Bidirectional extracellular electron transfer pathways of Geobacter sulfurreducens biofilms: Molecular insights into extracellular polymeric substances.

The basis for bioelectrochemical technology is the capability of electroactive bacteria (EAB) to perform bidirectional extracellular electron transfer (EET) with electrodes, i.e. outward- and inward-EET. Extracellular polymeric substances (EPS) surrounding EAB are the necessary media for EET, but the biochemical and molecular analysis of EPS of Geobacter biofilms on electrode surface is largely lacked. This study constructed Geobacter sulfurreducens-biofilms performing bidirectional EET to explore the bidirectional EET mechanisms through EPS characterization using electrochemical, spectroscopic fingerprinting and proteomic techniques. Results showed that the inward-EET required extracellular redox proteins with lower formal potentials relative to outward-EET. Comparing to the EPS extracted from anodic biofilm (A-EPS), the EPS extracted from cathodic biofilm (C-EPS) exhibited a lower redox activity, mainly due to a decrease of protein/polysaccharide ratio and α-helix content of proteins. Furthermore, less cytochromes and more tyrosine- and tryptophan-protein like substances were detected in C-EPS than in A-EPS, indicating a diminished role of cytochromes and a possible role of other redox proteins in inward-EET. Proteomic analysis identified a variety of redox proteins including cytochrome, iron-sulfur clusters-containing protein, flavoprotein and hydrogenase in EPS, which might serve as an extracellular redox network for bidirectional EET. Those redox proteins that were significantly stimulated in A-EPS and C-EPS might be essential for outward- and inward-EET and warranted further research. This work sheds light on the mechanism of bidirectional EET of G. sulfurreducens biofilms and has implications in improving the performance of bioelectrochemical technology.

Anlotinib Monotherapy for Refractory Metastatic Colorectal Cancer: A Double-Blinded, Placebo-Controlled, Randomized Phase III Trial (ALTER0703).

BACKGROUND: Treatment options for refractory metastatic colorectal cancer (mCRC) were limited. Anlotinib is a novel multitarget tyrosine kinase inhibitor. ALTER0703 study was conducted to assess efficacy and safety of anlotinib for patients with refractory mCRC. MATERIALS AND METHODS: This was a multicenter, double-blinded, placebo-controlled, randomized phase III trial involving 33 hospitals in China. Patients had taken at least two lines of therapies were 2:1 randomized to receive oral anlotinib (12 mg/day; days 1-14; 21 days per cycle) or placebo, plus best supportive care. Randomization was stratified by previous VEGF-targeting treatments and time from diagnosis to metastases. The primary endpoint was overall survival (OS). The secondary endpoints were progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), quality of life (QoL), and safety. RESULTS: A total of 419 patients (anlotinib: 282; placebo: 137) were treated from December 2014 to August 2016. The median PFS was improved in anlotinib group (4.1 months; 95% confidence interval [CI], 3.4-4.5) over placebo group (1.5 months; 95% CI, 1.4-1.5), with a hazard ratio (HR) of 0.34 (95% CI, 0.27-0.43; p < .0001). However, median OS was similar between two groups (8.6 months; 95% CI, 7.8-9.7 vs. 7.2 months; 95% CI, 6.2-8.8; HR, 1.02; p = .870). Improvements of ORR and DCR were observed in anlotinib over placebo. The most common grade ≥ 3 anlotinib related adverse events were hypertension (20.92%), increased γ-GT (7.09%), and hand-foot skin reaction (6.38%). CONCLUSION: Anlotinib was tolerated in Chinese patients with refractory mCRC. Although OS did not reach significant difference, anlotinib still provided clinical benefits by substantially prolonged PFS in these patients. IMPLICATIONS FOR PRACTICE: In this randomized clinical trial that included 419 patients with refractory metastatic colorectal cancer, substantial prolonged in progression-free survival was noted in patients who received anlotinib compared with those given placebo. Improvements on objective response rate and disease control rate was also observed in anlotinib group. However, overall survival was similar between the two groups. In a word, in third-line or above treatment of Chinese patients with refractory metastatic colorectal cancer, anlotinib provided clinical benefit by significantly prolonged progression-free survival.

