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Background Mammography is widely used for breast cancer screening but suffers from a high false-positive rate. Here, we perform the largest comprehensive, multi-center study to date involving diverse ethnic groups, for the identification of circulating miRNAs for breast cancer screening. Methods This study had a discovery phase (n = 289) and two validation phases (n = 374 and n = 379). Quantitative PCR profiling of 324 miRNAs was performed on serum samples from breast cancer (all stages) and healthy subjects to identify miRNA biomarkers. Two-fold cross-validation was used for building and optimising breast cancer-associated miRNA panels. An optimal panel was validated in cohorts with Caucasian and Asian samples. Diagnostic ability was evaluated using area under the curve (AUC) analysis. Results The study identified and validated 30 miRNAs dysregulated in breast cancer. An optimised eight-miRNA panel showed consistent performance in all cohorts and was successfully validated with AUC, accuracy, sensitivity, and specificity of 0.915, 82.3%, 72.2% and 91.5%, respectively. The prediction model detected breast cancer in both Caucasian and Asian populations with AUCs ranging from 0.880 to 0.973, including pre-malignant lesions (stage 0; AUC of 0.831) and early-stage (stages I–II) cancers (AUC of 0.916). Conclusions Our panel can potentially be used for breast cancer screening, in conjunction with mammography.

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.

Astaxanthin is a natural pigment, known for its strong antioxidant activity and numerous health benefits to human and animals. Its antioxidant activity is known to be substantially greater than β-carotene and about a thousand times more effective than vitamin E. The potential health benefits have generated a growing commercial interest, and the escalating demand has prompted the exploration of alternative supply chain. Astaxanthin naturally occurs in many sea creatures such as trout, shrimp, and microalgae, some fungi, bacteria, and flowering plants, acting to protect hosts against environmental stress and adverse conditions. Due to the rapid growth and simple growth medium requirement, microbes, such as the microalga, Haematococcus pluvialis, and the fungus Xanthophyllomyces dendrorhous, have been developed to produce astaxanthin. With advances in metabolic engineering, non-carotenogenic microbes, such as Escherichia coli and Saccharomyces cerevisiae, have been purposed to produce astaxanthin and significant progress has been achieved. Here, we review the recent achievements in microbial astaxanthin biosynthesis (with reference to metabolic engineering strategies) and extraction methods, current challenges (technical and regulatory), and commercialization outlook. Due to greenness, sustainability, and dramatic cost reduction, we envision microbial synthesis of astaxanthin offers an alternative means of production (e.g. chemical synthesis) in the near future.

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 Mesenchymal stem cells (MSCs) driven gene directed enzyme prodrug therapy is a promising approach to deliver therapeutic agents to target heterogenous solid tumours. To democratize such a therapy, cryopreservation along with cold chain transportation is an essential part of the logistical process and supply chain. Previously, we have successfully engineered MSCs by a non-viral DNA transfection approach for prolonged and exceptionally high expression of the fused transgene cytosine deaminase, uracil phosphoribosyl transferase and green fluorescent protein (CD::UPRT::GFP). The aim of this study was to determine the effects of cryopreservation of MSCs engineered to highly overexpress this cytoplasmic therapeutic transgene. Methods Modified MSCs were preserved in a commercially available, GMP-grade cryopreservative—CryoStor10 (CS10) for up to 11 months. Performance of frozen-modified MSCs was compared to freshly modified equivalents in vitro. Cancer killing potency was evaluated using four different cancer cell lines. Migratory potential was assessed using matrigel invasion assay and flow cytometric analysis for CXCR4 expression. Frozen-modified MSC was used to treat canine patients via intra-tumoral injections, or by intravenous infusion followed by a daily dose of 5-flucytosine (5FC). Results We found that cryopreservation did not affect the transgene expression, cell viability, adhesion, phenotypic profile, and migration of gene modified canine adipose tissue derived MSCs. In the presence of 5FC, the thawed and freshly modified MSCs showed comparable cytotoxicity towards one canine and three human cancer cell lines in vitro. These cryopreserved cells were stored for about a year and then used to treat no-option-left canine patients with two different types of cancers and notably, the patients showed progression-free interval of more than 20 months, evidence of the effectiveness in treating spontaneously occurring cancers. Conclusion This study supports the use of cryopreserved, off-the-shelf transiently transfected MSCs for cancer treatment.

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.

