Conjugated Linolenic Acid (CLnA) vs Conjugated Linoleic Acid (CLA): A Comprehensive Review of Potential Advantages in Molecular Characteristics, Health Benefits, and Production Techniques.

Conjugated linoleic acid (CLA) has been extensively characterized due to its many biological activities and health benefits, but conjugated linolenic acid (CLnA) is still not well understood. However, CLnA has shown to be more effective than CLA as a potential functional food ingredient. Current research has not thoroughly investigated the differences and advantages between CLnA and CLA. This article compares CLnA and CLA based on molecular characteristics, including structural, chemical, and metabolic characteristics. Then, the in vivo research evidence of CLnA on various health benefits is comprehensively reviewed and compared with CLA in terms of effectiveness and mechanism. Furthermore, the potential of CLnA in production technology and product protection is analyzed. In general, CLnA and CLA have similar physicochemical properties of conjugated molecules and share many similarities in regulation effects and pathways of various health benefits as well as in the production methods. However, their specific properties, regulatory capabilities, and unique mechanisms are different. The superior potential of CLnA must be specified according to the practical application patterns of isomers. Future research should focus more on the advantageous characteristics of different isomers, especially the effectiveness and safety in clinical applications in order to truly exert the potential value of CLnA.

Metabolic, structure-activity characteristics of conjugated linolenic acids and their mediated health benefits.

Conjugated linolenic acid (CLnA) is a mixture of octadecenoic acid with multiple positional and geometric isomers (including four 9, 11, 13-C18:3 isomers and three 8, 10, 12-C18:3 isomers) that is mainly present in plant seeds. In recent years, CLnA has shown many promising health benefits with the deepening of research, but the metabolic characteristics, physiological function differences and mechanisms of different isomers are relatively complex. In this article, the metabolic characteristics of CLnA were firstly reviewed, with focus on its conversion, catabolism and anabolism. Then the possible mechanisms of CLnA exerting biological effects were summarized and analyzed from its own chemical and physical characteristics, as well as biological receptor targeting characteristics. In addition, the differences and mechanisms of different isomers of CLnA in anticancer, lipid-lowering, anti-diabetic and anti-inflammatory physiological functions were compared and summarized. The current results show that the position and cis-trans conformation of conjugated structure endow CLnA with unique physical and chemical properties, which also makes different isomers have commonalities and particularities in the regulation of metabolism and physiological functions. Corresponding the metabolic characteristics of different isomers with precise nutrition strategy will help them to play a better role in disease prevention and treatment. CLnA has the potential to be developed into food functional components and dietary nutritional supplements. The advantages and mechanisms of different CLnA isomers in the clinical management of specific diseases need further study.

Analysis of Chemical Composition and Antioxidant Activity of Idesia polycarpa Pulp Oil from Five Regions in China.

Idesia polycarpa pulp oil (IPPO) has the potential to become the new high-quality vegetable oil. The chemical parameters, fatty acid composition, bioactive ingredients, and antioxidant capacity of five Chinese regions of IPPO were studied comparatively, with significant differences among the regions. The oils were all abundant in unsaturated fatty acids, including linoleic acid (63.07 ± 0.03%-70.69 ± 0.02%), oleic acid (5.20 ± 0.01%-7.49 ± 0.03%), palmitoleic acid (4.31 ± 0.01%-8.19 ± 0.01%) and linolenic acid (0.84 ± 0.03%-1.34 ± 0.01%). IPPO is also rich in active substances such as tocopherols (595.05 ± 11.81-1490.20 ± 20.84 mg/kg), which are made up of α, ß, γ and δ isomers, ß-sitosterol (1539.83 ± 52.41-2498.17 ± 26.05 mg/kg) and polyphenols (106.77 ± 0.86-266.50 ± 2.04 mg GAE/kg oil). The free radical scavenging capacity of IPPO varies significantly depending on the region. This study may provide important guidance for the selection of Idesia polycarpa and offer insights into the industrial application of IPPO in China.

Innovative natural antimicrobial natamycin incorporated titanium dioxide (nano-TiO2)/ poly (butylene adipate-co-terephthalate) (PBAT) /poly (lactic acid) (PLA) biodegradable active film (NTP@PLA) and application in grape preservation.

