehalose concentrations in cassava leaves increased 2.3–5.5 folds in response to osmotic stress simulated by 20% PEG 6000. Through database mining, 24 trehalose pathway genes, including 12 trehalose-6-phosphate synthases (TPS), 10 trehalose-6-phosphate phosphatases (TPP), and two trehalases were identified in cassava. Phylogenetic analysis indicated that there were four cassava TPS genes (MeTPS1–4) that were orthologous to the solely active TPS gene (AtTPS1 and OsTPS1) in Arabidopsis and rice, and a new TPP subfamily was identified in cassava, suggesting that the trehalose biosynthesis activities in cassava had potentially been enhanced in evolutionary history. RNA-seq analysis indicated that MeTPS1 was expressed at constitutionally high level before and after osmotic stress, while other trehalose pathway genes were either up-regulated or down-regulated, which may explain why cassava accumulated high level of trehalose under normal conditions. MeTPS1 was then transformed into tobacco (Nicotiana benthamiana). Results indicated that transgenic tobacco lines accumulated significant level of trehalose and possessed improved drought stress tolerance. In conclusion, cassava accumulated significantly high amount of trehalose under normal conditions due to multiplied trehalose biosynthesis gene families and constant expression of the active MeTPS1 gene. High levels of trehalose subsequently contributed to high drought stress tolerance.Trehalose is a nonreducing α,α-1,1-disaccharide in a wide range of organisms, and has diverse biological functions that range from serving as an energy source to acting as a protective/signal sugar. However, significant amounts of trehalose have rarely been detected in higher plants, and the function of trehalose in the drought-tolerant crop cassava ( Manihot esculenta Crantz) is a perennial dicotyledonous crop belonging to the family of Euphorbiaceae. The starchy tuberous roots have global importance as food and feed, and are stable food for nearly 700 million people in the tropical and subtropical regions [Cassava  under dehydration condition [Arabidopsis thaliana in trace amount by using validamycin A, a trehalase inhibitor in the culture medium [−1 fresh weight (FW) of trehalose in seven-day-old plantlets of wild type Arabidopsis [Oryza sativa) with a level of approximately 0.015 mg·g−1 FW [Nicotiana tabacum) with a level of 0.001 to 0.002 mg·g−1 FW [Some resurrection plants are able to accumulate trehalose in concentrations as high as 36 mg·gondition ,21. Thesondition ,23. Howeondition . Endogene medium . A laterbidopsis . Trehalog·g−1 FW  and wildg·g−1 FW . Becauseg·g−1 FW ,30. Howeg·g−1 FW , and theg·g−1 FW ,33,34.TPS) and trehalose-6-phosphate phosphatase (TPP) gene families and constant expression of a key active TPS gene.In this paper, we report unexpected high levels of trehalose in all tested cassava varieties, which is highly correlated with dehydration stress, and phylogenetic and transcriptional analysis of trehalose biosynthesis gene family. The high level accumulation of trehalose in cassava may be explained by multiplied trehalose-6-phosphate synthase (High-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD) was used to measure soluble sugar contents in cassava. Preliminary results showed that soluble sugars were well separated using our protocols and parameters, with retention times of approximately 4.098, 4.520, 5.530, and 6.687 min for fructose, glucose, sucrose, and trehalose, respectively, in leaf samples A. Subseq−1 FW, respectively. At 1:00 p.m., trehalose contents reached the highest level. At 6:00 p.m., trehalose contents declined to similar level as in the morning  was detected in banana, tobacco, and sweet potato leaves  between trehalose concentration and water retaining capacity of detached leaves  contained similar high levels of trehalose as varieties SC5 and SC124 , althoug−1 FW A. Regresd leaves A. The re−1 FW (p < 0.05)  B–D. ThesTo investigate the response of trehalose level to osmotic stress, cassava plants were treated with 20% PEG 6000 solution in the pots. Trehalose levels in leaves displayed the most rapid and significant increase, which were 2.3 and 5.5 folds of the control level, respectively, in 12 and 24 h of treatments. Trehalose contents in stems increased 2.5 folds in 24 h. However, trehalose contents in root remained almost unchanged A. The otTPS) genes, ten putative trehalose-6-phosphate phosphatase (TPP) genes, and two trehalase (TRE) genes from the cassava genome databases  genes  were identified. Cassava4.1_004491m.g encoded a full-length CDS and was designated as TPS, TPP, and TRE genes were transcribed in cassava leaves and root, except for MeTPP2 and MeTPP9, which were absent in the transcriptomes of leaves and root  were the major TPS genes expressed in the control samples of leaves including full expanded leaf (FEL) that was active in photosynthesis, bottom leaf (BL) that was at the bottom of canopy and usually aged, and folded leaf (FL) that was newly emerged and not expanded, while MeTPS9, MeTPS12, MeTPS11, and MeTPS5 were highly expressed in the control samples of root (RT). The expression level of MeTPS1 was almost constant and high before and after treatment with 20% PEG, suggesting that MeTPS1 served as a housekeeping TPS enzyme and may explain the high trehalose level in cassava under normal conditions. The other supposed active TPS genes MeTPS2 and MeTPS4 had lower expression levels under normal conditions compared to MeTPS1, but were up-regulated in functional leaf after treated with PEG for 3 h. Interestingly, MeTPS8, the most abundant TPS in FEL0 and BL0 , the expression level of MeTRE2 was dominant over MeTRE1, and it was up-regulated in FEL24 and FL24. The changes of the expression levels of MeTRE1 in leaves were small  expression was most abundant in root compared to leaves , and the two trehalase genes re small .MeTPS1 seemed to be the most important TPS in cassava according to the phylogenetic and RNA-seq data. However, MeTPS1, unlike other Class I TPS , used an aspartic acid (D121) instead of a conserved glycine residue as the first binding site to the ribose moiety in UDPG  was constructed under the control of the CaMV 35S promoter (Nicotiana benthamiana Domin) using Agrobacterium tumefaciens GV3103. The presence of MeTPS1 in tobacco genome was confirmed by PCR  into the Arabidopsis genome, the trehalose concentration was increased to 0.026 mg·g−1 FW, and then dehydration tolerance was enhanced [TPS gene accumulated a maximum of 0.015 mg·g−1 FW trehalose, and showed greater drought resistance compared to the wild-type [−1 FW trehalose was also enhanced in abiotic stress tolerance, including drought [Jatropha, and castor bean were much higher than the levels in transgenic Arabidopsis, tobacco, and rice, supporting the role of trehalose in natural drought tolerance in cassava, Jatropha, and castor bean.The role of trehalose in drought stress tolerance in higher plants has been demonstrated in many species ,49,50,51g·g−1 FW . When trild-type . Transge drought . Thus, t drought . The levJatropha, and castor bean were below the levels in some resurrection plants, even if trehalose level was increased up to approximately 2.6 mg·g−1 FW upon osmotic stress by PEG. In contrast, some resurrection plants, such as Myrothamnus flabellifolius or Selaginella lepidophylla, easily accumulated up to 10 mg·g−1 FW [However, the levels of trehalose in cassava, g·g−1 FW ,52. TherWe observed circadian changes of trehalose level in cassava leaves C, and trWe measured the soluble sugar contents in 41 cassava varieties using HPLC-ELSD. High levels of trehalose were recorded in all cassava varieties . It is dHowever, we managed to prove the importance of trehalose in dehydration tolerance by analyzing the correlation between soluble sugar contents and water retaining capacities in leaves of different varieties. Leaf morphology and surface area are diversified among cassava varieties. Our preliminary experiments indicated that leaf morphology and size had notable influence on water loss of detached leaves, which is consistent with previous research in other crops ,56. To fPEG 6000 has been widely used to simulate drought stress and stimulates a series of physiological changes . We usedTPS genes, ten putative TPP genes, and two TRE genes from the cassava genome databases.The biosynthesis pathway of trehalose involves a two-step reaction in plants ,52,57. IArabidopsis, paddy rice, and maize were classified into two clades [Arabidopsis and paddy rice, respectively, while the remaining TPS proteins lack TPS activity [Arabidopsis  [Arabidopsis were classified into two clades [Arabidopsis, rice, and maize, and formed an independent clade, designated as Clade C. Moreover, the three motifs required for TPP activity in TPP proteins of cassava were more conserved than those in Arabidopsis, rice, and maize  . The TPPo clades ,58. Howend maize , especiaTaken together, the gene families in cassava trehalose biosynthesis pathway are special, and the biosynthesis of trehalose in cassava may have been enhanced in the evolutionary history by growing in arid and marginal areas.Cassava varieties SC124, SC5, and 39 other randomly selected varieties were obtained from the Institute of Tropical Crops Genetic Resources, Chinese Academy of Tropical Agricultural Sciences , and planted in a field at the Institute of Tropical Bioscience and Biotechnology, CATAS located in Haikou, Hainan Province.Manihot esculenta, variety SC124), Jatropha curcas variety 18, rubber tree , castor bean (Ricinus communis), tobacco (Nicotiana benthamiana), sweet potato (Ipomoea batatas), paddy rice (Oryza sativa) variety Teyou458, Cavendish banana (variety Williams), and sugarcane  were grown in the field of Haikou campus, Chinese Academy of Tropical Agricultural Sciences (CATAS). Healthy mature leaves were randomly collected from six-month-old plants of cassava, Jatropha, rubber tree, banana, sugarcane, and castor bean, and/or from three-month-old plants of tobacco, potato, and paddy rice in the morning, when the weather was fine and the temperature was about 30 °C. The samples were collected in three replicates and stored at −80 °C until use.To study soluble sugars in different plant species, cassava , three leaves  were collected from each plant. Leaf samples of all 39 varieties were collected in the morning, when the weather was fine and the temperature was about 30 °C. The samples were collected in three replicates and wrapped in aluminum foil, then