Mesenchymal stem cells (MSCs) driven gene-directed enzyme prodrug therapy has emerged as a potential strategy for cancer treatment. The tumour-nesting properties of MSCs enable these vehicles to target tumours and metastases with effective therapies. A crucial step in engineering MSCs is the delivery of genetic material with low toxicity and high efficiency. Due to the low efficiency of current transfection methods, viral vectors are used widely to modify MSCs in preclinical and clinical studies. We show, for the first time, the high transfection efficiency (> 80%) of human adipose tissue derived-MSCs (AT-MSCs) using a cost-effective and off-the-shelf Polyethylenimine, in the presence of histone deacetylase 6 inhibitor and fusogenic lipids. Notably, the phenotypes of MSCs remained unchanged post-modification. AT-MSCs engineered with a fused transgene, yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) displayed potent cytotoxic effects against breast, glioma, gastric cancer cells in vitro. The efficiency of eliminating gastric cell lines were effective even when using 7-day post-transfected AT-MSCs, indicative of the sustained expression and function of the therapeutic gene. In addition, significant inhibition of temozolomide resistant glioma tumour growth in vivo was observed with a single dose of therapeutic MSC. This study demonstrated an efficient non-viral modification process for MSC-based prodrug therapy.

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.

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.

The Singapore Integrative Omics Study provides valuable insights on establishing population reference measurement in 364 Chinese, Malay, and Indian individuals. These measurements include > 2.5 millions genetic variants, 21,649 transcripts expression, 282 lipid species quantification, and 284 clinical, lifestyle, and dietary variables. This concept paper introduces the depth of the data resource, and investigates the extent of ethnic variation at these omics and non-omics biomarkers. It is evident that there are specific biomarkers in each of these platforms to differentiate between the ethnicities, and intra-population analyses suggest that Chinese and Indians are the most biologically homogeneous and heterogeneous, respectively, of the three groups. Consistent patterns of correlations between lipid species also suggest the possibility of lipid tagging to simplify future lipidomics assays. The Singapore Integrative Omics Study is expected to allow the characterization of intra-omic and inter-omic correlations within and across all three ethnic groups through a systems biology approach., The Singapore Genome Variation projects characterized the genetics of Singapore’s Chinese, Malay, and Indian populations. The Singapore Integrative Omics Study introduced here goes further in providing multi-omic measurements in individuals from these populations, including genetic, transcriptome, lipidome, and lifestyle data, and will facilitate the study of common diseases in Asian communities.

Astaxanthin is a natural pigment, known for its strong antioxidant activity and numerous health benefits to human and animals. Its antioxidant activity is known to be substantially greater than β-carotene and about a thousand times more effective than vitamin E. The potential health benefits have generated a growing commercial interest, and the escalating demand has prompted the exploration of alternative supply chain. Astaxanthin naturally occurs in many sea creatures such as trout, shrimp, and microalgae, some fungi, bacteria, and flowering plants, acting to protect hosts against environmental stress and adverse conditions. Due to the rapid growth and simple growth medium requirement, microbes, such as the microalga, Haematococcus pluvialis, and the fungus Xanthophyllomyces dendrorhous, have been developed to produce astaxanthin. With advances in metabolic engineering, non-carotenogenic microbes, such as Escherichia coli and Saccharomyces cerevisiae, have been purposed to produce astaxanthin and significant progress has been achieved. Here, we review the recent achievements in microbial astaxanthin biosynthesis (with reference to metabolic engineering strategies) and extraction methods, current challenges (technical and regulatory), and commercialization outlook. Due to greenness, sustainability, and dramatic cost reduction, we envision microbial synthesis of astaxanthin offers an alternative means of production (e.g. chemical synthesis) in the near future.

Additional file 8. Scaling up MSC modification process on microcarrier culture. AD-MSCs were seeded at 28,000/cm2 on Cytodex® 3 microcarriers (total surface area of 1.9 cm2) in 24-well non-adherent plates, with agitation speed of 50 rpm for 24 h before transfection. Similar to flat-bed transfection, the polymer and DNA complex (at 200-300 ng of pDNA) were added to the cell culture using a dropwise manner after 15 min incubation. Cells were transfected in the presence or absence of the enhancer. Two-day post transfection, a the fluorescent images were captured at 10x magnification. Representative images are shown. b After which, cells were harvested through trypsinization. To separate the cells from microcarrier, the suspension cells were filtered through 70 μm cell strainer. The transfection efficiencies were determined through flow cytometry analysis. Cell viability based on PI exclusion was determined with NC-3000 automated cell counter. The combination chart presents % of GFP+ cells (graph bar) and cell viability (mark type) Data of biological triplicates are expressed as mean + SD. c For microcarriers with total surface area of 1.9 cm2 and 9.5 cm2, cells were transfected in 24-well non-adherent plates. To further scale up the transfection process, AD-MSCs were seeded on Cytodex® 3 (total surface area of 47.5 cm2) at 28,000 cells/cm2 in 125 mL Erlenmeyer flasks. One day later, cells were transfected in the presence of Enhancer. The amount of DNA was fixed at 150 ng/cm2 of microcarriers. Two-day post transfection, transfection efficiency was analysed. The scatter plot represents absolute number of GFP+ cells collected from each culture vessel; deduced from the readout of cell counting and flow cytometry analysis of the GFP expression. Data are expressed as mean + SD, n = 3.