Poly (butylene adipate-co-terephthalate) (PBAT)/polylactic acid (PLA) blended with compatibilizers (polycaprolactone, PCL; poly (ethylene glycol), PEG; titanium dioxide, nano-TiO2) (TP@PLA composites) were developed by melt processing. Natamycin incorporated into TP@PLA blend composites formed NTP@PLA films, which exhibited high tensile strength (24.1-43.5 MPa) and elongation at break (85.8-258.2 %), and exhibited good oxygen permeability, water vapor permeability, surface hydrophobicity and biodegradability. The in vitro results revealed that inhibition of Penicillium expansum cell growth of the NTP@PLA films with addition of 1.0 wt% natamycin reached 95.72 %. The NTP@PLA film with natamycin effectively reduced incidence of decay (1.52 %) on grapes, maintained their quality, and inhibited the growth of pathogenic fungi to up to 0.42 log cfu·g-1. This study generates new insights into the preservation properties of antimicrobial NTP@PLA film, which endow it with great application potential as a novel and eco-friendly packaging material for the food industry.

Plasma Copy Number Alteration-Based Prognostic and Predictive Multi-Gene Risk Score in Metastatic Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) accounted for more than 34,000 deaths in US males [...].

An Improved Detection of Circulating Tumor DNA in Extracellular Vesicles-Depleted Plasma.

Circulating tumor DNA (ctDNA) in plasma has been used as a biomarker for cancer detection and outcome prediction. In this study, we collected the five precipitates (fractions 1-5) and leftover supernatant plasma component (fraction 6) by a sequential centrifugation in plasma samples from nine small cell lung cancer (SCLC) patients. The fractions 3, 5 and 6 were large vesicles, exosomes and extracellular vesicles (EVs)-depleted plasma, respectively. Fragment size analysis using DNAs from these fractions showed dramatical differences from a peak of 7-10 kb in fraction 1 to 140-160 bp in fraction 6. To determine ctDNA content, we performed whole genome sequencing and applied copy number-based algorithm to calculate ctDNA percentage. This analysis showed the highest ctDNA content in EV-depleted plasma (average = 27.22%), followed by exosomes (average = 22.09%) and large vesicles (average = 19.70%). Comparatively, whole plasma, which has been used in most ctDNA studies, showed an average of 23.84% ctDNA content in the same group of patients. To further demonstrate higher ctDNA content in fraction 6, we performed mutational analysis in the plasma samples from 22 non-small cell lung cancer (NSCLC) patients with known EGFR mutations. This analysis confirmed higher mutation detection rates in fraction 6 (14/22) than whole plasma (10/22). This study provides a new insight into potential application of using fractionated plasma for an improved ctDNA detection.

Multiplex Digital PCR to Detect Amplifications of Specific Androgen Receptor Loci in Cell-Free DNA for Prognosis of Metastatic Castration-Resistant Prostate Cancer.

Amplification of androgen receptor (AR) is a common genomic event in metastatic castration-resistant prostate cancer (mCRPC). To evaluate the prognostic value of the amplifications of specific loci in the AR gene in cell-free DNA, we developed a multiplex digital PCR (dPCR) assay that targeted AR enhancer (AR-En), AR exon 1 (AR-E1), AR exon 8 (AR-E8) and OPHN1 (downstream of AR). We selected three relatively stable genes, C2orf16, FAM111B, and GRIA3, as reference controls for copy number normalization. One hundred and eight mCRPC patients were recruited to test the association of specific AR loci amplification with clinical outcome. Using a normalized ratio ≥ 1.92 as cutoff, amplification of AR-En, AR-E1, AR-E8 and OPHN1 was observed in 28, 25, 24 and 19 of 108 mCRPC patients, respectively. Among the 41 patients with AR region amplification, 9 (21.9%) showed amplification at all four selected regions and 15 (36.6%) showed amplification at AR-En, AR-E1, and AR-E8. Six (14.6%) patients showed independent AR-En amplification, while the remaining 3 (7.3%) demonstrated AR-E8 amplification only. Kaplan-Meier analysis showed overall survival's association with the amplification of AR-En (p = 0.02, HR = 1.68 (1.07-2.65)), AR-E8 (p = 0.02, HR = 1.78 (1.08-2.92)) and AR-En-E8 (the combination of AR-En and AR-E8 (p = 0.009, HR = 1.77 (1.15-2.73)). Multivariate models that included AR-En-E8 amplification and clinical factors significantly improved prognostic performance (p = 0.0001). With further validation, the multiplex dPCR assay may assist in prognostication of mCRPC patients.

