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ObjectiveAn unmet need exists for a non-invasive biomarker assay to aid gastric cancer diagnosis. We aimed to develop a serum microRNA (miRNA) panel for identifying patients with all stages of gastric cancer from a high-risk population.DesignWe conducted a three-phase, multicentre study comprising 5248 subjects from Singapore and Korea. Biomarker discovery and verification phases were done through comprehensive serum miRNA profiling and multivariant analysis of 578 miRNA candidates in retrospective cohorts of 682 subjects. A clinical assay was developed and validated in a prospective cohort of 4566 symptomatic subjects who underwent endoscopy. Assay performance was confirmed with histological diagnosis and compared with Helicobacter pylori (HP) serology, serum pepsinogens (PGs), ‘ABC’ method, carcinoembryonic antigen (CEA) and cancer antigen 19–9 (CA19-9). Cost-effectiveness was analysed using a Markov decision model.ResultsWe developed a clinical assay for detection of gastric cancer based on a 12-miRNA biomarker panel. The 12-miRNA panel had area under the curve (AUC)=0.93 (95% CI 0.90 to 0.95) and AUC=0.92 (95% CI 0.88 to 0.96) in the discovery and verification cohorts, respectively. In the prospective study, overall sensitivity was 87.0% (95% CI 79.4% to 92.5%) at specificity of 68.4% (95% CI 67.0% to 69.8%). AUC was 0.848 (95% CI 0.81 to 0.88), higher than HP serology (0.635), PG 1/2 ratio (0.641), PG index (0.576), ABC method (0.647), CEA (0.576) and CA19-9 (0.595). The number needed to screen is 489 annually. It is cost-effective for mass screening relative to current practice (incremental cost-effectiveness ratio=US$44 531/quality-of-life year).ConclusionWe developed and validated a serum 12-miRNA biomarker assay, which may be a cost-effective risk assessment for gastric cancer.Trial registration numberThis study is registered with ClinicalTrials.gov (Registration number: NCT04329299).

Minimally invasive testing for early detection of lung cancer to improve patient survival is a major unmet clinical need. This study aimed to develop and validate a serum multi-microRNA (multimiR) panel as a minimally invasive test for early detection of nonsmall cell lung cancer (NSCLC) regardless of smoking status, gender, and ethnicity. Our study included 744 NSCLC cases and 944 matched controls, including smokers and nonsmokers, male and female, with Asian and Caucasian subjects. Using RT-qPCR and a tightly controlled workflow, we quantified the absolute expression of 520 circulating microRNAs (miRNAs) in a Chinese cohort of 180 early stage NSCLC cases and 216 healthy controls (male smokers). Candidate biomarkers were verified in two case-control cohorts of 432 Chinese and 218 Caucasians, respectively (including females and nonsmokers). A multimiR panel for NSCLC detection was developed using a twofold cross-validation and validated in three additional Asian cohorts comprising 642 subjects. We discovered 35 candidate miRNA biomarkers, verified 22 of them, and developed a five-miR panel that detected NSCLC with area under curve (AUC) of 0.936-0.984 in the discovery and verification cohorts. The panel was validated in three independent cohorts with AUCs of 0.973, 0.916, and 0.917. The sensitivity of five-miR test was 81.3% for all stages, 82.9% for stages I and II, and 83.0% for stage I NSCLC, when the specificity is at 90.7%. We developed a minimally invasive five-miR serum test for detecting early stage NSCLC and validated its performance in multiple patient cohorts independent of smoking status, gender, and ethnicity.

Objectives To evaluate cost-effectiveness of a novel screening strategy using a microRNA (miRNA) blood test as a screen, followed by endoscopy for diagnosis confirmation in a 3-yearly population screening program for gastric cancer. Methods A Markov cohort model has been developed in Microsoft Excel 2016 for the population identified to be at intermediate risk (Singaporean men, aged 50-75 years with Chinese ethnicity). The interventions compared were (1) initial screening using miRNA test followed by endoscopy for test-positive individuals and a 3-yearly follow-up screening for test-negative individuals (proposed strategy), and (2) no screening with gastric cancer being diagnosed clinically (current practice). The model was evaluated for 25 years with a healthcare perspective and accounted for test characteristics, compliance, disease progression, cancer recurrence, costs, utilities, and mortality. The outcomes measured included incremental cost-effectiveness ratios, cancer stage at diagnosis, and thresholds for significant variables. Results The miRNA-based screening was found to be cost-effective with an incremental cost-effectiveness ratio of $40 971/quality-adjusted life-year. Key drivers included test costs, test accuracy, cancer incidence, and recurrence risk. Threshold analysis highlights the need for high accuracy of miRNA tests (threshold sensitivity: 68%; threshold specificity: 77%). A perfect compliance to screening would double the cancer diagnosis in early stages compared to the current practice. Probabilistic sensitivity analysis reported the miRNA-based screening to be cost-effective in >95% of iterations for a willingness to pay of $70 000/quality-adjusted life-year (approximately equivalent to 1 gross domestic product/capita) Conclusions The miRNA-based screening intervention was found to be cost-effective and is expected to contribute immensely in early diagnosis of cancer by improving screening compliance.