Additional file 14. AD-MSCs generated with PEI plus Enhancers outperformed other methods. AD-MSCs were transfected with CD::UPRT::GFP plasmid using various protocols. Twenty-four hours post-transfection, CD::UPRT::GFP_AD-MSCs were cocultured with U-251MG in DMEM supplemented with 2% FBS, in the presence or absence of 100 μg/mL 5FC. The therapeutic cells were mixed at ratios of 1 CD::UPRT::GFP_AD-MSC to 25, 10, 5, 1 cancer cells. Seven days later, cell viability in the treatment conditions was evaluated spectrophotometrically by MTS assay. Cell viability was defined as sample/control × 100%. Conditions without 5FC treatment served as controls. No MSC condition suggests lack of 5FC cytotoxicity in cancer cells. Data presents mean + SD of the biological quadruplicates. Statistical differences between Lipofectamine 3000 and other methods were calculated using two tailed Student’s t-test. **, P

Additional file 9. Representative images and FACS profile of AD-MSCs transfected with 300 ng of CD::UPRT::GFP plasmid, in the presence or absence of the Enhancer.

Additional file 6. Supplementation of Enhancer to improve transfection in AD-MSCs. One day post seeding of 50,000 AD-MSCs, cells were transfected by 150-500 ng of PF463-CMV-GFP complexed with PEI at 1 μg pDNA to 3 μL PEI, in the presence or absence of Enhancer. a Two days post-transfection, the fluorescent images were captured at 4x magnification. Representative images are presented. b After which, cells were harvested for FACS analysis. Untransfected MSC served as negative control for gating. Data represents mean ± SD, n = 3 (Bar graph).

Additional file 2. Flow cytometry profiles of the cell cycle of the TMZ sensitive and resistant cell lines in the presence and absence of TMZ treatment. The number of cells acquired during flow cytometry measurement is 5000 per sample. The analysis of the cell cycle is presented in Fig. 1c and d.

Additional file 7. Scaling out MSC modification process on flat-bed culture. AD-MSCs were seeded in 6-well plate, T25, T75 and T175 flasks at 20,000/cm2. One day post seeding, cells were transfected with 150 ng PF463-CMV-GFP/ cm2 complexed at 1 μg pDNA to 3 μL PEI. Two-day post transfection, cells were harvested for analysis. Total number of cells harvested from each culture vessels were determined by the automated cell counter NC-3000. After which, the % of GFP+ cells measured with flow cytometry. a The scatter plot represents absolute number of GFP+ cells collected from each culture vessel; deduced from the readout of cell counting and flow cytometry of the GFP expression. Data of biological triplicates are expressed as mean + SD. b Representative images present the flow cytometry profile of unmodified and modified AD-MSCs harvested from T175 flasks.

Additional file 5. Determination of the transfection efficiencies of various commercial carriers in AD-MSCs. One day post seeding of 50,000 AD-MSCs, cells were transfected with different gene carriers at various GFP encoded plasmid (PF463-CMV-GFP, PlasmidFactory) and gene carrier amounts. AD-MSCs were transfected with a Lipofectamine 3000, b Polyfect, c Transficient, and d Turbofect according to the manufacturer’s instructions. e For cells transfected with PEI, the protocol is detailed in methods and materials section. Two-day post transfection, AD-MSCs were harvested. Transfection efficiency and cell viability (Propidium Iodide exclusion assay) were determined using NucleoCounter®NC-3000™. For each gene carrier, the left and right graph bar presents % of GFP+ cells and % of cell viability (% of PI- population), respectively. Data represents mean ± SD, n = 3.