rapidly frozen in liquid nitrogen, and stored at −80 °C prior to analysis.To study the distribution of trehalose in cassava plant, leaves, stems, and roots were collected from three-month-old plants of cassava variety SC5; To study the circadian oscillation of trehalose contents, leaves were collected from three-month-old plants of cassava varieties SC124 and SC5 at 8:00 a.m., 1:00 p.m., and 6:00 p.m. The samples were collected in three replicates and stored at −80 °C as described above.g for 10 min. The supernatant was transferred to a clean tube. The pellet was extracted twice with 2 mL hot distilled water. The supernatants were combined and dried at 80 °C. The residue was dissolved in 50% (v/v) acetonitrile to a final volume of 10 mL, and stored at 4 °C until use. The samples were filtered with 0.22 µm MillexTM membrane  three times before loading into the HPLC column.Plant samples were ground in liquid nitrogen. Approximately 1 g powder was transferred into a 10 mL centrifuge tube, and 5 mL of hot distilled water was immediately added to the powder. The tubes were boiled at 100 °C for 15 min, and cooled to room temperature, and then centrifuged at 10,000× v/v) acetonitrile to a volume of 50 mL. These solutions were diluted to prepare 0.1, 0.2, 0.5, 1.0, 2.0, and 4.0 mg·mL−1 standard solutions, and stored at 4 °C until use. To prepare the standard curve for identification of sucrose, 1, 2, 5, 10, 15, and 20 mg·mL−1 standard solutions were prepared. Ten microliters of each standard solution was loaded onto a Waters e2695 High Performance Liquid Chromatography instrument fitted with Empower software . The column was an XBridge-NH2 , and the column temperature was set to 25 °C. The mobile phase was acetonitrile: water  supplemented with 0.1% (w/v) NH4OH, and the flow rate was set to 1 mL·min−1. The eluate was detected and measured with an Alltech 3300 Evaporative Light Scattering Detector . The ELSD drift-tube temperature was set to 85 °C, and the nitrogen flow velocity was 2 L·min−1. The regression equations and the correlation coefficients (2R) for the sugar quantity and the peak area were calculated using Excel 2007  Co., Ltd., Shanghai, China). To measure the concentrations of soluble sugars in the experimental samples, 10 µL of each sample was loaded onto the column and analyzed as described above. The concentrations of fructose, glucose, sucrose, and trehalose were calculated according to the regression equations.To prepare the standard curves for identification of glucose, fructose, and trehalose, 0.2 g standard substances  were dissolved in 50% , and placed separately on a long bench to allow equal atmospheric exposure. Room temperature was set to 25 °C, and humidity was 75%. The leaves were weighed at 30 min intervals (Wi) until withered. The leaves were finally dried to constant weight (Wt) in a constant-temperature dryer DKM812C  at 80 °C. Water retaining capacity was calculated as relative water content in the detached leaves at 3 h after detachment, using the equation (Wi − Wt)/(Wb − Wt) × 100%. The experiments were replicated three times. The significance of the correlations between soluble sugar contents and water retaining capacities of detached leaves was analyzed using IBM SPSS Statistics Version 24.0, and the correlation curves in ftp://ftp.jgi-psf.org/pub/compgen/phytozome/v9.0/Mesculenta/). A total of 25 putative trehalose pathway genes, including 12 putative trehalose-6-phosphate synthase (TPS) genes, ten putative trehalose-6-phosphate phosphatase (TPP) genes, and three trehalase (TRE) genes were identified from the cassava genome databases  and Gramene (available at: http://ensemble.gramene.org) databases. The nomenclature for the genes in Arabidopsis, rice, and maize was adopted from previous publications [The gene family members involved in trehalose biosynthesis and hydrolysis were mined in the Phytozome cassava genome database Version 4.1 accessed on 14 May 2015 , total RNA was extracted from leaves of cassava variety SC8 using a Plant RNA Extraction Kit . mRNA was purified using a Dynabeads mRNA purification kit . First-strand cDNA was synthesized using a 5′ Full RACE kit with TAP (TaKaRa) accordingly to the manufacturer’s protocol. Two rounds of RACE PCR were performed using a gene specific primer and a universal primer each provided in the kit. For the first round PCR, the MeTRE2 gene specific primer was 5′-CAATTCGGGTTGCTTCCTGACTTCG-3′ that was designed with MacVector 14.0 . For the second round PCR, the MeTRE2 gene specific primer was 5′-CTTAGGCAAGAACCCATCAGGCTC-3′. The PCR reactions were carried out using the parameters suggested by the manufacturer.To determine 5′ end of a truncated trehalase gene The influence of PEG-simulated drought stress on cassava was studied previously, and a large transcriptome dataset of leaves  and roots before and after 20% PEG treatment was generated using RNA-seq method , and a nMeTPS1 coding sequence was amplified with primers 5′-CGGGATCCATGCCTGGAAACCAGTATAACG-3′ and 5′-GCGTCGACTCAAGAAGATGCCCTGGCTAG-3′, and cloned in the pEASY-T1 Simple cloning vector . The plasmid with correct sequence was digested with Bam HI and Sal I, and ligated into pCAMBIA2300-35S-OCS [MeTPS1. After confirmation by sequencing, the vector was transformed into Agrobacterium tumerfaciens GV3103 via electroporation. Seeds of Nicotiana benthamiana were sterilized for 30 s in 70% (v/v) ethanol, and then 5 min in 5% bleach, followed by rinsing with sterile distilled water for three times. The seeds were then sown on half strength semi-solid MS medium supplemented with 30 g·L−1 sucrose and 6 g·L−1 agar . Plants were incubated at 16 h light per day with light intensity of 3000 Lux. Twenty-day-old seedlings were used for transformation using leaf discs as explants according to previously described [−1 6-benzylaminopurine (6-BA), 0.5 mg·L−1 indole-3-acetic acid (IAA), 10 mg·L−1 hygromycin, and 500 mg·L−1 carbenicillin. The explants were transferred to fresh selection medium once every two weeks. Shoots resistant to hygromycin were transferred to the rooting medium (MS supplemented with 15 mg·L−1 hygromycin and 250 mg·L−1 carbenicillin). Plants with high quality roots were used to test the presence of foreign gene by PCR.-35S-OCS , resultiescribed . The infThe putative transgenic plants were transplanted in a mixed soil (50% soil and 50% coconut husk) and incubated in the green house until they flowered and set seeds (T1). The T1 seeds were then sterilized and grown on selection medium again to screen for plants that inherited the foreign gene. The selected plants were grown in green house until seeds of T2 generation were set. The T2 seeds were sterilized and sown in selection medium, and the lines with 100% transgenic seeds were considered homozygous and were kept for further analysis.MeTPS1 gene-specific primers 5′-ATGCCTGGAAACCAGTATAACG-3′ and 5′-TCAAGAAGATGCCCTGGCTAG-3′. Amplifications were performed using a T-1 Thermocycler  at 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, and a final extension at 72 °C for 5 min. The PCR products were visualized with a Tanon 4100 Gel Imaging System  after electrophoresis.Genomic DNA was isolated from transgenic and wild type tobacco plants using a Universal Genomic DNA Extraction Kit . Total RNA was extracted according to a Plant RNA Extraction Kit . The quality of total RNA was evaluated with a 2100 Bioanalyzer . The mRNA was reverse transcribed to cDNA using a Superscript III First-Strand Synthesis System . PCR was performed using


Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane ( Sugarcane, accumulating large quantities of sucrose in stem tissues, is one of the most important sugar crops and has also been developed into an important energy crop , while the formula for Badila is Copies/genome = 10Xt/[25 × 10−9 g × 6.02 × 1023/(7880 (M bp) × 106 × 660)]. Then, an estimated copy number was generated for each gene , one S. spontaneum SES208 and several different genotypes of S. officinarum including Black Cheribon , Black Cheribon , Loethers, LA Purple and Crystalina were chosen for further testing. In addition, modern sugarcane varieties Q117 and YCE05-179 were included, of which Q117 was one of the sugarcane varieties used in the study by Casu et al. (2012) and YCE05-179 was a newly derived sugarcane line with outstanding yield-related traits and disease resistance. The estimated copy numbers for these sugarcane genotypes were listed in P4H, APRT and CYC were sugarcane-specific in low copy number through several sugarcane cultivars.Due to the exact genome size and ploidy of modern sugarcane being unknown, it is necessary to verify the stability of selected primers P4H-3, APRT-2 and CYC-2 in more sugarcane genotypes with a different genetic background. Considering the ploidy and chromosome composition of 2.2.3.P4H, APRT and CYC, in a uniform background as well as to overcome the problem of suitable positive plant material and avoid differences of DNA extraction, a multi-target reference plasmid, carrying the primer sequences  corresponding to the above three genes, was constructed and assigned as pMD18-PAC with a size of 3005 bp. The three standard curves, which were suitable for analyzing the dosages of P4H, APRT and CYC genes, were established individually based on the 10-fold dilution with a dosage range of 1.0 × 106–1.0 × 102 copies per μL. The estimated copy numbers of P4H, APRT and CYC genes were about 3, 2 and 4 in S. spp. hybrid ROC22, and were 2, 2 and 3 in S. officinarum Badila, respectively. Using APRT gene as the internal control, the relative abundance of P4H/APRT, APRT/APRT and CYC/APRT were 1.5, 1.0 and 2.0 in ROC22, respectively. Similarly, the corresponding relative abundance values present in S. officinarum Badila were 1.0, 1.0 and 1.5, respectively  were close to 1.0 for all three genes P4H, APRT and CYC (Ct). The PCR efficiencies (E) of P4H, APRT and CYC were all also close to 1.0 . At the same time, both P4H and APRT genes showed even lower copies or less dosages compared to CYC in the genomes of ROC22 and Badila, while the relative abundance values of P4H and CYC were higher than that of APRT. These results indicated that the APRT gene was a specific amplicon within the detection using the primer pairs APRT-2, and thus this gene was assumed to be the most suitable one as an endogenous reference gene in sugarcane.For the technical performance of qPCR, the mean squares of coefficient of determination , as all the primers were designed from sugarcane ESTs; Thirdly, a practically