Circulating miRNAs are promising liquid biopsy biomarkers for noninvasive cancer detection. However, detection of subtle, but meaningful differences in circulating miRNA quantities between diseased and healthy samples remains a key challenge in clinical settings because biomarker signal/noise ratios are often low. Because extracellular vesicles (EVs) are key sources of circulating miRNAs in serum, it was hypothesized that isolating EVs would enrich miRNA biomarkers, leading to enhanced diagnostic ability and improved biomarker performance. This research assessed the performance of EV-miRNAs against serum miRNAs as biomarkers for gastric cancer (GC). It was first determined that polymer-based precipitation (PBP) gave the highest EV-miRNA recovery when compared with ultracentrifugation, column affinity, peptide affinity, and immunobead affinity EV purification. Four PBP reagents were used to isolate EV-miRNAs from 15 GC and 15 healthy controls and 133 GC-related miRNAs were profiled from EV fractions and total serum using real-time quantitative PCR. A PBP reagent that generated the most EV-miRNA biomarkers was selected and used to validate 11 EV-miRNAs in an independent set of 20 GC and 20 controls. Eight of these EV-miRNA biomarkers were found to give better GC detection accuracy (area under the curve, approximately 0.8). Overall, data suggest that EV miRNAs can improve GC detection performance compared with serum miRNAs and led to the identification of eight EV-miRNAs as potential noninvasive biomarkers for GC.

Background Clinicians need improved tools to better identify nonacute heart failure with preserved ejection fraction (HFpEF). Objectives The purpose of this study was to derive and validate circulating microRNA signatures for nonacute heart failure (HF). Methods Discovery and validation cohorts (N = 1,710), comprised 903 HF and 807 non-HF patients from Singapore and New Zealand (NZ). MicroRNA biomarker panel discovery in a Singapore cohort (n = 546) was independently validated in a second Singapore cohort (Validation 1; n = 448) and a NZ cohort (Validation 2; n = 716). Results In discovery, an 8-microRNA panel identified HF with an area under the curve (AUC) 0.96, specificity 0.88, and accuracy 0.89. Corresponding metrics were 0.88, 0.66, and 0.77 in Validation 1, and 0.87, 0.58, and 0.74 in Validation 2. Combining microRNA panels with N-terminal pro–B-type natriuretic peptide (NT-proBNP) clearly improved specificity and accuracy from AUC 0.96, specificity 0.91, and accuracy 0.90 for NT-proBNP alone to corresponding metrics of 0.99, 0.99, and 0.93 in the discovery and 0.97, 0.96, and 0.93 in Validation 1. The 8-microRNA discovery panel distinguished HFpEF from HF with reduced ejection fraction with AUC 0.81, specificity 0.66, and accuracy 0.72. Corresponding metrics were 0.65, 0.41, and 0.56 in Validation 1 and 0.65, 0.41, and 0.62 in Validation 2. For phenotype categorization, combined markers achieved AUC 0.87, specificity 0.75, and accuracy 0.77 in the discovery with corresponding metrics of 0.74, 0.59, and 0.67 in Validation 1 and 0.72, 0.52, and 0.68 in Validation 2, as compared with NT-proBNP alone of AUC 0.71, specificity 0.46, and accuracy 0.62 in the discovery; with corresponding metrics of 0.72, 0.44, and 0.57 in Validation 1 and 0.69, 0.48, and 0.66 in Validation 2. Accordingly, false negative (FN) (81% Singapore and all NZ FN cases were HFpEF) as classified by a guideline-endorsed NT-proBNP ruleout threshold, were correctly reclassified by the 8-microRNA panel in the majority (72% and 88% of FN in Singapore and NZ, respectively) of cases. Conclusions Multi-microRNA panels in combination with NT-proBNP are highly discriminatory and improved specificity and accuracy in identifying nonacute HF. These findings suggest potential utility in the identification of nonacute HF, where clinical assessment, imaging, and NT-proBNP may not be definitive, especially in HFpEF.