Plasma cell-free DNA-based predictors of response to abiraterone acetate/prednisone and prognostic factors in metastatic castration-resistant prostate cancer.

BACKGROUND: The combination of abiraterone acetate and prednisone (AA/P) is used to treat metastatic prostate cancer, but molecular predictors of treatment response are not well elucidated. We evaluated plasma circulating tumor DNA- (ctDNA-) based copy number alterations (CNAs) to determine treatment-related predictive and prognostic biomarkers for metastatic castration-resistant prostate cancer (mCRPC). METHODS: Serial plasma specimens were prospectively collected from 88 chemotherapy-naive mCRPC patients before and after 12 weeks of AA/P treatment. Sequencing-based CNA analyses were performed on 174 specimens. We evaluated CNA-associated 12-week responses for primary resistance, time to treatment change (TTTC) for secondary resistance, and overall survival for prognosis (P < 0.05). Associations with primary resistance were analyzed using the Fisher exact test. Kaplan-Meier survival curves and Cox regression analyses were used to determine the associations of CNAs with acquired resistance and overall survival. RESULTS: ctDNA reduced by 3.89% in responders and increased by 0.94% in nonresponders (P = 0.0043). Thirty-one prostate cancer-related genes from whole genome CNAs were tested. AR and AR enhancer amplification were associated with primary resistance (P = 0.0039) and shorter TTTC (P = 0.0003). ZFHX3 deletion and PIK3CA amplification were associated with primary resistance (P = 0.026 and P = 0.017, respectively), shorter TTTC (P = 0.0008 and P = 0.0016, respectively), and poor survival (P = 0.0025 and P = 0.0022, respectively). CNA-based risk scores combining selected significant associations (AR, NKX3.1, and PIK3CA) at the univariate level with TTTC were predictive of secondary resistance (P = 0.0002). and established prognoses for survival based on CNAs in ZFHX3, RB1, PIK3CA, and OPHN1 (P = 0.002). Multigene risk scores were more predictive than individual genes or clinical risk factors (P < 0.05). CONCLUSION: Plasma ctDNA CNAs and risk scores can predict mCRPC-state treatment and survival outcomes.

Combination of Low Fluctuation of Temperature with TiO2 Photocatalytic/Ozone for the Quality Maintenance of Postharvest Peach.

Chilling injury, tissue browning, and fungal infection are the major problems of peach fruit during post-harvest storage. In this study, a precise temperature control cold storage with low-temperature fluctuation (LFT) and internal circulation flow system is designed. An ozone (O3) generator and a (titanium dioxide) TiO2 photocatalytic reactor were applied to cold storage to investigate the variation of LFT combined with ozone fumigation and a TiO2 photocatalytic reactor in the efficiency of delaying ripening and maintaining peach fruit quality. Results showed that the temperature fluctuation with the improved control system was only ±0.1 to ±0.2 °C compared with that of ±0.5 to ±1.0 °C in conventional cold storage. LFT significantly reduced the chilling injury of peach fruit during storage. Although LFT combined with fumigation of 200 mg m-3 ozone periodical treatment slightly damaged the peach fruit after 40 d of storage, its combination with the TiO2 photocatalytic system significantly improved the postharvest storage quality of the fruit. This treatment maintained higher titratable acidity (TA), total soluble solids (TSS), better firmness, color, microstructure, and lower decay rate, polyphenol oxidase (PPO) activities, total phenol accumulation, respiratory intensity, ethylene production, and malondialdehyde (MDA) content during 60 d of storage. All the results show that LFT combined with the TiO2 photocatalytic system might be a promising technology for quality preservation in peach fruit storage.

Bioinformatics Analysis for Circulating Cell-Free DNA in Cancer.

Molecular analysis of cell-free DNA (cfDNA) that circulates in plasma and other body fluids represents a "liquid biopsy" approach for non-invasive cancer screening or monitoring. The rapid development of sequencing technologies has made cfDNA a promising source to study cancer development and progression. Specific genetic and epigenetic alterations have been found in plasma, serum, and urine cfDNA and could potentially be used as diagnostic or prognostic biomarkers in various cancer types. In this review, we will discuss the molecular characteristics of cancer cfDNA and major bioinformatics approaches involved in the analysis of cfDNA sequencing data for detecting genetic mutation, copy number alteration, methylation change, and nucleosome positioning variation. We highlight specific challenges in sensitivity to detect genetic aberrations and robustness of statistical analysis. Finally, we provide perspectives regarding the standard and continuing development of bioinformatics analysis to move this promising screening tool into clinical practice.