Additional file 3. Gene expression of genes related in 5FU resistance pathway. Fold change of expression for DPYD, TYMS and UMPS in a U-251MG and b U-87MG TMRZ cell lines were calculated in relative to their respective parental cell lines. Similarly, changes in the expression of ABCC5 transporter were calculated accordingly for c U-251MG and d U-87MG TMZR cell lines. All samples were analysed in triplicates. The fold change in the expressions of the gene of interest was calculated after normalization to the house keeping gene GAPDH. Line graph shows the average fold change in gene expression, mean + SD (n = 3). Significant differences between parental and TMZR cells were calculated using unpaired, two-tailed Student’s t-test. p-value

Additional file 4. Gene expression z-score of patient derived cell lines for 5-FU pathway. DPYD, TYMS, UMPS, TYMP and ABCC5 expression levels are shown. The information was obtained from www.hgcc.se Dated: 3 Jun 2019.

Cationic polymers remain attractive tools for non-viral gene transfer. The effectiveness of these vectors rely on the ability to deliver plasmid DNA (pDNA) into the nucleus of cells. While we have previously demonstrated the potential of Lignin-PGEA-PEGMA as a non-viral gene delivery vector, alterations of cellular phenotype and cytotoxicity were observed post transfection. The present study aims to explore transfection conditions for high efficiency and low toxicity of the Lignin-PGEA-PEGMA based gene delivery system. Cellular toxicity was significantly reduced by using the centrifugation protocol, which enables rapid deposition of DNA complexes. Replacement of media post centrifugation resulted in minimal exposure of cells to excess polymers, which were toxic to cells. At an optimized DNA amount (500–750 ng) and molar ratios of nitrogen (N) in polymer to phosphate (P) in pDNA (N/P = 30–40), with the use of a novel transfection enhancer that facilitates endosomal escape and nuclear trafficking, the efficiency of gene delivery was increased significantly 24 h post transfection. The present study demonstrated an appropriately optimized protocol that enabled the utility of a novel cationic polymer blend with a mixture of fusogenic lipids and a histone deacetylate inhibitor in non-viral transfection, thereby providing an attractive alternative to costly commercial gene carriers.

A silicon nanowire platform prepared by glancing angle deposition and metal assisted chemical etching (GLAD-MACE) was used for oligonucleotides hybridization. The limit of detection of this platform was enhanced due to the large amount of probe molecules that can be accommodated on the nanowire surface and pores on the sidewall. In contrast to conventional substrates, the GLAD-MACE nanowires can accept 100 times more probes without showing probe steric hindrance. Compared to the detection of oligonucleotides with fluorescent reporters on a traditional substrate, even those facilitated by a microfluidic mixing chamber, at least a 10 times lower LoD could be reached with a passive hybridization strategy. For a device built with GLAD-MACE nanowires, it is clear that one important factor to optimize the system performance was to design an apparatus that can accelerate the diffusion process of antisense DNAs.

One-pot multienzyme biosynthesis is an attractive method for producing complex, chiral bioactive compounds. It is advantageous over step-by-step synthesis, as it simplifies the process, reduces costs and often leads to higher yield due to the synergistic effects of enzymatic reactions. In this study, dihydroartemisinic acid (DHAA) pathway enzymes were overexpressed in Saccharomyces cerevisiae, and whole-cell biotransformation of amorpha-4,11-diene (AD) to DHAA was demonstrated. The first oxidation step by cytochrome P450 (CYP71AV1) is the main rate-limiting step, and a series of N-terminal truncation and transcriptional tuning improved the enzymatic activity. With the co-expression of artemisinic aldehyde dehydrogenase (ALDH1), which recycles NADPH, a significant 8-fold enhancement of DHAA production was observed. Subsequently, abiotic conditions were optimized to further enhance the productivity of the whole-cell biocatalysts. Collectively, approximately 230 mg/L DHAA was produced by the multi-step whole-cell reaction, a ~50% conversion from AD. This study illustrates the feasibility of producing bioactive compounds by in vitro one-pot multienzyme reactions.

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.

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.

Neurturin (NRTN), a member of the GDNF family of ligands (GFL), is currently investigated in a series of clinical trials for Parkinson's disease. NRTN signals through its cognate receptor GFRα2 and co-receptor RET to induce neurite outgrowth, but the underlying mechanism remains to be better understood. STAT3 was previously shown to be activated by oncogenic RET, independent of ligand and GFRα. In this study, we demonstrated that NRTN induced serine727 but not tyrosine705 phosphorylation of STAT3 in primary cortical neuron and neuronal cell lines. Remarkably, STAT3 phosphorylation was found to be mediated specifically by GFRα2c and RET9 isoforms. Furthermore, serine but not tyrosine dominant negative mutant of STAT3 impaired NRTN induced neurite outgrowth, indicative of the role of STAT3 as a downstream mediator of NRTN function. Similar to NGF, the NRTN induced P-Ser-STAT3 was localized to the mitochondria but not to the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NRTN induced neurite outgrowth. Collectively, these findings demonstrated the hitherto unrecognized and novel role of specific GFRα2 and RET isoforms in mediating NRTN activation of STAT3 and the transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 mediated NRTN induced neurite outgrowth.