Simple Summary Breast cancer screening by mammography suffers from high rates of false positivity, resulting in unnecessary investigative imaging and biopsies. There is an unmet need for biomarkers that can distinguish between malignant and benign breast lesions. We performed miRNA profiling on 638 patients with abnormal mammograms and 100 healthy controls. A six-miRNA panel was identified and validated in an independent cohort that had an AUC of 0.881 when differentiating between cases versus those with benign lesions or healthy individuals with normal mammograms. In addition, biomarker panel scores increased with tumor size, stage and number of lymph nodes involved. This study demonstrates that circulating miRNAs can potentially be used in conjunction with mammography to differentiate between patients with malignant and benign breast lesions. Abstract Mammography is extensively used for breast cancer screening but has high false-positive rates. Here, prospectively collected blood samples were used to identify circulating microRNA (miRNA) biomarkers to discriminate between malignant and benign breast lesions among women with abnormal mammograms. The Discovery cohort comprised 72 patients with breast cancer and 197 patients with benign breast lesions, while the Validation cohort had 73 and 196 cancer and benign cases, respectively. Absolute expression levels of 324 miRNAs were determined using RT-qPCR. miRNA biomarker panels were identified by: (1) determining differential expression between malignant and benign breast lesions, (2) focusing on top differentially expressed miRNAs, and (3) building panels from an unbiased search among all expressed miRNAs. Two-fold cross-validation incorporating a feature selection algorithm and logistic regression was performed. A six-miRNA biomarker panel identified by the third strategy, had an area under the curve (AUC) of 0.785 and 0.774 in the Discovery and Validation cohorts, respectively, and an AUC of 0.881 when differentiating between cases versus those with benign lesions or healthy individuals with normal mammograms. Biomarker panel scores increased with tumor size, stage and number of lymph nodes involved. Our work demonstrates that circulating miRNA signatures can potentially be used with mammography to differentiate between patients with malignant and benign breast lesions.

Optimization of metabolic pathways consisting of large number of genes is challenging. Multivariate modular methods (MMMs) are currently available solutions, in which reduced regulatory complexities are achieved by grouping multiple genes into modules. However, these methods work well for balancing the inter-modules but not intra-modules. In addition, application of MMMs to the 15-step heterologous route of astaxanthin biosynthesis has met with limited success. Here, we expand the solution space of MMMs and develop a multidimensional heuristic process (MHP). MHP can simultaneously balance different modules by varying promoter strength and coordinating intra-module activities by using ribosome binding sites (RBSs) and enzyme variants. Consequently, MHP increases enantiopure 3S,3′S-astaxanthin production to 184 mg l−1 day−1 or 320 mg l−1. Similarly, MHP improves the yields of nerolidol and linalool. MHP may be useful for optimizing other complex biochemical pathways., Achieving high titer yield and productivity of target chemicals in industrial organism depends on multidimensional pathway optimization. Here, the authors use a refined modular method called multidimensional heuristic process to improve production of astaxanthin, nerolidol and linalool in E. coli.

Apocarotenoids, such as α-, β-ionone and retinol, have high commercial values in the food and cosmetic industries. The demand for natural ingredients has been increasing dramatically in recent years. However, attempts to overproduce β-ionone in microorganisms have been limited by the complexity of the biosynthetic pathway. Here, an Escherichia coli-based modular system was developed to produce various apocarotenoids. Incorporation of enzyme engineering approaches (N-terminal truncation and protein fusion) into modular metabolic engineering strategy significantly improved α-ionone production from 0.5 to 30 mg/L in flasks, producing 480 mg/L of α-ionone in fed-batch fermentation. By modifying apocarotenoid genetic module, this platform strain was successfully re-engineered to produce 32 mg/L and 500 mg/L of β-ionone in flask and bioreactor, respectively (> 80-fold higher than previously reported). Similarly, 33 mg/L of retinoids was produced in flask by reconstructing apocarotenoid module, demonstrating the versatility of the "plug-n-play" modular system. Collectively, this study highlights the importance of the strategy of simultaneous modular pathway optimization and enzyme engineering to overproduce valuable chemicals in microbes. This article is protected by copyright. All rights reserved

The preservation of nucleic acids from clinical samples is critical to facilitate accurate molecular diagnosis. The use of a paper matrix, Flinders Technology Associates (FTA) Elute cards, to archive DNA and viral RNA is well-documented. However, the feasibility of FTA Elute cards for archiving serum and serum exosomal microRNAs (miRNAs) remains unclear. Here, we performed a comprehensive evaluation of FTA Elute cards for miRNA storage and recovery in different pre-analytical conditions. The recovery of serum miRNA dry-spotted on FTA Elute cards by direct elution with water at high temperature was poor. However, serum miRNAs dry-spotted on the cards were isolated with about 40% yield when using QIAzol lysis reagent and recovery was improved remarkably (>80%) upon extraction from cards pre-treated with trehalose. miRNAs stored on the cards remained stable at room temperature and can be kept for prolonged periods. Furthermore, miRNAs could be similarly recovered from serum exosomes dry-spotted on the cards. Importantly, when using sera from gastric cancer (GC) patients, the miRNAs were efficiently recovered from trehalose pre-treated cards without affecting their representation. Collectively, we have demonstrated the potential of FTA Elute cards to archive serum and serum exosomal miRNAs, making it useful for biomarker discovery and diagnostics.