useful plasmid was constructed with multi-target genes for qPCR, which resulted in the detection under the same background. Therefore, this study should serve as a solid foundation for establishing an efficient technique to estimate copy numbers of target genes in sugarcane.The innovative points in this study are: Firstly, an endogenous reference gene ScMV-CP transgenic sugarcane [GAN transgenic sugarcane [Hs 1 pro-1 of nematode-resistant transgenic sugarcane [Bt transgenic sugarcane [Since the first GM plant, an antibiotic-resistant tobacco, was produced in 1982 , an incrugarcane , GAN traugarcane , Hs 1 prugarcane , and Bt ugarcane . In addiP4H, APRT, ENOL, CYC, TST and PRR, for their suitability as endogenous reference genes for transgenic detection in sugarcane. We began on the basis of preliminary estimations, by comparing copy numbers of the above six genes among eight different sugarcane genotypes. We then selected the genes with low copy numbers to construct a plasmid with multi-target reference genes for further tests. With the development of new GM crops, more and more reference plasmids have been employed simultaneously to detect multiple foreign genes [APRT, with the feature of low copies, can be considered as a reliable reference one among various sugarcane genotypes as the primer pairs APRT-2 has been tested in several sugarcane varieties with different genetic background.The advances in PCR instrumentation and fluorescence chemistry have made it possible to precisely quantify the specific amplification products . Comparegn genes . Based ogn genes ,46. The 4.4.1.Saccharum spp. hybrids ROC22 and YCE05-179, S. officinarum Badila and LA Purple and S. spontaneum SES208 were collected from the Key Laboratory of Sugarcane Biology and Genetic Breeding of the Ministry of Agriculture; Leaf samples of sugarcane variety Q117, S. officinarum Black Cheribon , Black Cheribon , Loethers and Crystalina were provided by the National Sugarcane Germplasm Nursery, Yunnan, China.Leaf samples of sugarcane cultivars 4.2.Taq from TaKaRa Biotechnology Co., Ltd., ; competent cell DH5α and DNA Markers from TianGen Biotechnology Co., Ltd., .SYBR Green Master (ROX) was purchased from Roche ; Plasmid Mini Kit and Gel Extraction Kit from Omega Bio-Tek ; pMD 18-T vector and Ex PCR Tubes, PCR Strip Tubes, Optical 96-Well Reaction Plate and other PCR related consumables were from Applied Biosystems  unless specified.4.3.et al. [Genomic DNA was extracted according to the CTAB-based protocol described by Paterson et al. . DNA con4.4.P4H, APRT, ENOL, CYC, TST and PRR, with source DNA sequences from Sorghum bicolor reported by Casu et al. [Six primer pairs from putative reference genes, u et al. , were se4.5.Taq polymerase, 1.0 μL (10 μmol/L) of each gene-specific forward and reverse primer and 17.375 μL ddH2O. PCR was performed under the following conditions: 98 °C for 30 s (1 cycle); 98 °C for 15 s, 60 °C for 15 s; 72 °C for 15 s (35 cycles). All PCR products were separated by electrophoresis on a 3.0% agarose gel with an Amersham Pharmacia EPS301 electrophoresis apparatus  in 1× TAE buffer. A Bio-Rad Gel imaging system  was used for observation of the electrophoresis products.DNA fragments were amplified by PCR based on 25 ng genomic DNA of sugarcane cultivar ROC22 or Badila. A final volume of 25 μL was used, containing 2.5 μL 10× PCR Buffer, 2.0 μL (2.5 mM) of dNTPs, 0.125 μL (5 U/μL) Ex 4.6.E. coli (DH5α) and 100 μL of transformed culture was spread onto LB plates containing ampicillin (50 μg/mL), X-gal (20 mg/mL) and IPTG (50 mg/mL). Transformed (white) colonies were picked up and processed for plasmid isolation and sequencing. Plasmids were extracted using a Plasmid Omega Bio-Tek Mini kit  according to the manufacturer’s instructions.PCR-amplified fragments were purified with a gel purification kit from Omega Bio-Tek  and cloned into the pMD18-T vector from TaKaRa Biotechnology Co., Ltd. , according to the manufacturer’s instructions. The recombinant plasmid was transformed into competent 4.7.Plasmid DNA concentration was estimated by measuring the absorbance at 260 nm as described above. Plasmid copy number was calculated according to the following formula:4.8.All qPCR assays were performed on an ABI PRISM 7500 Sequence Detection System  in 25 μL reaction volume containing 12.5 μL of 2× SYBR Green PCR Master mix , 1.0 μL of diluted genomic DNA (25 ng) and 1.0 μL (10 μmol/L) each of a gene-specific forward and reverse primer. The following standard PCR reaction conditions were used for all transcripts: 50 °C 2 min; 95 °C 10 min; 45 cycles of 95 °C 15 s, 60 °C 1 min; 1 cycle of 95 °C 15 s, 60 °C 15 s, 95 °C 15 s. The last cycle provided dissociation curves for each sample, allowing for assessment of the specificity of amplification. For each sample test, each PCR reaction had three replicates and the experiment was repeated three times.4.9.8 to 1.0 × 101 copies/μL with each step differing by 10-fold. It’s necessary to suspend well by pipetting 30 times when diluting. Assays of qPCR were performed using 25 ng/μL (working concentration) DNA and water as control, with three replicates. After reaction, the values of threshold cycles are achieved. Each standard curve is established by plotting the threshold cycle (Ct) on the Y-axis and the natural log of concentration (copies/μL) on the X-axis, and the equation y = k × x + b, coefficient of determination (R2) and percentage of variance in copy numbers were achieved.Each of the purified plasmid was diluted with sterile deionized water to obtain a standard series from 1.0 × 104.10.E) can be calculated according to the equation as follow [The copy number for putative reference gene in sugarcane genome DNA can be calculated against its established standard curve. After reaction, the values of threshold cycles are achieved. From the slope of a standard curve, PCR amplification efficiency (s follow :Xt) of each endogenous reference gene is calculated by relating the Ct value (Yt) to its corresponding standard curve, then the single cell copy number (n) of each endogenous reference gene in the sugarcane samples can be calculated by the following formula:The total copy number (10 −9 g is the amount of DNA template (measured by UV absorption) used in each qPCR reaction system, 6.02 × 1023 is Avogadro’s constant, the genome size of sugarcane can be determined by flow cytometry [MW) of a DNA base pair is 660 daltons.Notes: 25 × 10ytometry , and ave4.11.6 to 1.0 × 102 copies of reference plasmids, and the validated primer pairs in the above experiments were employed for qPCR with templates of 25 ng/μL sugarcane genomic DNA of ROC22 and Badila. In addition, one of the above genes was used as the internal control to calculate the relative abundance values to compare the gene dosage [In order to create a reference plasmid containing all the test sequences corresponding to the putative reference genes for further test in the same background, all the tested sequences were synthesized by Sangon Biotech Co., Ltd.  and constructed onto the same vector to obtain a multi-target reference plasmid . The stae dosage . The rel5.APRT, as an endogenous reference gene, was identified from a set of six putative potential “single copy” ones, P4H, APRT, ENOL, CYC, TST and PRR in sugarcane. The primer pair APRT-2, corresponding to the APRT gene, was screened out from nineteen initial primer pairs that corresponded to the above six reference genes. The selected gene of APRT was considered as “single copy” based on: (1) low heterogeneity among different sugarcane species; and (2) low copy number. This conclusion was derived from testing on all eight sugarcane genotypes with different genetic background including S. officinarum, S. spontaneum and the modern varieties (S. spp. hybrids). In addition, the copy number was calculated based on the comparison to a multi-target gene plasmid standard curve. Therefore, APRT can be used as a reliable endogenous reference gene; when combined with the developed primer pair, it is suitable for transgenic sugarcane precise detection based on absolute quantification using qPCR, which hopefully will accelerate sugarcane genetic engineering and the commercialization of GM sugarcane.The present study succeeded in selection of endogenous reference genes for transgenic sugarcane detection. The


Glycine max (L.) Merr.) isoflavone is important for human health and plant defense system. To identify novel quantitative trait loci (QTL) and epistatic QTL underlying isoflavone content in soybean, F5:6, F5:7 and F5:8 populations of 130 recombinant inbred (RI) lines, derived from the cross of soybean cultivar ‘Zhong Dou 27′ (high isoflavone) and ‘Jiu Nong 20′ (low isoflavone), were analyzed with 95 new SSR markers. A new linkage map including 194 SSR markers and covering 2,312 cM with mean distance of about 12 cM between markers was constructed. Thirty four QTL for both individual and total seed isoflavone contents of soybean were identified. Six, seven, ten and eleven QTL were associated with daidzein (DZ), glycitein (GC), genistein (GT) and total isoflavone (TI), respectively. Of them 23 QTL were newly identified. The qTIF_1 between Satt423 and Satt569 shared the same marker Satt569 with qDZF_2, qGTF_1 and qTIF_2. The qGTD2_1 between Satt186 and Satt226 was detected in four environments and explained 3.41%-10.98% of the phenotypic variation. The qGTA2_1, overlapped with qGCA2_1 and detected in four environments, was close to the previously identified major QTL for GT, which were responsible for large a effects. QTL  between Satt144-Satt569 were either clustered or pleiotropic. The qGCM_1, qGTM_1 and qTIM_1 between Satt540-Sat_244 explained 2.02%–9.12% of the phenotypic variation over six environments. Moreover, the qGCE_1 overlapped with qGTE_1 and qTIE_1, the qTIH_2 overlapped with qGTH_1, qGCI_1 overlapped with qDZI_1, qTIL_1 overlapped with qGTL_1, and qTIO_1 overlapped with qGTO_1. In this study, some of unstable QTL were detected in different environments, which were due to weak expression of QTL, QTL by environment interaction in the opposite direction to a effects, and/or epistasis. The markers identified in multi-environments in this study could be applied in the selection of soybean cultivars for higher isoflavone content and in the map-based gene cloning.Soybean ( Soy food is considered as the functional food because it contains health beneficial molecules such as isoflavone –2. HumanTraditional methods of genetic improvement of quantitative traits were