Exosomal miRNAs as Novel Pharmacodynamic Biomarkers for Cancer Chemopreventive Agent Early Stage Treatments in Chemically Induced Mouse Model of Lung Squamous Cell Carcinoma.

BACKGROUND: Chemopreventive agent (CPA) treatment is one of the main preventive options for lung cancer. However, few studies have been done on pharmacodynamic biomarkers of known CPAs for lung cancer. MATERIALS AND METHODS: In this study, we treated mouse models of lung squamous cell carcinoma with three different CPAs (MEK inhibitor: AZD6244, PI-3K inhibitor: XL-147 and glucocorticoid: Budesonide) and examined circulating exosomal miRNAs in the plasma of each mouse before and after treatment. RESULTS: Compared to baselines, we found differentially expressed exosomal miRNAs after AZD6244 treatment (n = 8, FDR < 0.05; n = 55, raw p-values < 0.05), after XL-147 treatment (n = 4, FDR < 0.05; n = 26, raw p-values < 0.05) and after Budesonide treatment (n = 1, FDR < 0.05; n = 36, raw p-values < 0.05). In co-expression analysis, we found that modules of exosomal miRNAs reacted to CPA treatments differently. By variable selection, we identified 11, 9 and nine exosomal miRNAs as predictors for AZD6244, XL-147 and Budesonide treatment, respectively. Integrating all the results, we highlighted 4 miRNAs (mmu-miR-215-5p, mmu-miR-204-5p, mmu-miR-708-3p and mmu-miR-1298-5p) as the key for AZD6244 treatment, mmu-miR-23a-3p as key for XL-147 treatment, and mmu-miR-125a-5p and mmu-miR-16-5p as key for Budesonide treatment. CONCLUSIONS: This is the first study to use circulating exosomal miRNAs as pharmacodynamic biomarkers for CPA treatment in lung cancer.

Prognostic association of plasma cell-free DNA-based androgen receptor amplification and circulating tumor cells in pre-chemotherapy metastatic castration-resistant prostate cancer patients.

BACKGROUND: The prognostic significance of plasma cell-free DNA (cfDNA) androgen receptor amplification (ARamp) in metastatic castration-resistant prostate cancer (mCRPC) compared with circulating tumor cell (CTC) counts is not known. METHODS: As part of correlative aims of a prospective study in mCRPC, concurrent and serial collections of plasma and CTCs were performed. Specimen collections were performed at baseline after progression on androgen deprivation therapy and then 12 weeks later. QuantStudio3D digital PCR system was used to determine plasma cfDNA AR copy number variations and Cell search assay for enumerating CTC counts. Association of baseline cfDNA ARamp status/CTC counts with overall survival (OS) (primary goal) was evaluated using Kaplan-Meier method and log-rank test (p ≤ 0.05 for significance) and receiver operator curves (ROCs) for ARamp status and CTC counts ≥5. A multivariate analysis was performed using Cox regression models that included ARamp, CTC counts, and other clinical factors. RESULTS: ARamp was detected in 19/70 patients at baseline. At the time of analysis, 28/70 patients had died (median follow-up 806 days; interquartile range: 535-966). ARamp was associated with poor OS (2-year OS of 35% in ARamp vs. 71% in non-ARamp; log-rank p value ≤0.0001). Baseline CTC counts ≥5 (vs. <5) was also associated with poor survival (2-year OS of 44 vs. 74%; log-rank p = 0.001). ROC analysis demonstrated area under the curve of 0.66 for ARamp-based prognosis and 0.68 for CTC count-based prognosis (p = 0.84 for difference). The best two variables included for multivariable analysis were ARamp and CTC counts ≥5; however, the two-factor model was not significantly better than using ARamp alone for predicting survival (hazard ratio = 5.25; p = 0.0002). CONCLUSIONS: CTCs and plasma cfDNA ARamp were observed to have equal prognostic value in mCRPC. Larger cohorts that incorporate molecular and clinical factors are needed to further refine prognosis in CRPC.