Circular RNA hsa_circ_0004277 contributes to malignant phenotype of colorectal cancer by sponging miR-512-5p to upregulate the expression of PTMA.

In recent years, extensive reports have been published concerning the molecular mechanism underlying the occurrence and progression of colorectal cancer. Circular RNAs (circRNAs) have been identified as important modulators in the biological processes of colorectal cancer. Microarray analysis unveiled that differential circ-0004277 expression was identified in tissue samples of colorectal cancer. High circ-0004277 expression was then verified in tissue samples and cell lines of colorectal cancer via qRT-PCR. Kaplan-Meier analysis was used for identifying the association between circ-0004277 expression and the overall survival rate of colorectal cancer patients. A relationship existed between higher circ-0004277 expression and decreased overall survival rate of colorectal cancer patients. From a functional perspective, circ-0004277 knockdown accelerated cell apoptosis and restrained cell proliferation of colorectal cancer. From mechanistic perspective, circ-0004277 upregulated PTMA by sponging miR-512-5p. Rescue assay was used for verifying the roles of the circ-0004277-miR-512-5p-PTMA axis. Both miR-512-5p and PTMA participated in circ-0004277-mediated colorectal cancer cell proliferation based on experiments. In summary, our study showed that circ-0004277 promoted the proliferation of colorectal cancer cells as a miR-512-5p sponge to upregulate the PTMA expression.

CircHIPK3 promotes colorectal cancer cells proliferation and metastasis via modulating of miR-1207-5p/FMNL2 signal.

Increasing evidences demonstrate that circular RNAs (circRNAs) are extensively implicated in various cancers including colorectal cancer (CRC). In the present study, we found that circRNA HIPK3 (circPIK3) was upregulated in CRC. We identified that circHIPK3 was closely related with unfavorable clinicopathological features in patients with CRC. Functional transwell assay and proliferation assay indicated that circHIPK3 served as an oncogene and promoted CRC cells migration, invasion and proliferation. Meanwhile, we found that formin like 2 (FMNL2) was a key downstream molecule in circHIPK3-induced metastasis and proliferation in CRC cells. We further verified that circHIPK3 was mainly located at cytoplasm through an immunofluorescence assay. An online bioinformatics screening and a GEO datasets analysis showed that microRNA 1207-5p (miR-1207-5p) was downregulated in CRC. Also, we found that miR-1207-5p shared a similar miR-1207-5p response elements (MREs-1207-5p). Meanwhile, we showed that miR-1207-5p suppressed CRC cells migration, invasion and proliferation via directly targeting of FMNL2. Even further, via a constructed luciferase assay, we indicated that circHIPK3 was another target of miR-1207-5p. Functionally, we proved that circHIPK3 enhanced FMNL2 mediated promotion of migration, invasion and proliferation by sponging of miR-1207-5p in CRC cells. In summary, the outcomes of this study illustrated that circHIPK3 promoted CRC cells migration, invasion and proliferation modulating of FMNL2 by sponging of miR-1207-5p. Our findings indicated that circHIPK3/miR-1207-5p/FMNL2 axis might be a new strategy in molecular treatment of CRC.

Enhanced Current Production by Exogenous Electron Mediators via Synergy of Promoting Biofilm Formation and the Electron Shuttling Process.