mainly dependent on phenotypic information , which w2 value). Major QTL will account for a relatively large amount of R2 value (R2 > 10%), while minor QTL will usually account for a relatively small amount of R2 value (R2 < 10%) [An individual QTL is described as ‘major’ or ‘minor’ is based on the proportion of the phenotypic variation explained by a QTL (based on the R2 < 10%) . Sometim2 < 10%) . BecauseTo boost QTL detection, appropriate crosses need to be selected to generate sufficient genetic variations in genomic level . AlthougOver fifty QTLs underlying individual and/or individual and total isoflavone content have been reported , 31–37. Epitasis is also thought to contribute to isoflavone variation . The resZeng et al.  identifi5:6, F5:7, F5:8 recombinant inbred lines (RIL) that were advanced by single-seed-descent from the cross between ‘Zhong Dou 27′  and ‘Jiu Nong 20′ . ‘Zhong Dou 27′ contained high individual and total isoflavone (TI) contents , whereas ‘Jiu Nong 20′ had low individual and TI contents . The 130 RILs of F5:6, F5:7, F5:8 and parents were planted at Harbin, Hulan and Suihua in 2005, 2006 and 2007, respectively [The mapping populations of 130 Fhttp://www.soybease.org), were used to detect polymorphisms. The PCR reactions were 94°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 52°C, 30 s at 72°C and 5 min at 72°C after the last cycle. The amplified PCR products were mixed with loading buffer and denatured for 5 min at 94°C and then kept on ice for 5 min. The denatured PCR products were separated on 6% (w/v) denaturing polyacrylamide gel and visualized by silver staining [Total DNA of the parents and each RI line were isolated from dried leaf tissues by CTAB method . A totalstaining . The polstaining .P≤ 0.05). The genetic linkage map was constructed using Mapchart 2.1 [Polymorphic markers were identified and mapped on the 20 linkage groups by Mapmaker 3.0b with the Kosambi mapping function . WinQTLChart 2.1 . QTL genhart 2.1 . The nomij − Tj / Sj = λ1ζi1τj1 + λ2ζi2τj2 + εij, where Tij was the mean value of QTL i for environment j; Tj was the mean value of environment j over all QTL, Sj was the standard deviation of environment j among QTL mean; ζi1 and ζi2 were the PC1 (first principle component) and PC2 (second principle component) scores respectively, for QTL mean i; τj1 and τj2 were the PC1 and PC2 scores respectively, for environment j; and εij was the residual of the model associated with QTL i, challenged with environment j.GGE Bioplot methodology  was emplIn this study, a total of 500 new SSR markers were used to detect polymorphisms between the two parents, 95 of which were integrated into the linkage map constructed by Zeng et al . This liIn this study, the identification of QTL was based on multi-environmental phenotypic data of Zeng et al. . Thirty Three QTL  associated with DZ, GT and TI were located on Gm13 (LG F) between Satt144-Satt569. They explained 1.87%–17.03% of the phenotypic variation in seven environments over three years. Another three QTL associated with GC, GT and TI were located on GM07 (LG M) between Satt540-Sat_244. They explained 2.02%–9.12% of the phenotypic variation across seven environments.Among the 34 QTL, 23 QTL were newly identified. Most of them were detected in one or two environments and explained 1.43%-17.05% of the phenotypic variation. The qTIF_1 in the interval between Satt423 and Satt569 shared the same marker Satt569 with qDZF_2, qGTF_1 and qTIF_2. The qGTD2_1 between Satt186 and Satt226 was detected in four environments and explained 3.41%–10.98% of the phenotypic variation. The qGTA2_1 was detected in four environments, which shared the same marker interval of Sat_040-Satt233 with qGCA2_1.Interestingly, six pairs of QTL overlapped with each other and shared the same marker interval , Fig. 1.a) and/or additive × environment interaction effect (ae) at certain environments  associated with DZ, one QTL (qGCI_1) associated with GC, two QTL  associated with GT and three QTL  associated with TI, contributed to the allele that decreased individual and total isoflavone through significant a effects, respectively.Six, seven, ten and eleven QTL associated with DZ, GC, GT and TI respectively, had additive main effect  associated with DZ, four QTL  associated with GC, four QTL  associated with GC and four QTL  associated with TI, had only significant ae effects rather than significant a effects. Other QTL in seven environments had both significant a effects and significant ae effects.The impact of QTL aa effects in different environments, respectively. One, one, one and two epistatic pairs of QTL decreased DZ, GC, GT and TI content, respectively, through significant aa effects in different environments  was an important component of QTL × environment (QE) interaction effects. One QTL and one pairs of QTL were found with only epistatic effects (aa), which was associated with DZ and TI content. Two, four, two and four pairs of QTL were found with only epistatic effects (aa), which was associated with DZ, GC, GT and TI content, respectively.The epistasis × environment interaction effect  among seven environments in our previous report . Meng eta effects (a effects are more likely to be stable across multiple environments. Most of the QTL discussed above with higher a effects (Significant at P = 0.01) were stable across at least three environments Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Long program/erase endurance and data retention at high temperature under low-current operation are also reported in published literature. So far, bilayered TaOx with inert electrodes (Pt and/or Ir) or single layer TaOx with semi-reactive electrodes (W and Ti/W or Ta/Pt) is proposed for real RRAM applications. It is found that the memory characteristics at current compliance (CC) of 80 μA is acceptable for real application; however, data are becoming worst at CC of 10 μA. Therefore, it is very challenging to reduce the operation current (few microampere) of the RRAM devices. This study investigates the switching mode, mechanism, and performance of low-current operated TaOx-based devices as compared to other RRAM devices. This topical review will not only help for application of TaOx-based nanoscale RRAM devices but also encourage researcher to overcome the challenges in the future production.Resistive switching memories (RRAMs) are attractive for replacement of conventional flash in the future. Although different switching materials have been reported; however, low-current operated devices (<100 μA) are necessary for productive RRAM applications. Therefore, TaO Generally, it needs high voltage for program and erase operations (>׀10 ׀V) [15 cycles), these memories show insufficient scalability [2/Cu structure. Ultra-high-speed operation of RRAM using atomic layer deposited HfO2 switching material is reported by Lee et al. [12 cycles is also demonstrated in TaOx-based crossbar device [x-based RRAM devices have been also reported extensively in the literature and shown good resistive switching performance. It is expected that this TaOx-based RRAM device has strong potential for production in near future. However, the TaOx-based RRAM devices with prospective and challenges have not been reviewed in literature yet.Semiconductor memory is an essential component of today’s electronic systems. It is used in any equipment that uses a processor such as computers, smart phones, tablets, digital cameras, entertainment devices, global positioning systems, automotive systems, etc. Memories constituted 20% of the semiconductor market for the last 30 years and are expected to increase in the coming years . General>׀10 ׀V) , magneti>׀10 ׀V) , phase-c>׀10 ׀V) , and res>׀10 ׀V) -25. The >׀10 ׀V) . All thelability . Moreovelability . In prinlability  has demoe et al. , where ae et al.  also deme et al.  requiredr device . Other ix-based devices as compared to other RRAMs in literature. Long program/erase endurance and data retention of >85°C with high yield have a greater prospective of TaOx-based nanoscale RRAM devices; however, lower current (few microampere) operation is very challenging for practical application, which is reviewed in detail here.This topical review investigates the switching mode, mechanism, and performances of the TaOResistance switching effect was first reported by Hickmott in 1962  and had The resistance of a RRAM device can be modulated in two ways as shown by the current/voltage (I-V) curves in Figure Generally, depending on the conduction path, the switching mechanism can be classified as (1) filamentary-type and (2) interface-type, as shown in Figure + ions generated from metallic Cu due to anodic dissolution. These ions then diffused through the solid electrolyte due to electric field and reached to the BE where these ions reduced to become metallic Cu and electro-crystallize on the BE. As a result, a conducting filament grew preferentially from the BE and finally bridge the BE and TE. Consequently, the device switched to the LRS. That is the reason that ECM devices were also called conducting bridge RAM. When negative voltage was applied on the TE electrode, the Cu filament broken due to electrochemical dissolution reaction initiated by an electronic current through the metallic bridge, and, in parallel, an electrochemical current and the device came into HRS. In recent years, many solid electrolyte materials such as GeSex[2S [2S [2O5[2[2[2[2[x[x/GdOx[x/TaSiOy[x/TaOx[2O3[x[2-) migration to form conducting filament, as shown in Figure 2- ions occurs in the switching material due to chemical bond breaking. The generated O2- ions migrate toward the TE under the external bias, and oxygen gas evolution at the anode due to anodic reaction are also reported in literature. To maintain the charge neutrality, the valance state of the cations changes. Therefore, it is called VCM memory. Due to O2- ion generation and anodic reaction, oxygen vacancy conducting path generates in the switching material between TE and BE, and device switches to LRS. The electroforming conditions strongly depend on the dimension of the sample, in particular, the switching material thickness. In addition, thermal effects play an essential role in the electroforming, and it sometimes damage the devices by introducing morphological changes [Further, depending on the switching material and electrodes, the resistive switching memory can be divided into two types: cation-based switching called electrochemical metallization (ECM) memory and anion-based switching called valance change memory (VCM) . In cati as GeSex,59,60, G as GeSex,62, Cu2SGeSex[2S , Ag2S [6x[2S [2S , Ta2O5[6 [2S [2O5,66, SiO22S [2O5[2, TiO2[682O5[2[2[2, HfO2[702[2[2[2[x, MoOx/Gd[x[x/GdOx, TiOx/Ta[x/TaSiOy, GeSex/TOy[x/TaOx, CuTe/Al/TaOx[2O3, and Ti/ changes ,21. Part changes . Thus, tx[x[x[2O [3[2[x[x[x[x[x[0.7Ca0.3MnO3[x[x)-based devices [2O5 and TaO2 with large solubility of O (71.43 to 66.67 at.