Metabolic engineering aims at altering cellular metabolism to produce valuable products at high yields and titers. Achieving high titers and productivity can be challenging if final products are largely accumulated intracellularly. A potential solution to this problem is to facilitate the export of these substances from cells by membrane transporters. Amorphadiene, the precursor of antimalarial drug artemisinin, is known to be secreted from Escherichia coli overexpressing the biosynthetic pathway. In order to assess the involvement of various endogenous efflux pumps in amorphadiene transport, the effects of single gene deletion of 16 known multidrug-resistant membrane efflux transporters were examined. The outer membrane protein TolC was found to be intimately involved in amorphadiene efflux. The overexpression of tolC together with ABC family transporters (macAB) or MFS family transporters (emrAB or emrKY) enhanced amorphadiene titer by more than threefold. In addition, the overexpression of transporters in the lipopolysaccharide transport system (msbA, lptD, lptCABFG) was found to improve amorphadiene production. As efflux transporters often have a wide range of substrate specificity, the multiple families of transporters were co-expressed and synergistic benefits were observed in amorphadiene production. This strategy of screening and then rationally engineering transporters can be used to improve the production of other valuable compounds in E. coli. Biotechnol. Bioeng. 2016;113: 1755-1763. © 2016 Wiley Periodicals, Inc.

Background: Survival outcomes of breast cancer patients can be significantly improved by early detection and treatment. Implementation of mammogram-based screening has significantly improved early detection of breast cancer in the west. However, the use of screening mammography is less prevalent in Asia partly due to social and cultural reasons. The aim of this study was to determine if a serum microRNA (miRNA) panel could be used as blood-based biomarkers to assist in the early detection of breast cancer. Methods: We carried out a multi-center, multi-ethnic study to identify and validate miRNA biomarkers for the early detection of breast cancer. A total of 1042 subjects including 540 breast cancer cases (predominantly stage 1 and 2 cases) and 502 matched controls from 6 independent sources were included in this study. Among these, there were 750 American and European subjects recruited by biobanks and 292 Singaporean Asian Subjects recruited at the National Cancer Centre Singapore and the National University Hospital. The study was conducted in 3 phases in which sera of 289 European Caucasian serum samples (Discovery Cohort) were first interrogated to identify differentially expressed miRNAs between early-stage breast cancer cases and matched controls among 520 circulating miRNA candidates by quantitative RT-PCR using MiRXES assays. The remaining 753 subjects from 5 independent sources were assigned into two groups for biomarker optimization/algorithm development (Optimization Cohort, n=374) and validation (Validation Cohort, n=379). Results: Among the 520 circulating miRNAs measured, 241 were quantified in absolute copy numbers for 289 subjects in the Discovery Cohort. Thirty-three candidate miRNAs were identified. These miRNAs consistently showed differential expression between cancer and control subjects in the Optimization Cohort. A multi-variant panel of 8 miRNAs and an algorithm was developed using the Discovery and Optimization Cohort, with an AUC of 0.981 and 0.918 respectively. When validated in the independent Validation Cohort, the panel demonstrated an AUC of 0.915. Conclusion: We developed and validated a serum miRNA panel that is both sensitive (72.2 - 77.4%) and specific (90.2 - 91.5%) in detecting early stage of breast cancer in both Caucasian and Asian populations. Citation Format: Lihan Zhou, Ruiyang Zou, Sau Yeen Loke, Mikael Hartman, Patrick C.K Goo, Ann S.G. Lee-Lim, Heng-Phon Too. Identification and validation of a serum microRNA panel for detection of early-stage breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-02.

To date, low transfection efficiency remains the major drawback of polymer based gene delivery. Many cell types including stem cells, fibroblast and neurons are known to be poorly transfected with polymer based gene carriers and the high toxicity severely restrict their utility in gene delivery. Continual efforts are made to identify cellular barriers to efficient transfection as these carriers have low immunogenicity, ease of manufacturing and scalability. Here, we summarize the current status of understanding on uptake mechanism of polymer-DNA complexes (polyplexes), their endosomal escape, cytosolic transport and nuclear entry of pDNA.

Deep brain stimulation (DBS) is an established therapy for movement disorders such as Parkinson's disease (PD). Although the efficacy of DBS is clear, its precise molecular mechanism remains unknown. The glial cell line derived factor (GDNF) family of ligands has been shown to confer neuroprotective effects on dopaminergic neurons, and putaminal infusion of GDNF have been investigated in PD patients with promising results. Despite the potential therapeutic role of GDNF in alleviating motor symptoms, there is no data on the effects of electrical stimulation on GDNF-family receptor (GFR) expression in the basal ganglia structures. Here, we report the effects of electrical stimulation on GFRα1 isoforms, particularly GFRα1a and GFRα1b. Wistar rats underwent 2 hours of high frequency stimulation (HFS) at the globus pallidus interna nucleus. A control group was subjected to a similar procedure but without stimulation. The HFS group, sacrificed 24 hours after treatment, had a threefold decrease in mRNA expression level of GFRα1b (p=0.037), but the expression level reverted to normal 72 hours after stimulation. Our preliminary data reveal the acute effects of HFS on splice isoforms of GFRα1, and suggest that HFS may modulate the splice isoforms of GFRα1a and GFRα1b to varying degrees. Going forward, elucidating the interactions between HFS and GFR may shed new insights into the complexity of GDNF signaling in the nervous system and lead to better design of clinical trials using these signaling pathways to halt disease progression in PD and other neurodegenerative diseases.