Exogenous electron mediators (EMs) can facilitate extracellular electron transfer (EET) via electron shuttling processes, but it is still unclear whether and how biofilm formation is affected by the presence of EMs. Here, the impacts of EMs on EET and biofilm formation were investigated in bioelectrochemical systems (BESs) with Shewanella oneidensis MR-1, and the results showed that the presence of five different EMs led to high density current production. All the EMs substantially promoted biofilm formation with 15-36 times higher total biofilm DNA with EMs than without EMs, and they also increased the production of extracellular polymeric substances, which was favorable for biofilm formation. The current decreased substantially after removing EMs from the medium or by replacing electrodes without biofilm, suggesting that both biofilm and EMs are required for high density current production. EET-related gene expression was upregulated with EMs, resulting in the high flux of cell electron output. A synergistic mechanism was proposed: EMs in suspension were quickly reduced by the cells and reoxidized rapidly by the electrode, resulting in a microenvironment with sufficient oxidized EMs for biofilm formation, and thus, besides the well-known electron shuttling process, the EM-induced high biofilm formation and high Mtr gene expression could jointly contribute to the EET and subsequently produce a high density current. This study provides a new insight into EM-enhanced current production via regulating the biofilm formation and EET-related gene expression.

A novel long noncoding RNA, LINC00483 promotes proliferation and metastasis via modulating of FMNL2 in CRC.

Long non-coding RNAs (lncRNAs) are extensively involved in multiple malignancies including colorectal cancer (CRC). In the present study, we found a novel lncRNA, long intergenic non-protein coding RNA 483 (LINC00483), which was upregulated in CRC. We also illustrated that upregulated LINC00483 was correlated with poor clinicopathological features of patients with CRC. Functionally, we displayed that a knockdown of LINC00483 suppressed LOVO and HT29 cells proliferation and metastatic ability. We further illustrated that miR-204-3p was involved in LINC00483 induced proliferation and metastasis. An overexpression of miR-204-3p could attenuate the facilitative effect which LINC00483 presented. Through a luciferase assay, we showed the direct binding effect between LINC00483 and miR-204-3p. Even further, we revealed that LINC00483 and formin like 2 (FMNL2) shared a similar miR-204-3p response elements (MREs-204-3p). FMNL2 was a direct target of miR-204-3p. FMNL2 was a downstream gene of LINC00483 and participated in LINC00483 mediated proliferation and metastasis. Lastly, we proved that LINC00483 promoted proliferation and metastasis via modulating of FMNL2 in LOVO and HT29 cells. In summary, the outcomes of this study illustrated that LINC00483 promoted CRC cells proliferation and metastasis via modulating of FMNL2 by acting as a ceRNA of miR-204-3p. LINC00483/miR-204-3p/FMNL2 axial might be a novel target in molecular treatment of CRC.

Circular RNA PVT1 promotes metastasis via miR-145 sponging in CRC.

Circular RNAs (circRNAs) are a class of covalently closed non-coding RNAs and are widely involved in various cancers including colorectal cancer (CRC). Circular RNA PVT1 (circPVT1) was reported in several malignancies but the role it plays in CRC remains unclear. In our current research, we focused on the expression and function of circPVT1) works in CRC. We found that circPVT1 was upregulated in CRC. Also, we illustrated that the upregulated circPVT1 was closely correlated with poor prognosis and bad clinicopathological features of patients with CRC. Through a loss of function experiment, we showed that a downregulation of circPVT1 suppressed CRC cells metastasis. Through online prediction, we found that circPVT1 had a microRNA response element (MRE) for miR-145. Additionally, we demonstrated that miR-145 was downregulated in CRC. Even further, we showed that miR-145 was involved in circPVT1 mediated facilitation of CRC metastasis. In a further mechanical study, we demonstrated that circPVT1 could target miR-145. Lastly, we revealed that the metastasis-promoting role of circPVT1 in CRC was partially achieved via miR-145 sponging. In brief, the findings of the present study illustrated that circPVT1, working as an oncogene, promotes metastasis via miR-145 sponging in CRC. CircPVT1/miR-145 axial might be a novel point in targeting treatment of CRC.

Prognostic and clinicopathological significance of long noncoding RNA CTD-2510F5.4 in gastric cancer.