%) above 1,000°C in its phase diagram [x is important in order to achieve long switching endurance  reaction of TaOx is low which shows its better stability [Resistance switching can originate from a variety of defects that alter electronic transport rather than a specific electronic structure of insulating materials, and consequently, almost all insulating oxides exhibit resistance switching behavior. Over the years, several materials in different structures have been reported for RRAM application to have better performance. The switching materials of anion-based devices include transition metal oxides, complex oxides, large bandgap dielectrics, nitrides, and chalcogenides. Table x-76, TiOxx[x-81, HfOxx[x[x,38,82-86x[x[x[2O , SrTiO3[x[x[2O [3,88, ZrO2x[2O [3[2-92, WOx[2O [3[2[x,30,93, A [3[2[x[x-97, ZnOx3[2[x[x[x,98-101, 2[x[x[x[x,103, GdOx[x[x[x[x,105, Pr0Ca0.3MnO3,106, GeO0.3MnO3[x,108, and devices ,109-128  devices , chemica diagram . This prd device ). It hasx is reported by Wei et al. [x are stable owing to small difference of Gibbs free energy in between LRS and HRS, and the barrier height between these states is quite high. Due to these benefits of TaOx switching material, it is important to design RRAM for real application. That is why this material has been studied in this review below.A schematic potential energy curve for TaOi et al. . This im2 of the W/Ti/TaOx/W and W/TaOx/W structures was fabricated [2O5 film with a thickness of ≈7 nm was then deposited by an e-beam evaporator. Then, a thin Ti (≈3 nm) interfacial layer by rf sputtering was deposited. The final devices were obtained after a lift-off process. Memory device structure and thicknesses of all layers were observed by transmission electron microscopy (TEM) with an energy of 200 keV. Figure x/W structure. The device size is 150 × 150 nm2. The thickness of TaOx layer is 6.8 nm  film. This also prevents oxidation of the W TE at the TaOx/W interface owing to the migration of oxygen from the underlying films toward the Ti film, which contributes to the improved resistive switching memory performance as described below.A small via size of 150 × 150 nmbricated . A high-at 300 K , the Ti x/TaOx/W device exhibits >1,000 consecutive repeatable dc switching cycles with a better resistance ratio of 102 under a low CC of 80 μA, the W/TaOx/W device shows few switching cycles with a higher CC of 300 μA [2-) migrate from the switching material toward W TE, and this has a lesser possibility to form an oxygen-rich layer at the W TE/TaOx interface, leading to the formation of multi-conduction filaments. However, the insertion of a thin (≈3 nm) Ti layer in between the W and TaOx layers in the W/TiOx/TaOx/W device makes a vast difference because Ti can be used as an oxygen reservoir. A repeatable switching of >10,000 cycles is also observed [2- rather than oxygen vacancies will migrate from TaOx toward the TE, resulting in a TiO2 layer which controls the conducting vacancy filament diameter in the TaOx layer by controlling current overflow and producing a tighter distribution of the LRS. Owing to this series resistance, the devices exhibit non-ohmic current. It is true that the conducting filament is formed through the TaOx film. When negative voltage is applied to the TE, oxygen ions are pushed from the TiO2 layer toward the conducting filament where they recombine with oxygen vacancies or oxidize the conducting filament. The device will be in HRS. Control of oxygen-deficient filament formation and rupture is facilitated by insertion of the thin Ti layer at the TE/TaOx interface, which results in repeatable and reproducible resistive switching characteristics, which has very good prospective of TaOx-based resistive switching memory in a W/TiOx/TaOx/W structure for real application. Some other reported results have been explained below.Resistive switching memory characteristics are explained here. Figure f 300 μA . In thisobserved . Under 'x/Ta device with a diameter of 100 μm, where Pt was grounded and external bias was on the Ta electrode. Long program/erase (P/E) endurance of 1.5 × 1010 cycles with a pulse width of 1 μs is reported. Further, a comparison of endurance characteristics made between TiOx and TaOx-based devices  solid solution. The energy-filtered TEM (EFTEM) zero-loss images and oxygen map of the switching region confirm also the reduction of TaOx thickness by half in the active region, and the oxygen content in the reduced region is found as low as that in the Ta electrode. The switching phenomenon is believed to be due to oxygen vacancies and ions through nano-ionic transport and a redox process, and this can be called VCM [2Ox5- layer contact while in the insulating state, and an ohmic contact is formed in the LRS. This suggests that oxygen ion movement under external bias leads to the LRS to HRS or HRS to LRS. Lee et al. [x-based crossbar resistive switching memory device. Figure x switching layer with insulating Ta2Ox5- layer near TE and TaOx2- near BE as can be seen in the cross-section TEM image presented in Figure 2 to 30 × 30 nm2. All size devices have shown self-compliance bipolar switching with small set/rest voltage of -1.0/2.0 V. The switching current of 50 × 50 μm2 device was >200 μA and for 30 × 30 nm2 device was approximately 40 μA, respectively  or single-layer TaOx with semi-reactive electrodes (W and Ti/W or Ta/Pt) are reported; however, conducting nano-filament formation/rupture is controlled by oxygen ion migration through bilayered or interfacial layer design under external bias. Further, high-density memory with a small size of 30 × 30 nm2 could be designed using crossbar architecture [x-based resistive switching memory devices is investigated, as compared to other switching materials in different RRAMs. This topical review shows good prospective; however, it needs to overcome the challenges for future production of the TaOx-based nanoscale RRAM application.It is reviewed that TaOof 80 μA ,109, whiterature ,110. So itecture . It is fThe authors declare that they have no competing interests.AP and DJ reviewed the papers under the instruction of SM. AP wrote the first draft and DJ prepared Tables


Enterobacteriaceae causing severe infections are necessary.Transplant recipients are at high risk of infections caused by multidrug resistant microorganisms. Due to the limited therapeutic options, innovative antimicrobial combinations against carbapenem-resistant Stenotrophomonas maltophilia and pan-sensitive Escherichia coli, successfully treated with antimicrobial therapy. On postoperative day 22, diagnosis of surgical site infection and nosocomial pneumonia with concomitant bacteremia due to a Klebisella pneumoniae carbapenemase-producer E coli was made. The patient was treated with the double-carbapenem regimen (high dose of meropenem plus ertapenem) and a potent synergistic and bactericidal activity of this un-conventional therapeutic strategy was observed in vitro. Despite a microbiological response with prompt negativity of blood cultures, the patient faced a worse outcome because of severe hemorrhagic shock.A 61-year-old woman with a history of congenital solitary kidney underwent renal transplantation. The postoperative course was complicated by nosocomial pneumonia due to The double-carbapenem regimen might be considered as a rescue therapy in those subjects, including transplant recipients, in whom previous antimicrobial combinations failed or when colistin use might be discouraged. Performing in vitro synergy testing should be strongly encouraged in cases of infections caused by pan-drug resistant strains, especially in high-risk patients. Enterobacteriaceae (CRE) are of great concern worldwide because they are associated with high mortality rates.2The emergence and global spread of infections caused by carbapenem-resistant 3Resistance to carbapenems is mainly mediated by 2 mechanisms: production of extended spectrum beta-lactamases combined with porin loss or production of carbapenem-hydrolyzing enzymes, namely carbapenemases.4 However, increasing rates of polymixin resistance have been recently reported.5In the presence of carbapenemases, carbapenems at standard dosage might be ineffective; thus, polymixin-based antimicrobial combinations have emerged as the milestone of CRE treatment.Klebsiella pneumoniae carbapenemase (KPC)-producer K pneumoniae, which represents the most common CRE reported in the literature.9In this setting, an innovative approach based on double-carbapenem combination (ertapenem [ERT] followed by high dose of meropenem [MEM]) has been shown to be effective against 10Given extensive healthcare contact before and after transplant and the need for lifelong immunosuppression, transplant patients are vulnerable to several infections, including those caused by multidrug resistant (MDR) organisms.11 Meanwhile, patients waiting for transplantation might become colonized with nosocomial microorganisms and eventually develop systemic infections due to these challenging pathogens early after transplantation.11The risk of infection after transplantation changes over time and is a function of the state of immunosuppression.Escherichia coli in a renal transplant patient treated with the double-carbapenem regimen.Herein, we describe a case of bloodstream infection (BSI) caused by a KPC-producer Stenotrophomonas maltophilia and a pan-sensitive E coli. An abdominal computed tomography scan displayed a prevesical hematoma (9 × 5 × 7 cm) requiring drainage placement. Cytomegalovirus (CMV)-DNA was 44,400 copies/mL (detection limit <200 copies/mL). According to creatinine clearance (37 mL/min), a postantibiogram antimicrobial therapy consisting of MEM 1 g every 12 h, trimethoprim/sulfamethoxazole (TMP/SMX) 320/1600 mg divided every 8 h and levofloxacin 750 mg every 48 h was started, with clinical (defervescence) and radiological (disappearance of pulmonary consolidations) responses. Furthermore, intravenous therapy with ganciclovir 150 mg every 24 h was started, with a prompt undetectability of CMV viremia.A 61-year-old woman with a history of congenital solitary kidney