Genomic alterations of plasma cell-free DNAs in small cell lung cancer and their clinical relevance.

OBJECTIVES: To identify genomic variations in cell-free DNA (cfDNA) and evaluate their clinical utility in small cell lung cancer (SCLC). MATERIALS AND METHODS: We performed whole genome sequencing using plasma cfDNAs derived from 24 SCLC patients for copy number variation (CNV) analysis, and targeted sequencing using 17 pairs of plasma cfDNA and their matched gDNA for mutation analysis. We defined somatic mutations by comparing cfDNA to its matched gDNA with 5% variant alleles as the cutoff for mutation calls. We applied Kaplan-Meier to correlate the genomic alterations with overall survival (OS) and progression-free survival (PFS). RESULTS: We observed widespread somatic copy-number alterations and mutations, including amplification of MYC at 8q24, FGF10 at 5p13, SOX2 at 3q26 and FGFR1 at 8p12, as well as deletion of TP53 at 17p13, RASSF1 at 3p21.3, RB1 at 13q14.2, FHIT at 3p14, and PTEN at 10q23. The most frequent mutations were genes involved in chromatin regulation (KMT2D, ARID1A, SETBP1 and PBRM1), PI3K/MTOR pathway(MTOR,PIK13G), Notch1 signalling pathway (NOTCH1), and DNA repair related gene ATRX. Kaplan-Meier analysis revealed poor OS and PFS in patients with somatic mutations in gene SETBP1 (P = 0.0061/0.0264, HR = 4.785/3.841, 95% CI = 2.014-28.25/1.286-16.58) and PBRM1 (P = 0.0276/0.0286, HR = 3.532/3.506, 95% CI = 1.275 to 25.34/1.26-24.87). Poor OS was also associated with somatic mutations in ATRX (P = 0.0099, HR = 4.024, 95% CI = 1.926-42.95), EP300 (P = 0.025/0.0622, HR = 3.382/2.891, 95% CI = 1.448-27.76/1.013-17.29), while poor PFS was associated with ATM mutation (P = 0.0038, HR = 4.604, 95% CI = 2.211-40.93). The mutation index produced by summing up the number of mutations in the five genes was significantly associated with the poor OS/PFS (P = 0.0185/0.0294) after adjusting the effect of the stage. CONCLUSIONS: Our result supports blood plasma as a promising sample source for the genomic analysis in SCLC patients whose tumor tissues are scarcely available and demonstrates potential clinical utilities of cfDNA-based liquid biopsy for clinical management of this deadly disease.

High-throughput screening of prostate cancer risk loci by single nucleotide polymorphisms sequencing.

Functional characterization of disease-causing variants at risk loci has been a significant challenge. Here we report a high-throughput single-nucleotide polymorphisms sequencing (SNPs-seq) technology to simultaneously screen hundreds to thousands of SNPs for their allele-dependent protein-binding differences. This technology takes advantage of higher retention rate of protein-bound DNA oligos in protein purification column to quantitatively sequence these SNP-containing oligos. We apply this technology to test prostate cancer-risk loci and observe differential allelic protein binding in a significant number of selected SNPs. We also test a unique application of self-transcribing active regulatory region sequencing (STARR-seq) in characterizing allele-dependent transcriptional regulation and provide detailed functional analysis at two risk loci (RGS17 and ASCL2). Together, we introduce a powerful high-throughput pipeline for large-scale screening of functional SNPs at disease risk loci.

Plasma exosomal miRNAs-based prognosis in metastatic kidney cancer.