BACKGROUND: Compelling studies have demonstrated the correlation between aberrant expressed lncRNAs and human cancers, and revealed promise of these lncRNAs as biomarkers in predicting patients' survival and outcome. METHODS: We downloaded the RNA-seq data from the Cancer Genome Atlas, and screened out DEGs and DELs between gastric cancer tissues and normal gastric tissues. By bioinformatics analysis, we identified CTD-2510F5.4 was a malignant phenotype associated lncRNA. The expression levels of CTD-2510F5.4 in tissues were detected by ISH, and the relationships between CTD-2510F5.4 expression and clinicopathological characteristics were analyzed by statistical analysis. RESULTS: By bioinformatics analysis and functional analysis, we identified CTD-2510F5.4 was a malignant phenotype associated lncRNA of gastric cancer that potentially regulated cell cycle and apoptosis. CTD-2510F5.4 expression was significantly higher in gastric cancers, and was correlated with pathological grade, vascular or nerve invasion, AJCC TNM stage and OS. Moreover, gastric cancer patients with high CTD-2510F5.4 expression showed significantly shorter MST. High CTD-2510F5.4 expression was a independent risk factor for gastric cancers at pathological grade < III and without vascular or nerve invasion. CONCLUSIONS: We identified CTD-2510F5.4 was a malignant phenotype associated lncRNA potentially involved in the pathogenesis of gastric cancer. Our data also supported the clinical potential of CTD-2510F5.4 being a diagnostic and prognostic biomarker for gastric cancer.

Paclitaxel reversed trastuzumab resistance via regulating JUN in human gastric cancer cells identified by FAN analysis.

AIM: In this study, we aim to use bioinformatics approach to identify paclitaxel-targeted modulators potentially involved in the process of reversing the trastuzumab resistance. Materials & methods: We extracted data from GSE77346 to identify potential trastuzumab resistance-related genes, used bioinformatics analysis and functional/activity network approach to find genes involved in trastuzumab resistance reversal. RESULTS: We identified hub differentially expressed genes related to trastuzumab resistance, trastuzumab targeting and paclitaxel targeting, respectively. We then found C-Jun may be critical in trastuzumab resistance reversal.  This process may involve transcriptional activation of DUSP1 by JUN, which lead to regulation of DUSP1-related signaling pathways. CONCLUSION: The present study revealed paclitaxel may reverse the trastuzumab resistance by JUN, which possibly in turn regulated DUSP1 and DUSP1-related signaling pathways.

An LC-MS/MS method for simultaneous determination of 1,5-dicaffeoylquinic acid and 1-O-acetylbritannilactone in rat plasma and its application to a pharmacokinetic study.

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantitative determination of 1,5-dicaffeoylquinic acid (1,5-DCQA) and 1-O-acetylbritannilactone (1-O-ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB-C18 column using isocratic mobile phase consisting of methanol-water-formic acid (70:30:0.1, v/v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple-quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850-213 ng/mL for 1,5-DCQA, and 0.520-130 ng/mL for 1-O-ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.

A genetic engineering strategy to enhance outer membrane vesicle-mediated extracellular electron transfer of Geobacter sulfurreducens.

Bioelectrochemical systems (BESs) are unique devices that harness the metabolic activity of electroactive microorganisms (EAMs) to convert chemical energy stored in organic substrates into electrical energy. Enhancing electron transfer efficiency between EAMs and electrodes is the key to practical implementation of BESs. Considering the role of outer membrane vesicles (OMVs) in mediating electron transfer of EAMs, a genetic engineering strategy to achieve OMVs overproduction was explored to enhance electron transfer efficiency and the underlying mechanisms were investigated. This study constructed a mutant strain of Geobacter sulfurreducens that lacked the ompA gene encoding an outer membrane protein. Experimental results showed that the mutant strain produced more OMVs and possessed higher electron transfer efficiency in Fe(III) reduction, dye degradation and current generation in BESs than the wild-type strain. More cargoes such as c-type cytochromes, functional proteins, eDNA, polysaccharides and signaling molecules that might be favorable for electron transfer and biofilm formation were found in OMVs produced by ompA-deficient anodic biofilm, which possibly contributed to the improved electron transfer efficiency of ompA-deficient biofilm. The results indicate that overproduction of OMVs in EAMs might be a potential strategy to enhance BESs performance.