Characterization of the rheological properties of heterogeneous biopolymers is important not only to understand the effect of substrate elasticity on cell behaviors, but also to provide insights into mechanical changes during cellular remodeling of the environment. Conventional particle-tracking microrheology (PTM) techniques are compromised by probe–network slippage and cage-hopping problems, and require a priori knowledge of network mesh size in order to determine a suitable probe size. We demonstrated here the usefulness of covalently bound probes for PTM of biopolymers to overcome the above limitations. We showed that, in a well-defined system like polyacrylamide gels, surface-modified probe particles using a zero-length crosslinker provided more reliable measurements of network mechanics as compared to standard carboxylated probes. We further demonstrated that appropriate surface modification of microspheres for PTM circumvented the requirement of using microspheres larger than the network mesh, an approach typically considered to be ideal. Using the method presented in this study, we found the local network at the leading edge of a typical C6 glioma cell to be stiffer as compared to the side. Our findings established that permanent interaction between the probe and network is crucial to reliably measure the local network mechanics in reconstituted, heterogeneous networks using PTM.

Efficient non-viral gene delivery is highly desirable but often unattainable with some cell-types. We report here that non-viral DNA polyplexes can efficiently transfect differentiated neuronal and stem cells. Polyplex transfection centrifugation protocols was enhanced by including a simultaneous treatment with a DOPE/CHEMS lipid suspension and a microtubule inhibitor, Tubastatin A. Lipoplex transfection protocols were not improved by this treatment. This mechanism of action was unravelled by systematically identifying and rationally mitigating barriers limiting high transfection efficiency, allowing unexpected improvements in the transfection of mesenchymal stem cells (MSC), primary neuron and several hard-to-transfect cell types beyond what are currently achievable using cationic polymers. The optimized formulation and method achieved high transfection efficiency with no adverse effects on cell viability, cell proliferation or differentiation. High efficiency modification of MSC for cytokine overexpression, efficient generation of dopaminergic neuron using neural stem cells and enhanced genome editing with CRISPR-Cas9 were demonstrated. In summary, this study described a cost-effective method for efficient, rapid and scalable workflow for ex vivo gene delivery using a myriad of nucleic acids including plasmid DNA, mRNA, siRNA and shRNA.

New types of asymmetric functionalizations of alkenes are highly desirable for chemical synthesis. Here, we develop three novel types of regio- and enantioselective multiple oxy- and amino-functionalizations of terminal alkenes via cascade biocatalysis to produce chiral α-hydroxy acids, 1,2-amino alcohols and α-amino acids, respectively. Basic enzyme modules 1–4 are developed to convert alkenes to (S)-1,2-diols, (S)-1,2-diols to (S)-α-hydroxyacids, (S)-1,2-diols to (S)-aminoalcohols and (S)-α-hydroxyacids to (S)-α-aminoacids, respectively. Engineering of enzyme modules 1 & 2, 1 & 3 and 1, 2 & 4 in Escherichia coli affords three biocatalysts over-expressing 4–8 enzymes for one-pot conversion of styrenes to the corresponding (S)-α-hydroxyacids, (S)-aminoalcohols and (S)-α-aminoacids in high e.e. and high yields, respectively. The new types of asymmetric alkene functionalizations provide green, safe and useful alternatives to the chemical syntheses of these compounds. The modular approach for engineering multi-step cascade biocatalysis is useful for developing other new types of one-pot biotransformations for chemical synthesis., Singapore-MIT Alliance, Singapore. Agency for Science, Technology and Research (A*STAR research grant Project No. 1021010026), National University of Singapore (Synthetic Biology for Clinical and Technological Innovation (SynCTI) program)

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.