Plasma exosomal miRNAs were evaluated for prognosis in an initial set of 44 metastatic renal cell cancer (mRCC) patients by RNA sequencing. Among ∼3.49 million mappable reads per patient, miRNAs accounted for 93.1% of the mapped RNAs. 227 miRNAs with high abundance were selected for survival analysis. Cox regression analysis identified association of 6 miRNAs with overall survival (OS) (P<0.01, False discovery rate (FDR) < 0.3). Five of the associated miRNAs were quantified in an independent follow-up cohort of 65 mRCC patients by TaqMan-based miRNA assays. Kaplan-Meier analysis confirmed the significant OS association of three miRs; miR-let-7i-5p (P=0.018, HR=0.49, 95% CI=0.21-0.84), miR-26a-1-3p (P=0.025, HR=0.43, 95% CI=0.10-0.84) and miR-615-3p (P=0.0007, HR=0.36, 95% CI=0.11-0.54). A multivariate analysis of miR-let-7i-5p with the clinical factor-based Memorial Sloan-Kettering Cancer Center (MSKCC) score improved survival prediction from an area under the curve (AUC) of 0.58 for MSKCC score to an average AUC of 0.64 across 48-month follow-up time. The multivariate model was able to define a high-risk group with median survival of 14 months and low risk group of 39 months (P=0.0002, HR=3.43, 95%CI, 2.73-24.15). Further validation of miRNA-based prognostic biomarkers are needed to improve current clinic-pathologic based prognostic models in patients with mRCC.

Molecular characterization of cell-free eccDNAs in human plasma.

Extrachromosomal circular DNAs (eccDNAs) have been reported in most eukaryotes. However, little is known about the cell-free eccDNA profiles in circulating system such as blood. To characterize plasma cell-free eccDNAs, we performed sequencing analysis in 26 libraries from three blood donors and negative controls. We identified thousands of unique plasma eccDNAs in the three subjects. We observed proportional eccDNA increase with initial DNA input. The detected eccDNAs were also associated with circular DNA enrichment efficiency. Increasing the sequencing depth in an additional sample identified many more eccDNAs with highly heterogenous molecular structure. Size distribution of eccDNAs varied significantly from 31 bp to 19,989 bp. We found significantly higher GC content in smaller eccDNAs (<500 bp) than the larger ones (>500 bp) (p < 0.01). We also found an enrichment of eccDNAs at exons and 3'UTR (enrichment folds from 1.36 to 3.1) as well as the DNase hypersensitive sites (1.58-2.42 fold), H3K4Me1 (1.23-1.42 fold) and H3K27Ac (1.33-1.62 fold) marks. Junction sequence analysis suggested fundamental role of nonhomologous end joining mechanism during eccDNA formation. Further characterization of the extracellular eccDNAs in peripheral blood will facilitate understanding of their molecular mechanisms and potential clinical utilities.

Cell-free DNA copy number variations in plasma from colorectal cancer patients.

To evaluate the clinical utility of cell-free DNA (cfDNA), we performed whole-genome sequencing to systematically examine plasma cfDNA copy number variations (CNVs) in a cohort of patients with colorectal cancer (CRC, n = 80), polyps (n = 20), and healthy controls (n = 35). We initially compared cfDNA yield in 20 paired serum-plasma samples and observed significantly higher cfDNA concentration in serum (median = 81.20 ng, range 7.18-500 ng·mL-1 ) than in plasma (median = 5.09 ng, range 3.76-62.8 ng·mL-1 ) (P < 0.0001). However, tumor-derived cfDNA content was significantly lower in serum than in matched plasma samples tested. With ~10 million reads per sample, the sequencing-based copy number analysis showed common CNVs in multiple chromosomal regions, including amplifications on 1q, 8q, and 5q and deletions on 1p, 4q, 8p, 17p, 18q, and 22q. Copy number changes were also evident in genes critical to the cell cycle, DNA repair, and WNT signaling pathways. To evaluate whether cumulative copy number changes were associated with tumor stages, we calculated plasma genomic abnormality in colon cancer (PGA-C) score by summing the most significant CNVs. The PGA-C score showed predictive performance with an area under the curve from 0.54 to 0.84 for CRC stages I-IV. Locus-specific copy number analysis identified nine genomic regions where CNVs were significantly associated with survival in stage III-IV CRC patients. A multivariate model using six of nine genomic regions demonstrated a significant association of high-risk score with shorter survival (HR = 5.33, 95% CI = 6.76-94.44, P < 0.0001). Our study demonstrates the importance of using plasma (rather than serum) to test tumor-related genomic variations. Plasma cfDNA-based tests can capture tumor-specific genetic changes and may provide a measurable classifier for assessing clinical outcomes in advanced CRC patients.

3C-digital PCR for quantification of chromatin interactions.