[Retracted] miR­885­5p upregulation promotes colorectal cancer cell proliferation and migration by targeting suppressor of cytokine signaling.

[This retracts the article DOI: 10.3892/ol.2018.8645.].

Significance of a minor pilin PilV in biofilm cohesion of Geobacter sulfurreducens.

Bacterial adhesion plays a vital role in forming and shaping the structure of electroactive biofilms that are essential for the performance of bioelectrochemical systems (BESs). Type IV pili are known to mediate cell adhesion in many Gram-negative bacteria, but the mechanism of pili-mediated cell adhesion of Geobacter species on anode surface remains unclear. Herein, a minor pilin PilV2 was found to be essential for cell adhesion ability of Geobacter sulfurreducens since the lack of pilV2 gene depressed the cell adhesion capability by 81.2% in microplate and the anodic biofilm density by 23.1 % at -0.1 V and 37.7 % at -0.3 V in BESs. The less cohesiveness of mutant biofilms increased the charge transfer resistance and biofilm resistance, which correspondingly lowered current generation of the pilV2-deficient strain by up to 63.2 % compared with that of the wild-type strain in BESs. The deletion of pilV2 posed an insignificant effect on the production of extracellular polysaccharides, pili, extracellular cytochromes and electron shuttles that are involved in biofilm formation or extracellular electron transfer (EET) process. This study demonstrated the significance of pilV2 gene in cell adhesion and biofilm formation of G. sulfurreducens, as well as the importance of pili-mediated adhesion for EET of electroactive biofilm.

GSTT1 null genotype contributes to increased risk of gastric cancer in Chinese population: evidence from a meta-analysis.

Previous studies suggested glutathione S-transferase T1 (GSTT1) null genotype might be a candidate genetic polymorphism with a role in the susceptibility to gastric cancer, but studies form Chinese population reported controversial findings. Thus, a meta-analysis was performed to clarify the effect of GSTT1 null genotype on gastric cancer risk in Chinese population. Eligible studies were searched in Medline, Embase, and China National Knowledge Infrastructure databases. Between-study heterogeneity was assessed using the I (2) statistic. Odds ratios (OR) with the corresponding 95 % confidence intervals (95 % CI) were pooled to assess the association. Twenty case-control studies involving a total of 3,204 gastric cancer cases and 5,462 controls were finally included in the meta-analysis. Meta-analysis of all 20 studies showed that GSTT1 null genotype was associated with an elevated risk of gastric cancer in Chinese population (OR=1.26, 95 % CI 1.09-1.46, P OR=0.002). The cumulative meta-analysis showed a trend of a more obvious association between GSTT1 null genotype and risk of gastric cancer in Chinese population as information accumulated gradually. Sensitivity analysis by omitting individual study, in turns, did not materially alter the pooled ORs. This meta-analysis provides a strong evidence for the significant association between GSTT1 null genotype and gastric cancer risk in Chinese population, and GSTT1 null genotype contributes to increased risk of gastric cancer.

Research progress of integrated traditional Chinese and Western medicine in the treatment of advanced gastric cancer.

Gastric cancer (GC) is a malignant tumor originating from the gastric epithelium, and its incidence and mortality rates rank third among all malignant tumors worldwide. It is also one of the most common cancers in China and is treated predominantly by Western medicine in clinical practice. However, with the advancements in medical technology and informatics, the values of traditional Chinese medicine (TCM) in preventing and treating GC and improving prognosis have increasingly been recognized. According to TCM, clinical manifestations of GC can be divided into Yege (dysphagia), regurgitation, stomach pain, and Zhengxia (abdominal mass). Due to the unbalanced distribution of health care resources in China, most GC patients already have progressive or advanced-stage disease at the first diagnosis. As a result, most GC patients have poor physical function, and surgery or chemotherapy alone will aggravate the impairment to the immune function and seriously affect the quality of life. In contrast, TCM therapies have shown promising efficacy in the management of these patients. Here we review the role of the integrated TCM and Western medicine in treating advanced GC.