MicroRNAs are small noncoding RNAs that serve as important regulators of eukaryotic gene expression and are emerging as novel diagnostic and therapeutic targets for human diseases. Robust and reliable detection of miRNAs is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Existing methods for miRNA quantification rely on fluorescent probes for optimal specificity. In this study, we developed a high-performance real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay that allows specific and rapid detection of mature miRNAs using a fast thermocycling profile (10 sec per cycle). This assay exhibited a wide dynamic range (>7 logs) and was capable of detecting miRNAs from as little as 1 pg of the total RNA or as few as 10 cells. The use of modified reverse-transcription oligonucleotides with a secondary structure and hemi-nested reverse PCR primers allowed excellent discrimination of mature miRNAs from their precursors and highly homologous family members using SYBR Green I. Using a novel approach involving uracil-DNA glycosylase treatment, we showed that carryover of the reverse transcription oligonucleotide to the PCR can be successfully eliminated and discrimination between miRNA homologs could be further enhanced. These assays were further extended for multiplexed detection of miRNAs directly from cell lysates without laborious total RNA isolation. With the robust performance of these assays, we identified several miRNAs that were regulated by glial cell-line-derived neurotrophic factor in human glioblastoma cells. In summary, this method could provide a useful tool for rapid, robust, and cost-effective quantification of existing and novel miRNAs.

The safety and toxicity of nanoparticles are of growing concern despite their significant scientific interests and promising potentials in many applications. The properties of nanoparticles depend not only on the size but also the structure, microstructure and surface coating. These in turn are controlled by the synthesis and processing conditions. The dependence of cytotoxicity on particle size and on the presence of oleic acid as surfactant on nickel ferrite particles were investigated in vitro using the Neuro-2A cell line as a model. For nickel ferrite particles without oleic acid prepared by ball milling, cytotoxicity was independent of particle size within the given mass concentrations and surface areas accessible to the cells. For nickel ferrite particles coated with oleic acid prepared by the polyol method, the cytotoxicity significantly increased when one or two layers of oleic acid were deposited. Large particles (150+/-50 nm diameter) showed a higher cytotoxicity than smaller particles (10+/-3 nm diameter).

An improved method to prepare alkylamine-stabilized Pt and Ru nanoparticles based on the original Brust’s procedure [J. Chem. Soc., Chem. Commun. (1994) 801] has been developed. The new method involves, firstly, mixing an aqueous solution of metal salts such as PtCl62−, PtCl42− or Ru3+ with an ethanol solution of dodecylamine; extracting the metal ions into a toluene layer; and finally reducing the metal ions to their zero valent states using NaBH4. Alkylamine-stabilized Pt nanoparticles prepared this way had a polyhedral or wormlike appearance, depending closely on the chemical nature of the metal precursor salts being used. On the contrary, dodecylamine-stabilized ruthenium nanoparticles were predominantly spherical. The particle formation and growth processes in the hydrocarbon layer could be influenced by the different ways dodecylamine was bound to H2PtCl6, K2PtCl4 or RuCl3 at the precursor stage.

Positively charged ruthenium nanoparticles were prepared by NaBH4 reduction at room temperature and at pH values lower than 4.9. The ruthenium nanoparticles were characterized by zeta potential measurement, TEM, XPS, and XRD. Particles with a mean diameter of 1.8 nm and a standard deviation of 0.40 nm could be obtained under the experimental conditions. The surface charge on the particles is believed to originate from hydrated proton adsorption. The positively charged ruthenium nanoparticles could be used as the starting material for further functionalization by PVP, ethylenediamine, and dodecylamine.

An alternative phase-transfer method has been developed to prepare alkylamine-stabilized Pt nanoparticles. This method involves preparing a Pt hydrosol and then mixing the Pt hydrosol with a toluene solution of dodecylamine. This is followed by the addition of 1 M NaOH or 1 × PBS buffer to adjust the pH of the hydrosol to suitable values. Dodecylamine-stabilized Pt nanoparticles prepared this way had a predominantly cubic appearance, uniform size distribution, and self-assembled on transmission electron microscopy copper grids with an interparticle spacing of 2.2 nm. These results were then used to derive a protocol for preparing oligonucleotide-stabilized Pt nanoparticles in the aqueous environment. The oligonucleotide-stabilized Pt nanoparticles formed this way were either of spherical or irregular cubic shape depending on whether H2PtCl6 or K2PtCl4 was used as the starting metal salt.

A novel method has been developed to prepare water-dispersible ethylenediamine (en)-stabilized ruthenium nanoparticles. The procedure involves the reduction of an en–RuCl 3 complex by sodium borohydride. The Ru nanoparticles so prepared are fairly stable in water. TEM imaging shows a mean diameter of about 2.1 nm for the particles and a narrow particle size distribution.