BACKGROUND: Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls. RESULTS: To demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genes MYC and CAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element and MYC gene promoter was 0.7 (95% CI 0.40-1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and two EcoRI fragments containing CAPG gene promoter were 1.9 copies (95% CI 1.41-2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76-1.92), respectively, while the interaction signals were reduced on either side of the promoter region of CAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145). CONCLUSIONS: Compared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate multiple and complex normalization controls and provide accurate calculation of proximity-based fragment ligation frequency. Therefore, we recommend 3C-dPCR as a preferred method for sensitive detection of low frequency chromatin interactions.

miR-375 induces docetaxel resistance in prostate cancer by targeting SEC23A and YAP1.

BACKGROUND: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically are centered on docetaxel-based chemotherapy. We previously reported that elevated miR-375 levels were significantly associated with poor overall survival of mCRPC patients. In this study, we evaluated if miR-375 induced chemo-resistance to docetaxel through regulating target genes associated with drug resistance. METHODS: We first compared miR-375 expression level between prostate cancer tissues and normal prostate tissues using data from The Cancer Genome Atlas (TCGA). To examine the role of miR-375 in docetaxel resistance, we transfected miR-375 using a pre-miRNA lentiviral vector and examined the effects of exogenously overexpressed miR-375 on cell growth in two prostate cancer cell lines, DU145 and PC-3. To determine the effect of overexpressed miR-375 on tumor growth and chemo-resistance in vivo, we injected prostate cancer cells overexpressing miR-375 into nude mice subcutaneously and evaluated tumor growth rate during docetaxel treatment. Lastly, we utilized qRT-PCR and Western blot assay to examine two miR-375 target genes, SEC23A and YAP1, for their expression changes after miR-375 transfection. RESULTS: By examining 495 tumor tissues and 52 normal tissues from TCGA data, we found that compared to normal prostate, miR-375 was significantly overexpressed in prostate cancer tissues (8.45-fold increase, p value = 1.98E-23). Docetaxel treatment induced higher expression of miR-375 with 5.83- and 3.02-fold increases in DU145 and PC-3 cells, respectively. Interestingly, miR-375 appeared to play a dual role in prostate cancer proliferation. While miR-375 overexpression caused cell growth inhibition and cell apoptosis, elevated miR-375 also significantly reduced cell sensitivity to docetaxel treatment in vitro, as evidenced by decreased apoptotic cells. In vivo xenograft mouse study showed that tumors with increased miR-375 expression were more tolerant to docetaxel treatment, demonstrated by greater tumor weight and less apoptotic cells in miR-375 transfected group when compared to empty vector control group. In addition, we examined expression levels of the two miR-375 target genes (SEC23A and YAP1) and observed significant reduction in the expression at both protein and mRNA levels in miR-375 transfected prostate cancer cell lines. TCGA dataset analysis further confirmed the negative correlations between miR-375 and the two target genes (r = -0.62 and -0.56 for SEC23A and YAP1, respectively; p < 0.0001). CONCLUSIONS: miR-375 is involved in development of chemo-resistance to docetaxel through regulating SEC23A and YAP1 expression. Our results suggest that miR-375 or its target genes, SEC23A or YAP1, might serve as potential predictive biomarkers to docetaxel-based chemotherapy and/or therapeutic targets to overcome chemo-resistance in mCRPC stage.

Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer.

Genetic profiling of urine cell free DNA (cfDNA) has not been evaluated in advanced prostate cancer. We performed whole genome sequencing of urine cfDNAs to identify tumor-associated copy number variations in urine before and after initiating androgen deprivation therapy in HSPC stage and docetaxel chemotherapy in CRPC stage. A log2 ratio-based copy number analysis detected common genomic abnormalities in prostate cancer including AR amplification in 5/10 CRPC patients. Other abnormalities identified included TMPRSS2-ERG fusion, PTEN gene deletion, NOTCH1 locus amplification along with genomic amplifications at 8q24.3, 9q34.3, 11p15.5 and 14q11.2, and deletions at 4q35.2, 5q31.3, 7q36.3, 12q24.33, and 16p11.2. By comparing copy number between pre- and post-treatment, we found significant copy number changes in 34 genomic loci. To estimate the somatic tumor DNA fraction in urine cfDNAs, we developed a Urine Genomic Abnormality (UGA) score algorithm that summed the top ten most significant segments with copy number changes. The UGA scores correlated with tumor burden and the change in UGA score after stage-specific therapies reflected disease progression status and overall survival. The study demonstrates the potential clinical utility of urine cfDNAs in predicting treatment response and monitoring disease progression.