Gliomas account for 70% of human malignant primary brain tumours. The most common form is glioblastoma multiforme, World Health Organization grade IV. Despite the implementation of post-operative adjuvant radiotherapy with concurrent temozolomide (TMZ), the disease’s overall prognosis remains dismal. TMZ is currently the only mono-chemotherapeutic agent for newly-diagnosed high-grade glioma patients and acquired resistance inevitably occurs in the majority of such patients, further limiting treatment options. Therefore, there is an urgent need to better understand the underlying mechanisms involved in TMZ resistance, a critical step to developing effective, targeted treatments. An emerging body of evidence suggests the intimate involvement of a novel class of nucleic acid, microRNA (miRNA), in tumorigenesis and disease progression for a number of human malignancies, including primary brain tumours. miRNA are short, single-stranded, non-coding RNA (∼22 nucleotides) that function as post-transcriptional regulators of gene expression. This review provides an overview of the key treatment obstacles faced in patients with high-grade gliomas, especially in the context of recurrent, chemoresistant tumours and the potential roles of miRNA in chemoresistance and management of this disease.

4065 Background: High mortality from gastric cancer is related to the late manifestation of its symptoms. A blood-based non-invasive biomarker with the ability to detect all stages of gastric cancer could significantly improve patient outcomes. We aimed to develop a novel serum miRNA assay for diagnosis of gastric cancer. Methods: We conducted a multi-center study involving 892 gastric cancer and control subjects from Singapore and Korea to develop a multi-target miRNA assay. Using RT-qPCR, we quantified the expressions of 578 serum miRNAs and constructed a 12-miR biomarker panel through multi-variant data analysis. The results were generated with the use of a logistic-regression algorithm, with the value of 40 or more considered to be positive. We subsequently validated this multi-miR assay in a large prospective cohort involving 4566 subjects and compared its performance with traditional markers such as H.Pylori and Pepsinogen. All participants underwent gastroscopy independent of the assay results. Results: Of the 4566 subjects that underwent gastroscopy and histopathological examination in the prospective cohort, 125 were diagnosed with gastric cancer. The 12-miR assay achieved an Area-Under-Curve (AUC) of 0.84, significantly outperforming (p-value < 0.01) that of H.Pylori (AUC of 0.64) and Pepsinogen (AUC of 0.62). The sensitivity of the miRNA assay in detecting early (stage 0-2) and late (stage 3-4) stage gastric cancer was 82.6% (95% CI, 68.6% to 92.2%) and 88.4% (95% CI, 78.4% to 94.9%) respectively at a specificity of 70.0% (95% CI, 67.8% to 71.9%). In comparison, H.Pylori showed a sensitivity of 80.4% at a specificity of 44.3% whereas the Pepsinogen showed sensitivity of 9.52% at a specificity of 95.3%. Using the miRNA assay as a pre-screening tool could potentially reduce number of endoscopy needed by 62% in detecting one case of gastric cancer. Conclusions: Our serum miRNA panel is a useful, non-invasive screening test for gastric cancer. It is cost-effective as it can reduce unnecessary diagnostic endoscopy.

3051 Background: The implementation of mammogram-based screening has significantly improved the early detection of breast cancer in the west. However, the use of screening mammography is less prevalent in Asia partly due to social and cultural reasons. The aim of this study was to determine if a serum microRNA (miRNA) panel could be used as biomarkers to assist in the early detection of breast cancer. Methods: We conducted a multi-center, multi-ethnic study to identify and validate miRNA biomarkers for the early detection of breast cancer. A total of 1070 subjects including 550 breast cancer cases (predominantly stage 1 and 2) and 520 matched controls from 6 independent sources were included in this study. Among these, there were 768 American and European subjects recruited by biobanks and 302 Singaporean Asian Subjects recruited at the National Cancer Centre Singapore and the National University Hospital. The study was conducted in 3 phases. First, 119 European Caucasian serum samples (Discovery Cohort) were interrogated to identify differentially expressed miRNAs between early-stage breast cancer cases and matched controls, among 520 circulating miRNA candidates by quantitative RT-PCR using MiRXES assays. The remaining 951 subjects from 5 independent sources were assigned into two groups for biomarker optimization/algorithm development (Optimization Cohort, n = 451) and validation (Validation Cohort, n = 500). Results: Among the 520 circulating miRNAs measured, 241 were quantified in absolute copy numbers for 119 subjects in the Discovery Cohort. Thirty-two candidate miRNAs were identified. These miRNAs consistently showed differential expression between cancer and control subjects in the Optimization Cohort. A multi-variant panel of 8 miRNAs and an algorithm was developed using the combined Discovery and Optimization Cohorts, with an AUC of > 0.90. When validated in the independent Validation Cohort, the panel demonstrated an AUC of > 0.85. Conclusions: We developed and validated a serum miRNA panel that is both sensitive (~70%) and specific (~90%) in detecting early-stage breast cancer.