underwent hemodialysis because of end-stage renal failure in 2004. In July 2014, she underwent deceased donor renal transplantation. Simultaneously, a double-pigtail stent was placed and an endoarterectomy of the left iliac artery was performed because of severe stenosis. The postoperatory course was complicated by peri-renal lymphocele and bleeding of the left iliac artery. Immuno-suppressive therapy with tacrolimus was started and its serum concentration was regularly measured. On postoperative day 10, the patient developed fever (T 38.5°C), chills, and dyspnea, with pain on palpation in the lower abdominal quadrants. A chest X-ray showed the presence of bilateral pulmonary consolidations and culture of bronchoalveolar lavage (BAL) grew E coli , antimicrobial treatment with ERT 500 mg every 24 h (1-h infusion) followed by high dose of MEM  was started, in the absence of adverse effects. Considering that the administration of the double-carbapenem regimen has not been included in the clinical recommendation so far, the patient gave informed written consent for this unconventional therapeutic approach; thus, ethical approval was not necessary.However, on postoperative day 22 the patient became again febrile (T 39.0°C). At the physical examination, the patient was in poor condition, obtunded. White blood cells were 6820 cells/mmc , blood urea nitrogen (BUN) 52.7 mg/dL (reference range 12–25 mg/dL), creatinine 1.4 mg/dL (reference range 0.4–1.0 mg/dL), C-reactive protein (CRP) 84,000 μg/L (reference range 0–6000 μg/L), and erythrocyte sedimentation rate (ESR) 72 mm/h (reference range 0–20 mm/h). A chest X-ray showed a new consolidation in the left pulmonary lobe. Purulent discharge was present at the site of the recent abdominal drainage placement. Urine culture was sterile whereas blood (n = 2), BAL, and abdominal drainage cultures grew carbapenem-resistant (CR) After 96 h of such therapy, the patient became afebrile and the general conditions improved. Blood (n = 3) and drainage cultures were sterile. However, a mechanical obstruction of the double pigtail stent and a leakage at the level of anastomosis between bladder and left ureter were then observed. Creatinine increased to 2.4 mg/dL, BUN was 78.6 mg/dL, and a percutaneous nephrostomy was required. Three days later the patient suddenly worsened, blood pressure was 70/40 mm Hg, heart rate (HR) 130 per min, respiratory rate 35 beats/min, red blood cells 2,900,000 cells/mmc , and hemoglobin 6.5 g/dL (reference range 11–14 g/dL). A radiological examination showed a massive bleeding at the level of surgical anastomosis. Despite a prompt vasopressor and inotropic support, the patient died  of ERT and MEM were determined by broth macrodilution method (BMD) in cation-adjusted Mueller–Hinton broth.13 For the molecular analysis, DNA was extracted from 1 colony of a fresh culture and then eluted in 100 μL of elution buffer . For amplification, the lyophilized PCR mix STATNAT DNA-Mix  containing 3.0 mM MgCl2, 0.8 mM each deoxynucleotide (dNTP), 2 U of hot-start Taq DNA polymerase, and reaction buffer was used. The mix was resuspended with 1 μL of the primer and probe mix and 1 μL of extracted DNA, with a final volume of 20 μL. The amplification was performed using a CFX 96 Real-Time System .14Diagnostic disks were used for the phenotypic determination of carbapenemases.5 CFU/mL. At 2, 4, 6, 8 and 24 h time points, the number of CFU was counted. The following concentrations were used: 1× MIC ERT, 1× MIC MEM, 2× MIC MEM, 0.5× MIC ERT + 0.5× MIC MEM, 1× MIC ERT + 0.5× MIC MEM, 1× MIC ERT + 1× MIC MEM, and 1× MIC ERT + 2× MIC MEM. Bactericidal activity was defined as a ≥3-log10 CFU/mL reduction of the initial bacterial count at each time point whereas synergy was defined as a ≥2-log10 decrease in CFU/mL between the combinations and its most active constituent after 24 h.Furthermore, the activity of MEM and ERT, alone and in combination, was investigated by time-kill studies using an initial inoculum of ∼5 × 10E coli MICs were 16 and 32 μg/mL for ERT and MEM, respectively. Phenotypic and molecular analyses showed that the strain was a KPC-producer.In the killing curves, despite an initial reduction in log CFU/mL a regrowth at 24 h was observed for 1× MIC MEM and, to a lesser extent, for 1× MIC ERT; in contrast, 2× MIC MEM showed an absence of growth at 24 h. When the double-carbapenem combination was tested at concentrations of 1× MIC MEM + 1× MIC ERT, 0.5× MIC MEM + 1× MIC ERT, and 2× MIC MEM + 1× MIC ERT, a bactericidal activity was achieved at 4, 6, and 8 h and maintained up to 24 h with an absence of bacterial growth  had in vitro bactericidal effect similar to that of 0.5 × MIC MEM + 1 × MIC ERT . However, the patient experienced a worse outcome due to a massive bleeding at the level of surgical anastomosis.S maltophilia and a pan-sensitive E coli might have contributed to the subsequent development of carbapenem resistance. Furthermore, although clinical outcomes with this regimen were favorable resulting in a rapid clearance of bacteremia, the patient died 9 days after the diagnosis for causes other than uncontrolled infection. Therefore, assessment of medium-term outcome and final definitive cure of the infection could not be allowed. Another limitation could have been obtaining synergistic antimicrobial in relation with the susceptibility profile of MEM and ERT, regarding the possibility of using carbapenems in cases with MIC values equal to or >32 μg/mL. However, throughout in vitro killing studies we were able to show how the combination 0.5 × MIC MEM + 1 × MIC ERT was bactericidal, supporting the hypothesis that ERT might have a prominent role in the combination by binding to carbapenemases and thus leading achievable serum concentration of MEM  to be effective.6The present report has some limitations. First, the inclusion of MEM in the treatment of the infection caused by E coli. This unconventional approach might be considered as a rescue therapy in those subjects, including transplant recipients, in whom previous antimicrobial combinations failed or when colistin use might be discouraged. Furthermore, this case outlines that performing in vitro synergy testing represents a useful strategy in order to select the best antimicrobial combinations, especially in cases of infections occurring in high-risk individuals such as transplant recipients.In conclusion, the present study showed the effectiveness of the double-carbapenem regimen in a transplant recipient with BSI due to CR


Acacia ampliceps , a leguminous shrub, has been introduced in salt-affected areas in the northeast of Thailand for the remediation of saline soils. However, the defense mechanisms underlying salt tolerance A. ampliceps are unknown. We investigated various physio-biochemical and morphological attributes of A. ampliceps in response to varying levels of salt treatment (200–600 mM NaCl). Seedlings of A. ampliceps (25 ± 2 cm in plant height) raised from seeds were treated with 200 mM (mild stress), 400 and 600 mM (extreme stress) of salt treatment (NaCl) under greenhouse conditions. Na+ and Ca2+ contents in the leaf tissues increased significantly under salt treatment, whereas K+ content declined in salt-stressed plants. Free proline and soluble sugar contents in plants grown under extreme salt stress (600 mM NaCl) for 9 days significantly increased by 28.7 (53.33 μmol g–1 FW) and 3.2 (42.11 mg g–1 DW) folds, respectively over the control, thereby playing a major role as osmotic adjustment. Na+ enrichment in the phyllode tissues of salt-stressed seedlings positively related to total chlorophyll (TC) degradation (R2 = 0.72). Photosynthetic pigments and chlorophyll fluorescence in salt-stressed plants increased under mild salt stress (200 mM NaCl). However, these declined under high levels of salinity (400–600 mM NaCl), consequently resulting in a reduced net photosynthetic rate (R2 = 0.81) and plant dry weight (R2 = 0.91). The study concludes that A. ampliceps has an osmotic adjustment and Na+ compartmentation as effective salt defense mechanisms, and thus it could be an excellent species to grow in salt-affected soils. HalophyK+ ratio . AlternaK+ ratio . In the tio (–1) , causingtio (–1) .Acacia ampliceps , a native of Australia, is a multipurpose plant species largely used for livestock feeding  than the other species: A. karroo (2.39 ton ha–1), A. stenophylla (0.53 ton ha–1), A. seyal (0.27 ton ha–1), and A. asak (0.18 ton ha–1)  . In Thaine soils . HoweverAcacia ampliceps Maslin) provided by Land Development Department, Ministry of Agriculture and Cooperatives, Thailand, were disinfected with 15% sodium hypochlorite for 30 min followed by washing three times with tap water and then with distilled water. Disinfected seeds were germinated in the plastic tray (30 × 60 × 5 cm) containing 1 kg of garden soil  under greenhouse conditions. 15-day old seedlings were transferred into plastic bag  containing garden soil (as above) and grown under controlled environmental conditions  for 7 weeks. The experiment was conducted in the greenhouse of the Thailand Science Park, Pathum Thani, Thailand (Latitude 14°01′12″N Longitude 100°31′12″E).Seeds of salt wattle , stomatal conductance (gs), and transpiration rate (E) in the phyllodes were measured (n = 6).Uniform-sized seedlings (25 ± 2 cm in plant height) were selected as initial material. Sodium chloride (NaCl) concentration in the soil solution was adjusted to 0 (control), 200 (mild stress), 400, and 600 mM (high stress). After 9 days, growth characters, NaShoot height (SH), root length (RL), number of phyllodes (NL), number of roots (NR), fresh weight (FW), dry weight (DW), and phyllode leaf area (LA) of seedlings were measured. Salt wattle seedlings were dried at 80°C in a hot-air oven for 2 days and then incubated in desiccator before the measurement of DW. Phyllode area was measured using a Root/Leaf Area Meter DT-scan .