Sub-MIC antibiotics affect microbial ferrihydrite reduction by extracellular membrane vesicles.

Environmental concentrations of antibiotics, usually below MIC, have significant biological effects on bacterial cells. Sub-MIC antibiotics exposure induces bacteria to produce outer membrane vesicles (OMVs). Recently, OMVs is discovered as a novel pathway for dissimilatory iron reducing bacteria (DIRB) to mediate extracellular electron transfer (EET). Whether and how the antibiotic-induced OMVs modulate iron oxides reduction by DIRB have not been studied. This study showed the sub-MIC antibiotics (ampicillin or ciprofloxacin) increased OMVs secretion in Geobacter sulfurreducens, and the antibiotic-induced OMVs contained more redox active cytochromes facilitating iron oxides reduction, especially for the ciprofloxacin-induced OMVs. Deduced from a combination of electron microscopy and proteomic analysis, the influence of ciprofloxacin on SOS response triggered prophage induction and led to the formation of outer-inner membrane vesicles (OIMVs) in, which was a first report in Geobacter species. While ampicillin disrupting cell membrane integrity resulted in more formation of classic OMVs from outer membrane blebbing. The results indicated that the different structure and composition of vesicles were responsible for the antibiotic-dependent regulation on iron oxides reduction. This newly identified regulation on EET-mediated redox reactions by sub-MIC antibiotics expands our knowledge about the impact of antibiotics on microbial processes or "non-target" organisms.

Nimotuzumab Plus Gemcitabine for K-Ras Wild-Type Locally Advanced or Metastatic Pancreatic Cancer.

PURPOSE: In a phase IIb trial of nimotuzumab plus gemcitabine, substantial clinical benefits were observed in patients with locally advanced or metastatic pancreatic cancer (PC). Therefore, we conducted a phase III clinical study to verify the efficacy and safety of this combination regimen in patients with K-Ras wild-type tumors (ClinicalTrials.gov identifier: NCT02395016). PATIENTS AND METHODS: Eligible patients were randomly assigned to receive nimotuzumab (400 mg once per week) or placebo followed by gemcitabine (1,000 mg/m2 on days 1, 8, and 15, once every 4 weeks) until disease progression or unacceptable toxicity. The primary end point was overall survival (OS) and the secondary end points were progression-free survival (PFS), response rates, and safety. RESULTS: A total of 480 patients were screened; 92 patients were enrolled and 82 patients with K-Ras wild-type tumors were eligible. In the full analysis set, the median OS was 10.9 versus 8.5 months, while the restricted mean survival time (RMST) was 18.05 versus 11.14 months for the investigational versus control arm (ratio of control v investigation = 0.62 [0.40-0.97]; P = .036). Median PFS was 4.2 versus 3.6 months in the investigational versus control arm (log-rank P = .04; hazard ratio, 0.60 [0.37-0.99]) and the restricted mean PFS time was 8.08 versus 4.76 months (RMST ratio, 0.58 [0.38-0.90]; P = .036). Both OS and PFS were longer in the nimotuzumab group than in the placebo group. The objective response rates and disease control rates were 7% versus 10% and 68% versus 63% for the investigational and control groups, respectively. The incidence of adverse events were comparable between the two groups. CONCLUSION: In patients with locally advanced or metastatic K-Ras wild-type PC, nimotuzumab plus gemcitabine significantly improved OS and PFS with a good safety profile.

Emerging roles of circular RNAs in non­small cell lung cancer (Review).