Background & Aim Mesenchymal stem cells (MSC) are emerging as promising vehicles for Gene-directed enzyme prodrug therapy (GDEPT). The inherent tumor-trophic migratory properties of MSC enable these vehicles to deliver effective, targeted therapy to tumors and metastatic diseases. A critical step in modifying MSC is the delivery of genes with high efficiency and low cytotoxicity. Due to the poor efficiency of transfection approaches, viral methods are used extensively to transduce MSC in preclinical and clinical studies. This study aims to develop a scalable non viral gene delivery method to efficiently modify MSC. Methods, Results & Conclusion Methods TrafEn (Trafficking Enhancer), to enhance transfection using off-the-shelf, cost-effective polymers. TrafEn was rationally developed to facilitate efficient trafficking of the genetic material inside cells. Polyplex transfection was enhanced by including a simultaneous treatment with DOPE/CHEMS lipid suspension and a microtubule stabilizer, a histone deacetylase-6 inhibitor. Result We demonstrate, for the first time, the efficient transfection (>90%) of human adipose tissue derived MSCs (AT-MSCs) using an off-the shelf and cost-effective cationic polymer, polyethylenimine, in the presence of fusogenic lipids and histone deacetylase 6 inhibitor (HDAC6i) (Figure 1). Importantly, the cell phenotypes of MSCs remained unchanged after modification, a key requisite for clinical application of MSCs. AT-MSCs modified with a fused transgene, yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT), exhibited strong cytotoxic effects towards glioma, breast and gastric cancer cells in vitro. The efficiency of killing of gastric MKN1 and MKN28 cell lines were greater than 80% even with 7 days post-transfected AT-MSCs, indicative that the expression period of the therapeutic gene was sustainable. Conclusion This study describes a useful tool for polymer based ex vivo MSC modification which is highly scalable and cost-effective. The proposed workflow bypasses the restriction in viral vector supply and expedites MSC modification, without compromising the quality and anti-cancer efficacy of the theranostic MSCs.

J. Neurochem. (2010) 115, 759–770. Abstract Malignant gliomas are highly invasive neuroepithelial tumors where the tendency to invade and migrate away from the primary tumor mass is thought to be a leading cause of tumor recurrence and treatment failures. Autocrine signals produced by secreted factors that signal through receptors on the tumor are known to contribute to the invasiveness. Glial cell line-derived neurotrophic factor and GDNF family receptor alpha 1 (GFRα1) are over-expressed in human gliomas. We have previously reported that human gliomas express high levels of GFRα1b, an alternatively spliced isoform of GFRα1. However, the functional significance of GFRα1b in glioma behaviors is currently unknown. In this study, we have designed isoform-specific small-interfering RNA to knockdown the highly homologous GFRα1a or GFRα1b isoform efficiently in malignant C6 glioma cells. Unexpectedly, the knockdown of GFRα1b but not GFRα1a induced cell elongation and inhibited C6 cell migration and invasion in vitro. In addition, GFRα1b was found to regulate the expression of RhoA small GTPase, which was required for migration of C6 cells. The decreases in RhoA expression and cell migration after GFRα1b knockdown were attenuated by small-interfering RNA -resistant GFRα1b but not GFRα1a, further demonstrating the specific role of GFRα1b in glioma migration. Interestingly, the knockdown of NCAM but not receptor tyrosine kinase Ret resulted in the reduction of RhoA expression and C6 cell migration. Taken together, these unanticipated results indicate that GFRα1b is involved in glioma migration through glial cell line-derived neurotrophic factor –GFRα1b-NCAM signaling complex and modulation of RhoA expression.

Glial cell-line derived neurotrophic factor (GDNF) is a neurotrophic factor known to promote neuronal survival of dopaminergic neurons in the embryonic midbrain as well as contribute to carcinogenesis in many cancers. Its ubiquitous presence in the central nervous system suggests a role in the mitogenesis of high-grade astrocytoma. GDNF is overexpressed in glioblastoma cell lines and human gliomas. GFRalpha1b is the predominant spliced receptor isoform in human gliomas and RET9 is the predominant co-receptor. Significantly there is differential overexpression of the GFRalpha1b spliced isoform compared to the GFRalpha1a spliced variant. Pre-treatment of glioblastoma cell lines with GDNF but not the alternative ligand neurturin, promoted mitogenic behaviour and conferred chemoresistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Signaling mapping of BCNU and GDNF suggest that the ability of GDNF to promote Akt activity and inhibit JNK activity may contribute to the increased cellular survival after BCNU chemotherapy.

Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study., Singapore-MIT Alliance for Research and Technology (SMART)

A stem–loop-based method to quantify the replicative strand of a model system, dengue virus, with high specificity and sensitivity is described. The high specificity of this approach is achieved at two levels: the use of a reverse transcription primer folded into a stem–loop structure with optimal energetics and the use of specific PCR primers to the loop structure. This approach has exceptional specificity to the replicative RNA as compared with the genomic sequence (>10 5 -fold difference), with a detection sensitivity of 10 copies. The high correlation to the biological “gold standard” plaque assay, used to quantify infectious virus, renders this method a useful quantitative tool that can replace the time-consuming, labor-intensive, and low-throughput plaque-based assays. The method has been extended to the detection of replicative strands of other RNA viruses ( West Nile virus and human respiratory syncytial virus ) with similar results. This real-time PCR method is reliable, simple to perform, and easily adaptable to different targets. The ability to detect and rapidly quantify replicating viruses is an important step in the elucidation of pathogenesis and is also useful for the evaluation of drugs designed to inhibit viral replication.

Four immunoreactive proteins, B.4, B.6, B.10, and B.M, with molecular weights ranging from 16,000 to 58,000, were observed from immunoblots of Mycobacterium tuberculosis total lysates screened with sera from individuals with active tuberculosis. These proteins were identified from microsequence analyses, and genes of proteins with the highest homology were PCR amplified and cloned into the pQE30 vector for expression studies. In addition, a 37.5-kDa protein, designated C17, was identified from a phage expression library of M. tuberculosis genomic DNA. Preliminary immunoblot assays indicated that these five resultant recombinant proteins could detect antibodies in individuals with active pulmonary and extrapulmonary tuberculosis. The overall ranges of sensitivities, specificities, positive predictive values, and negative predictive values for the recombinant antigens were 20 to 58, 88 to 100, 69 to 100, and 56 to 71%, respectively. The B.6 antigen showed preferential reactivity to antibodies in pulmonary compared to nonpulmonary tuberculosis serum specimens. All of these recombinant antigens demonstrated potential for serodiagnosis of tuberculosis. Tuberculosis (TB) is a major health problem in the developing world as well as a disease which is reemerging as a major health threat in the developed world (6). World Health Organization (WHO) statistics indicate that one-third of the world’s population is currently infected (34). TB is rated the second most common infectious disease and has the highest mortality rate of any infectious disease in the world. Currently there are 30 million cases of active TB worldwide, with approximately 8 million new cases and 3 million deaths reported annually. In addition, 50 million people may already be infected with multidrug-resistant (MDR) strains of Mycobacterium tuberculosis. A high prevalence of TB is also associated with human immunodeficiency virus (HIV) infection and AIDS and is now becoming the leading cause of death among HIV-positive individuals, with a fatality rate of 80% (34). Control of this disease revolves around good patient care and management. In particular, early detection and treatment of tuberculosis can limit transmission of the bacilli. Conventional tests for the diagnosis of tuberculosis include chest Xray, direct sputum smear for acid-fast bacilli, culture test, and the skin tuberculin PPD (purified protein derivative) test (29). Among these, the culture method is time-consuming but reliable. PCR and nucleic acid-based methods for detecting M. tuberculosis DNA sequences require complex equipment and highly skilled staff, and they are expensive and unsuitable for routine diagnostic testing in developing countries (5). Rapid serological diagnostic tests such as the enzyme-linked immunosorbent assay (ELISA) and membrane chromatography tests, in contrast, are simple and inexpensive, and the latter can be point-of-care devices (3). A major problem encountered in serological techniques is the specificity and reactivity of antigens used. A majority of M. tuberculosis antigens studied to date have homology with analogous proteins of environmental mycobacteria or other bacteria, resulting in unspecific reactivity to antibodies in patients with inactive TB or nontuberculous infections (8, 23). Hence, positive test results produced by these known antigens are generally unreliable, and supplementary tests are required to confirm tuberculosis infection. It was shown that the use of recombinant M. tuberculosis antigens of specific purity or particular epitopes may enhance the specificity and sensitivity of serological testing for TB when used in a panel of recombinant antigens (1). Rapid diagnostic tests that are specific and sensitive would be useful in both seroepidemiological and clinical studies pertaining to tuberculosis control and prevention. In this report, we describe the identification, isolation, and characterization of five recombinant antigens from M. tuberculosis for use as serodiagnostic markers for tuberculosis.

Background: Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results: In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion: This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli., Singapore-MIT Alliance

4060 Background: Gastric cancer is the 2nd most common cause of cancer deaths worldwide. Currently, endoscopy is the only reliable method for early diagnosis. However, the invasiveness and cost lim...