+, K+, and Ca2+ were assayed following the modified method of +. The tissues were ground into powder in liquid nitrogen, extracted with boiling distilled water, and centrifuged at 10,000 × g for 10 min. The supernatant was filtered through a 0.45 μm membrane filter . Cellular Na+, K+, and Ca2+ concentrations were determined using HPLC  coupled with 432 Conductivity Detector and WATER IC-PACK™ ion-exclusion column . Nanopure water was used as mobile phase with 0.6 mL min–1 flow rate. Na+, K+, and Ca2+  were used as standard.Na™, Vertical®) and stored at –20°C prior to the measurement of total soluble sugars content using HPLC. A volume of 40 μL of crude extracts was automatically injected into a Waters HPLC fitted with a Waters 600 pump using a MetaCarb 87°C column equipped with a guard column. Deionised water was used as the mobile phase at a flow rate of 0.5 mL min–1. The online detection was performed using a Waters 410 differential refractrometer detector, and the data was analyzed by Empower® software. Sucrose, glucose, and fructose  were used as standards.Total soluble sugars  in the second fully expanded phyllode (from shoot tip) were analyzed according to the modified method of w/v), and the solution was filtered through filter paper . The extracted solution was reacted with an equal volume of glacial acetic acid and ninhydrin reagent  and incubated at 95°C for 1 h. The reaction was terminated by placing the container in an ice bath. The reaction mixture was mixed vigorously with 2 mL of toluene. After cooling to 25°C, the chromophore was measured at 520 nm by UV-VIS spectro-photometer  using L-proline as a calibration standard.Free proline in the second fully expanded phyllode (from shoot tip) was extracted and analyzed according to the method of a), chlorophyll b (Chlb), and TC contents in the second fully expanded phyllode were analyzed following the method of ® to prevent evaporation and then stored at 4°C for 48 h. Chla and Chlb concentrations were measured at 662 nm and 644 nm using UV-VIS spectrophotometer against acetone (95.5%) as a blank.Chlorophyll a (Chl0) and maximum (Fm) fluorescence yields were measured under weak modulated red light (<0.5 μmol m–2 s–1) with 1.6 s pulses of saturating light (>6.8 μmol m–2 s–1 PAR) and calculated using FMS software for Windows®. The variable fluorescence yield (Fv) was calculated using the equation: Fv = Fm–F0. The ratio of variable to maximum fluorescence (Fv/Fm) was calculated as the maximum quantum yield of PSII photochemistry. The photon yield of PSII (ΦPSII) in the light was calculated as: ΦPSII = (Fm′-F)/Fm′ after 45 s of illumination, when steady state was achieved  in the pulse amplitude modulation mode . A phyllachieved .n; μmol m–2s–1), stomatal conductance , and transpiration rate  in the second fully expanded phyllode were measured using a portable photosynthesis system fitted with an infra-red gas analyzer . The gs and E were measured continuously by monitoring the content of the air entering and existing in the IRGA headspace chamber, according to –1 air-flow rate and 25°C of chamber temperature. The light intensity was adjusted to 1,000 μmol m–2 s–1 PPFD of 6400-02B red-blue LED light source.Net photosynthetic rate (Pn = 6). The mean values obtained were compared using Tukey’s HSD and analyzed by SPSS software.The experiment was arranged as Completely Randomized Block Design (CRBD) with six replicates (Leaf color of control (0 mM NaCl) and mild salt-stressed plants (200 mM NaCl) was green. In contrast, phyllodes and the bipinnate leaves (true older leaves) turned yellow in the plants grown under extreme salt stress (400–600 mM NaCl) for 9 days. The overall growth characters declined in plants subjected to extreme salt stress for 9 days Figure . SH, RL,+ and Ca2+ contents in the phyllode tissues of salt-stressed plants were continuously enriched, whereas K+ content declined significantly, depending on the NaCl concentration in the soil solution  was 16.1 and 9.5 mg g–1 DW, respectively.Na Figures . Na+/K+ s Figure . Under t–1 FW) and 28.7 fold (53.33 μmol g–1 FW) in plants growing under 400 and 600 mM NaCl, over that in the control plants  in plants grown under 600 mM NaCl  and photon yield of PSII (ΦPSII) declined when plants were subjected to extreme salt stress (400–600 mM NaCl) for 9 days. Under extreme salt stress, Fv/Fm and ΦPSII were diminished by 17.0 and 13.5%, respectively, compared to that in mild salt-stressed plants (Table n) in mild salt-stressed plants was increased and then declined significantly at greater salt stress in a concentration-dependent manner  and transpiration rate (E) in mild salt-stressed plants were peaked to 10.44 μmol H2O m–2 s–1 and 3.13 mmol H2O m–2 s–1, whereas these declined by 73.3 and 73.5%, respectively, when plants were exposed to 600 mM NaCl   was strongly related to decreased net photosynthetic rate (Pn)  with 127.2 cm SH, when grown in the saline soil habitat for 9 months (A. ampliceps grown under hydroponic culture was only 7% (retaining 38% DW of control) when plants were exposed to 1.5% NaCl (257 mM) and increased to 47% (with only 22% DW of control) under 2.0% NaCl (342 mM)  in hydroponic culture and then declined under severe salt stress (ECe = 20–30 dS m–1), especially at pH 9.5 , SH and the number of branch per plants were enhanced, depending on the cultivation period  . Our fint pH 9.5 . In contr 2 days . Also, Mton ha–1 . In the + and Ca2+ in phyllodes of A. ampliceps were continuously enriched depending on the salt concentration in the soil solution, whereas K+ was declined, thereby resulting in increased Na+/K+ ratio. In halophytic species, Na+ was enriched in vacuole organelles, relating to activities of Na+/H+ antiporter (NHXs) and vacuolar H+-ATPase (VH+-ATPase) localization on vacuolar membrane (+ content in new phyllode (young) of A. ampliceps grown under salt stress (ECe = 40 dS m–1 in summer season) was peaked and greater than that in older phyllodes (+ accumulation and K+ reduction) has been demonstrated in other acacia species  grown under salt stress  and then declined when subjected to severe salt stress (≥400 NaCl). Chla and Chlb pigments in salt-stressed A. auriculiformis were degraded in relation to the level of salinity (ranging from 1.2 to 10 dS m–1). Parallel to our findings, Fv/Fm, ΦPSII, Pn, gs, and E in Nipa palm  were promoted by a low salt concentration (ECe = 8.9 dS m–1) and then significantly dropped, especially at the high concentration (ECe = 57.2 dS m–1)  .+ and Ca2+ in the phyllodes of A. ampliceps under extreme salt stress (600 mM NaCl) were enriched, while K+ declined, consequently resulting in increased Na:K ratio. Na+ enrichment in phyllode tissues of salt-stressed plants may get stored in vacuolar organelles as Na+ compartmentation. Alternatively, free proline and soluble sugar were enriched in cellular levels of leaf tissues to play a key role in osmoregulation of salt defense mechanism to protect A. ampliceps from salt-induced toxicity. Therefore, chlorophyll degradation, chlorophyll fluorescence diminishing, and net photosynthetic rate reduction in A. ampliceps under severe salt stress were evidently observed, thereby leading to overall growth inhibition.NaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There are errors in the last sentence of the Materials and Methods under the subheading “Quantitative nephelometric assay.” The correct sentence should read: Normal ranges were defined as; 8,3–27,0 mg/l (λFLC), 6,7–22,4 mg/l (κFLC), 0,31–1,56 (FLC κ:λ ratio).There are errors in the second paragraph of the Results under the subheading “Prevalence of serum FLCs in SLE patients and healthy controls.” The correct paragraph should read: Fifty three percent (24 of 45) of examined SLE patients had an elevated level of serum κFLCs above the normal range compared to 2.5% (1 of 40) of examined healthy controls. Similarly, 38% (17 of 45) of SLE patients compared to 2.5% (1 of 40) of healthy controls showed λFLC levels above the normal range.There are multiple errors in the Discussion. The first sentence of the third paragraph should read: We observed that both κFLC and λFLC concentrations were elevated in sera from SLE patients compared to healthy controls with 53% compared to 2.5% having an elevated amount of κFLCs, and 38% compared to 2.5% having an elevated level of λFLCs. The second sentence of the fourth paragraph should read: 2.5% of healthy controls were found to have elevated levels of κFLC and λFLC, respectively. The first sentence in the last paragraph should read: In conclusion, serum FLC concentrations were elevated in SLE patients, especially κFLC levels with 53% of SLE patients having abnormally high concentrations.10.3410/f.717971156.793469463 should not appear in the published article. All subsequent references should be reduced by 1.Reference 46, Katzmann JA, Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR et al. (2002) Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem 48: 1437–1444. pmid:12194920 doi:


Eight additional subjects were treated with UAL (74 mg/m2) to evaluate multiple-dose pharmacokinetics. No ≥grade 3 adverse events (NCI-CTC) were observed. Sixty percent subjects achieved stable disease after 2 treatment cycles. Multiple-dose pharmacokinetic analysis suggested UAL does not accumulate in the body. This trial demonstrates that UAL was tolerable, had manageable toxicity, and could potentially improve patient remission rates. A large phase II study is recommended to confirm these results .Ursolic acid liposome (UAL), a new antitumor drug, has potential therapeutic value. However, limited clinical data exists regarding multiple-dose safety, antitumor activity, and the recommended dose (RD) of UAL for subjects with advanced solid tumors. All subjects were intravenously administered UAL for 14 consecutive days of a 21-day treatment cycle. Twenty-one subjects were enrolled in 1 of 3 sequential cohorts (56, 74, and 98 mg/m Eriobotrya japonica, Rosmarinus officinalis, and Glechoma hederacea [Ursolic acid (UA) is a natural hydroxy pentacyclic triterpene compound  isolatedederacea , 2. Prevederacea –5 and ceederacea , 7, inhiederacea , and inhederacea –11 in vaederacea . Thus, U2 [Liposomes have been utilized as a drug delivery system to overcome the poor solubility of UA, increase the therapeutic efficiency, reduce the side effects, and enhance the bioavailability of drugs that have been broadly applied –15. Curr2 . In actuThe primary objective of this study was to evaluate the tolerability of UAL treatment and the recommended dose (RD) in a multiple-dose administration phase II trial consisting of subjects with advanced solid tumors. The second objective was to perform a preliminary assessment of the antitumor activity of UALs.We performed a phase I, open-label, single center trial in subjects with advanced solid tumors. The SFDA of China and the Hospital Medical Ethics Committees approved the trial and it was conducted in accordance with the Declaration of Helsinki and the applicable local regulatory requirements and laws. A signed, written informed consent of the legal representatives and