Circular RNAs (circRNAs) are a class of novel endogenous transcripts with limited protein­coding abilities. CircRNAs have been demonstrated to function as critical regulators of tumor development and distant metastasis through binding to microRNAs (miRNAs) and interacting with RNA­binding proteins, thereby regulating transcription and translation. Emerging evidence has illustrated that certain circRNAs can serve as biomarkers for diagnosis and prognosis of cancer, and/or serve as potential therapeutic targets. Expression of functional circRNAs is commonly dysregulated in cancer and this is correlated with advanced Tumor­Node­Metastasis stage, lymph node status, distant metastasis, poor differentiation and shorter overall survival of cancer patients. Recently, an increasing number of studies have shown that circRNAs are closely associated with NSCLC. Functional experiments have revealed that circRNAs are intricately associated with the pathological progression of NSCLC. The present review provides an overview of the regulatory effect of circRNAs in the development and progression of NSCLC, taking into consideration various physiological and pathological processes, such as proliferation, apoptosis, invasion and migration, and their potential value as biomarkers and therapeutic targets.

The complete chloroplast genome of Cuscuta australis R. Br. (Convolvulaceae) and its phylogenetic implication.

The genus Cuscuta (Convolvulaceae) is an annual parasitic twining herb. There are about 200 species in this genus, which are widely distributed in tropical and subtropical areas. Cuscuta is mainly parasitic on crops bringing significant losses to the production of agriculture. Furthermore, dried seeds of C. chinensis and C. australis are used as a Chinese traditional herbal medicine. Despite the importance of Cuscuta species, it is difficult to distinguish these plants by the naked eye. Moreover, plastid sequence information available for Cuscuta species is limited. In this study, the complete chloroplast (cp) genome sequence of C. australis was determined using next-generation sequencing. The entire cp genome was determined to be 85,263 bp in length. It contained large single-copy (LSC) and small single-copy (SSC) regions of 50,384 and 6727 bp, respectively, which were separated by a pair of 14,076 bp inverted repeat (IR) regions. The genome contained 98 genes, including 61 protein-coding genes, 29 tRNA genes, and 4 rRNA genes. The overall GC content of the genome is 37.8%. A phylogenetic tree reconstructed by 26 chloroplast genomes reveals that C. australis is most related with Cuscuta pentagona in Convolvulaceae, with bootstrap support values of 100%.

Schedule-dependent synergistic interaction between gemcitabine and oxaliplatin in human gallbladder adenocarcinoma cell lines.

To define the most effective combination schedule of gemcitabine and oxaliplatin (L-OHP), we investigated the in-vitro interaction between these drugs in a panel of four human gallbladder adenocarcinoma cell lines (HAG-1, GB-d1, NOZ, and G-415). Cytotoxic activity was determined by the WST-1 assay. Different schedules of the two drugs were compared and evaluated for synergism, additivity, or antagonism with a quantitative method based on the median-effect principle of Chou and Talalay. Cell cycle perturbation and apoptosis were evaluated by flow cytometry. Simultaneous and sequential treatments of gemcitabine followed by L-OHP exhibited synergistic effects in all four cell lines, whereas the reverse sequence largely showed an antagonism. Gemcitabine exclusively arrested cells at the G0/G1 phase, and L-OHP at the G2/M phase, as measured by flow cytometric analyses. Apoptosis was most prominent when cells were treated simultaneously or in a sequence gemcitabine followed by L-OHP, producing apoptosis in treated cells (27-30%). In contrast, the reverse sequence yielded only 6-7% induction of apoptosis, the rate being not significantly different from those induced by each drug singly. Moreover, this sequence dependence was further confirmed by the experiment, which compared the number of HAG-1 cells 7 days after these combination schedules. These findings suggest that the interaction of gemcitabine and L-OHP is highly schedule dependent, with the most efficacious interaction observed in either simultaneous combination or in a sequence combination of gemcitabine followed by L-OHP.