Introduction Glioblastoma is the most common primary brain tumour with poor overall survival. Surgical resection followed by radiation and chemotherapy is the mainstay of treatment. The role of aggressive resection in improving overall survival remains contentious, although there is evolving data to suggest this trend. Definitive evidence will necessitate a well-designed randomized prospective trial, although it is not likely that this will be possible or feasible. One possible advantage of aggressive tumour resection is a rapid reduction in oncological burden which may augment the effect of adjuvant chemotherapy. Materials and methods Three glioblastoma cell lines were seeded in concentrations from 5000-20,000 cells per well onto 96-well plates. The cells were incubated for 24 hours before treatment with varying concentrations of 1,3-Bis(2- chloroethyl)-1-nitrosourea (BCNU) ranging from 25 to 175 μg/mL. After 24 hours of treatment with BCNU, the cells were then examined microscopically and subjected to a cell proliferation assay to determine cytotoxicity effects of BCNU. Results The drug concentration required to achieve greater than 90% growth inhibition (IC90) was taken as the reference for efficacy of chemotherapy dose. With tumour loading of 5000 cells per well, BCNU concentrations of 75–100 μg/mL resulted in greater than IC90, whereas BCNU concentration of 150–175 μg/mL was required with tumour loading of 20,000 cells per well. A higher concentration of chemotherapeutic agent is therefore required to bring about cell death in the presence of greater tumour burden. Conclusion Higher glioblastoma loading confers chemoresistance to BCNU. This is possibly secondary to complex interactions between tumour cells and neighbouring cells acting via autocrine or paracrine signaling pathways.

The presence of mRNA transcripts coding for preprotachykinin-A and the substance P receptor in cultured astrocytes is demonstrated by a combination of reverse transcription/polymerase chain reaction (PCR) and Southern blotting. These findings showed that astrocytes in culture are capable of synthesing both the precursor of substance P (preprotachykinin-A) and the cognate receptor, substance P receptor (NK1). The simultaneous presence of both the ligand (substance P) and the receptor (NK1) may indicate an autocrine nature of astrocyte communication.

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Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons.

Calcitonin gene-related polypeptide (CGRP) has been localised immunochemically within the rat and guinea pig anterior uvea to nerve fibres of trigeminal origin. As with substance P (1-3) the level of CGRP in the iris-ciliary body is depleted after thermal damage to the Gasserian ganglion and elevated in chronically sympathectically denervated eyes. Unlike substance P, a potent pupillary constrictor (4,5), CGRP has no notable miotic action, but does, however, cause an elevation of the intraocular pressure (IOP) accompanied by disruption of the blood-aqueous barrier. It is proposed that the diverse actions of these two sensory neuropeptides conjointly mediate the antidromic ocular injury response.

The presence of vasopressin-like immunoreactivity (VP-IR) in the rabbit eye was demonstrated by radioimmunoassay. Trigeminal nerve denervation resulted in a significant and selective decrease in the levels of VP-IR in the iris sphincter muscle and the cornea. The isolated iris sphincter muscle contracted in response to low concentrations of [Arg8]vasopressin (AVP) and related peptides. The V1 vasopressin receptor antagonist, d(CH2)5Tyr(Me)AVP, potently inhibited the contractile responses to AVP. AVP was found to induce an increase in the accumulation of inositol phosphates in the iris sphincter muscle but not in the dilator/ciliary body preparation in vitro. The present investigation demonstrates the presence of VP-IR in the rabbit eye and that this substance may be another sensory nerve-derived mediator acting on specific target sites in the anterior uvea.

The concentrations of tachykinins in rat spinal cord and dorsal root ganglia (DRGs) were measured using a combination of high performance liquid chromatography (HPLC) and radioimmunoassays (RIAs). Substance P-like immunoreactivity (SPLI) was found to be significantly higher than either substance K-like immunoreactivity (SKLI) or neuromedin K-like immunoreactivity (NMKLI) in both tissues. In the spinal cord, the concentration of SKLI was comparable to that of NMKLI. In DRGs, NMKLI is present at concentrations much lower than those of SKLI or SPLI. In addition to immunoreactive components co-eluting with the three mammalian tachykinins SP, SK and NMK, analyses using reverse-phase HPLC revealed an immunoreactive peak co-eluting with the C-terminal octapeptide of SK (SK3-10), and a yet to be identified peak eluting before SK. This study also demonstrates the use of a novel and highly specific RIA for NMK to measure NMKLI without the need of reverse-phase HPLC.

In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, we examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.

A novel and highly specific radioimmunoassay for the tachykinin peptide neuromedin K (NMK, also known as neurokinin beta, neurokinin B) has been developed and used to determine the distribution of this peptide in extracts of guinea pig tissues. In addition to immunoreactive components coeluting with the 3 mammalian tachykinins, substance P (SP), substance K (SK) and NMK, analyses using reverse-phase HPLC revealed immunoreactive peaks coeluting with the C-terminal octapeptide of SK (SK-(3-10], an N-terminally extended form of SK (gamma-preprotachykinin-(72-92)amide), and a yet unidentified peak eluting before NMK in the extracts of guinea pig brain and spinal cord. In contrast to the other tachykinins, SP and SK, which were present in high concentrations in extracts of all peripheral and central tissues examined, NMK-like immunoreactivity was detected only in extracts of central tissues. NMK-like immunoreactivity was not detected in extracts of terminal ileum and urinary bladder.

Background: Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results: Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion: This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites., Singapore–MIT Alliance for Research and Technology (Flagship Research Project)