the consent of each patient were obtained before any study procedure was performed. UALs were supplied by Wuhan Li Yuanheng Medicine Technology Co. Ltd  as a freeze-dried powder for infusion. Each glass vial contained 3 mg of active drug. It was uniformly dispersed in 250 mL of 5% glucose solution before administration.9/L, absolute neutrophil count (ANC) ≥ 2.0 × 109/L, platelet count ≥ 100 × 109/L, and hemoglobin ≥ 100 g/L); had adequate hepatic and renal function , aspartate transaminase (AST), and alkaline phosphatase (ALP) ≤ 2.5 the upper limit of normal (ULN) (or 5 × ULN for hepatic cancer/metastatic hepatic cancer), total bilirubin (TBIL) ≤ 1.5 × ULN, serum creatinine (CRE) levels of ≤ 1.5 × ULN, a creatinine clearance rate of ≤1.5 × ULN, and normal urea); and had normal pulmonary function.Eligible subjects were aged 18–75 years with cytologically or histologically confirmed advanced solid tumors; they either refused standard therapies or standard effective therapies did not exist; and they had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0–2, a Karnofsky score ≥ 60%, and a life expectancy ≥ 3 months; practiced adequate contraception; had adequate hematological function (white blood cell (WBC) ≥ 4.0 × 102, administered via a 14-day consecutive intravenous 4 h infusion and given a rest for 7-day per 21-day cycle. Each cohort consisted of at least 3 subjects. Once all enrolled subjects had been monitored for 2 weeks and had no higher than grade 3 nonhematological toxicity or grade 4 hematological toxicity, the next dose was administered. The trial was terminated when ≥1/3 of the subjects experienced DLT, a severe adverse event (AE), or tumor progression. The DLT was defined as grade 4 thrombocytopenia, grade 4 neutropenia lasting for ≥7 days, febrile neutropenia, grade 4 anemia, or grade 3/4 nonhematological toxicity. Evaluated subjects were required to complete at least 1 cycle of treatment. After that, if subjects needed to continue treatment because they could not gain any benefit from other treatments, additional cycles were administered until disease progression or unacceptable toxicity occurred, or if the patient refused further treatment. Additional subjects were recruited in order to evaluate the pharmacokinetics of UAL treatment. These subjects were administered a dose of 74 mg/m2 of UAL via a consecutive, 14-day, intravenous 4 h infusion.Subjects were assigned to 1 of 3 sequential dose cohorts of UAL: 56, 74, or 98 mg/m+, Na+, Ca2+, and Cl− were examined at screening and then once a week thereafter. Fibrinogen (Fbg) and prothrombin time (PT) were examined at screening and during the 3rd week. To further evaluate the immune functions of subjects after UAL administration, we measured CD4/CD8 and natural killer (NK) cell activity in the circulation both at screening and on the 14th day. AEs were evaluated according to the National Cancer Institute Common Terminology Criteria for AEs (NCI-CTCAE) version 3.0.Tolerability and toxicity were evaluated in all subjects treated with at least 1 cycle of UAL therapy. Vital signs including body temperature, respiration, pulsation, and blood pressure were examined at screening and once a day thereafter. Hematological parameters , urine routines , and stool routines  were tested, and an electrocardiogram was performed at screening and on the 14th day of the cycle. Blood biochemistries including ALT, AST, ALP, gamma-glutamyl transpeptidase (GGT), TBIL, direct-reacting bilirubin, total protein, GLU, lactate dehydrogenase, creatine kinase, bun urea nitrogen, CRE, UA, cholesterol, triglyceride (TG), high-density lipoprotein, low-density lipoprotein, KSerial randomly subjects treated with at least 2 cycles were selected to evaluate the therapeutic efficacy of UALs. The tumor response was examined by using computerized tomography, magnetic resonance imaging, chest radiography, or ultrasonography according to the response evaluation criteria in solid tumors (RECIST) at the scheduled times (baseline and 2 cycles later) either until the tumor progressed or until the final visit. Complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) were defined according to RECIST.Blood samples for pharmacokinetic analysis were collected into heparinized tubes on the 1st and 14th days of the study, at various time points including 0, 0.5, 1, 2, and 4 h during infusion and 5, 15, and 30 min and 1, 1.5, 2, 3, 4, 6, 8, and 12 h after the end of infusion. Plasma was separated using centrifugation and then stored at –20°C until analysis.8 column . The mobile phase consisted of acetonitrile and 10 mM ammonium formate  at a flow rate of 0.2 mL/min. The elution time was 3 m. Multiple-reaction monitoring was performed at m/z455.1 → 455.0 and m/z469.3 → 425.2 for UAL and glycyrrhetinic acid , respectively, in negative ion mode with an electrospray ionization source. Estimates of pharmacokinetic parameters for UAL were derived from individual concentration-time data sets by noncompartmental analysis.UAL concentrations were measured using validated ultra-performance liquid chromatography/tandem mass spectroscopy (UPLC/MS/MS) methods as described previously . In brieTolerability, toxicity, efficacy, and pharmacokinetic characteristics were explored and analyzed in detail. Noncompartmental pharmacokinetic parameters were determined from individual plasma concentration-time data using DAS version 2.1.1.Twenty-one subjects (7 men and 14 women), aged 19–68 years (median age: 54 years), were enrolled in the study, and their characteristics at baseline are listed in 2).Tolerability and toxicity were evaluated for all subjects. The vital sign data showed that all values fluctuated within the normal range at every time point among the 3 cohorts . All hemP > 0.05, 56 mg/m2; 0.82 ± 0.48 and 0.61 ± 0.24, P > 0.05, 74 mg/m2; 1.39 ± 0.96 and 1.23 ± 0.23, P > 0.05, 98 mg/m2). Significant differences in the NK cells were also not observed . These results suggested that the UAL did not affect patient immune function.Immune function tests showed no significant differences in CD4/CD8 at screening and on the 14th day  but then recovered after 2 h without any treatment (2 UAL experienced grade 2 GGT elevation. Two (10%) subjects treated with 56 and 74 mg/m2 UAL experienced grade 1 abdominal distention. Finally, 1 (5%) patient had grade 2 ALT elevation. Other mild symptoms including AST and TG elevation, pruritus, arthralgia, and hypokalemia were also observed. However, no National Cancer Institute common toxicity criteria (NCI-CTC) ≥ grade 3 treatment-related AEs were observed. The most frequent AEs included pyrexia, GGT elevation, and abdominal distention. These results suggested that UAL was tolerable and safe among 3 dose cohorts after administration via a consecutive 14-day intravenous 4 h infusion every 21 days. Therefore, a UAL dose of 98 mg/m2 was considered the RD for a phase II trial.In addition, 3 (14%) subjects treated with 56 mg/mreatment . Three  subjects preferred to receive and finish at least 2 cycles of UAL treatment, the evaluation of preliminary antitumor efficacy was limited. Three (60%) subjects achieved stable disease. One of these subjects had advanced renal carcinoma and had no significant change in the lesion after 2 cycles of treatment with 56 mg/m2 UAL. No CR or PR was observed, which could be because the subjects had advanced stage tumors and did not benefit from other prior treatment schemes. Another possible explanation is that the number of subjects that could be evaluated was too small. Regardless, UAL does have the potential to improve the patient remission rate. A phase II study of a large number of subjects is recommended to confirm this finding.Two additional subjects, 1 with primary non-Hodgkin lymphoma and the other with breast cancer, showed PD after 2-cycle treatment with 74 mg/mt1/2), maximum plasma concentration (Cmax), area under the plasma concentration time curve (AUCt0→), and AUC∞0→ during the 1st day were 4.58 ± 2.04 h, 1589 ± 635 ng/mL, 5172 ± 1136 ng·h/mL, and 5498 ± 1525 ng·h/mL, respectively. They were 4.00 ± 1.27 h, 1211 ± 204 ng/mL, 4705 ± 873 ng·h/mL, and 4834 ± 933 ng·h/mL, respectively, during the 14th day. There were no significant differences in the values of t1/2, Cmax, AUCt0→, and AUC∞0→ (P > 0.05) between days 1 and 14, suggesting that the pharmacokinetics were unaltered with multiple-daily dosing and that the UAL did not accumulate in the body. In addition, we found that there was a close relationship between the values of Cmax or AUC and AEs. The value of Cmax or AUC increased as the AEs  increased in seriousness.Eight additional subjects were enrolled in the trial in order to investigate the pharmacokinetics of UAL therapy. The pharmacokinetic data  followin2 was safe. The results are consistent with preclinical information [Cmax or AUC was associated with AEs. The value of Cmax or AUC was greater when the risk of AEs occurring in subjects was elevated. The reasons for this might be the following: when the value of Cmax is elevated, hepatocytes would be exposed to a high concentration of drug and would be stimulated to release serial enzymes including AST, ALT, and GGT. If the value of AUC was high simultaneously, the time of stimulation would be prolonged. Therefore, the risk of hepatotoxicity and gastrointestinal toxicity would become elevated. These results suggested that the manageable toxicity associated with UAL treatment could be further controlled via kinetic monitoring.This study demonstrated that UAL treatment of subjects with advanced solid tumors via multiple-dose and consecutive 14-day intravenous infusion every 21 days at doses of 56, 74, and 98 mg/mormation . In addi2. These results indicate UAL can potentially improve patient remission.UA has been widely reported to have antitumor activities in preclinical studies , 18–21. T1/2 was about 4.3 h [T1/2 of UAL was 4.00–4.58 h, suggesting the T1/2 value was low so that it could rapidly be eliminated from blood. This phenomenon suggested that UAL did not accumulate in the body and that UAL must be infused repeatedly to keep the plasma-drug concentration steady and further enhance its antitumor effect.The pharmacokinetic data of UA in animals showed that ut 4.3 h . In this2.In summary, the multiple-dose administration of UAL was tolerable with manageable toxicity. Further, the UAL did not accumulate in the body. We conclude that UAL has the potential to improve the patient remission rates. The recommended dose of UAL for a phase II clinical trial is 98 mg/m