Cell-free RNA Research Articles

LB 2: Early prediction of spontaneous preterm birth in a high-risk population using cfRNA profiling

Spontaneous preterm birth (sPTB) remains the main driver of childhood morbidity and mortality. There is a lack of predictive markers and targeted therapeutics due to an incomplete understanding of the molecular basis of this heterogenous syndrome. This study sought to determine if a cell free RNA (cfRNA) profile could reveal a molecular fingerprint in maternal blood months prior to the onset of sPTB.

Transcriptomic analysis of patient plasma reveals circulating miR200c as a potential biomarker for high-grade serous ovarian cancer

BackgroundHigh-grade serous tubo-ovarian cancer (HGSC) is the most common histological subtype of epithelial ovarian cancer, and highly lethal. Currently there is no effective screening test and prognosis is poor as the majority of cases are diagnosed at the advanced stage. Cell free RNAs including microRNAs (miRNAs) are dysregulated in ovarian cancer tissue and are detectable in the circulation. This study aimed to determine whether circulating cell free miRNAs may be potential biomarkers for the detection of HGSC. MethodsPlasma was collected from women with HGSC (Grade 3, n = 24), and benign ovarian masses (n = 24). RNA was extracted from patient plasma and subjected to miRNA targeted next generation sequencing (NGS). A subsequent validation cohort was assessed using plasma collected from women with HGSC (n = 14) and controls (with a benign ovarian mass; n = 15). RNA was extracted and assessed using quantitative RT-PCR. ResultsDifferential gene expression (DGE) of the NGS data revealed a significant increase in the miRNA, miR200c, in the circulation of women with HGSC (p less than 0.05) compared to controls. In the validation cohort miR200c expression by qPCR was found to also be increased in the circulation of women with HGSC compared to controls (p = 0.0023). ConclusionsCirculating miR200c may be a promising candidate biomarker for the detection of HGSC. Further larger cohort studies exploring earlier stages are needed to determine whether circulating miR200c may be a sensitive and specific biomarker of tubo-ovarian cancer.

Paraovarian Cyst in Repeat-breeder Buffalo heifers: Ultrasonographic, Antioxidants and Serum Cell-free RNA markers

This study aimed to (1) identify the status of reproductive tract of repeat-breeder heifers (RBH) ultrasonography, as well as (2) determinethe variations in steroid hormones, oxidative stress biomarkers, and cell-free RNA in serum.These culled animals from400 heifers at the breeding stationwere examined ultrasonography, and whole blood samples were collected. Theultrasonic diagnosed heifers as paraovarian cyst (POC, n=18) associated with genital pathological conditionswere compared with groups of control heifers at follicular(n=6), luteal (n=6), and early pregnant (n=10). The POC appeared as anechoic circumscribed masses located nearby, but separated from the ovary. Follicular number, corpus luteum size, serum cell-free RNA, and ovarian steroids were not affected by POC, although dominant follicle size tended to be lower (P= 0.069)in POC withCL absence (POC-CL). Heifers with POC-CLhad lower MDA levels than pregnant ones. Buffaloes that had POC in the presence of CL (POC+CL) were found to have higher SOD activity than luteal and pregnant cases. Total serum proteinslevels recorded lower valuesin POC+CL compared to that in follicular phase. There was a significant positive relationship between POCsize, and GPx activity and CL size, but negative correlations with dominate follicles size. The POC might interfere with ovarian function, via alterations in antioxidant levels.

Emerging on-chip electrokinetic based technologies for purification of circulating cancer biomarkers towards liquid biopsy: A review.

Early detection of cancer can significantly reduce mortality and save lives. However, the current cancer diagnosis is highly dependent on costly, complex, and invasive procedures. Thus, a great deal of effort has been devoted to exploring new technologies based on liquid biopsy. Since liquid biopsy relies on detection of circulating biomarkers from biofluids, it is critical to isolate highly purified cancer-related biomarkers, including circulating tumor cells (CTCs), cell-free nucleic acids (cell-free DNA and cell-free RNA), small extracellular vesicles (exosomes), and proteins. The current clinical purification techniques are facing a number of drawbacks including low purity, long processing time, high cost, and difficulties in standardization. Here, we review a promising solution, on-chip electrokinetic-based methods, that have the advantage of small sample volume requirement, minimal damage to the biomarkers, rapid, and label-free criteria. We have also discussed the existing challenges of current on-chip electrokinetic technologies and suggested potential solutions that may be worthy of future studies.

Current and Emerging Applications of Droplet Digital PCR in Oncology: An Updated Review.

In the era of personalized medicine and targeted therapies for the management of patients with cancer, ultrasensitive detection methods for tumor genotyping, such as next-generation sequencing or droplet digital polymerase chain reaction (ddPCR), play a significant role. In the search for less invasive strategies for diagnosis, prognosis and disease monitoring, the number of publications regarding liquid biopsy approaches using ddPCR has increased substantially in recent years. There is a long list of malignancies in which ddPCR provides a reliable and accurate tool for detection of nucleic acid-based markers derived from cell-free DNA, cell-free RNA, circulating tumor cells, extracellular vesicles or exosomes when isolated from whole blood, plasma and serum, helping to anticipate tumor relapse or unveil intratumor heterogeneity and clonal evolution in response to treatment. This updated review describes recent developments in ddPCR platforms and provides a general overview about the major applications of liquid biopsy in blood, including its utility for molecular response and minimal residual disease monitoring in hematological malignancies or the therapeutic management of patients with colorectal or lung cancer, particularly for the selection and monitoring of treatment with tyrosine kinase inhibitors. Although plasma is the main source of genetic material for tumor genomic profiling, liquid biopsy by ddPCR is being investigated in a wide variety of biologic fluids, such as cerebrospinal fluid, urine, stool, ocular fluids, sputum, saliva, bronchoalveolar lavage, pleural effusion, mucin, peritoneal fluid, fine needle aspirate, bile or pancreatic juice. The present review focuses on these "alternative" sources of genetic material and their analysis by ddPCR in different kinds of cancers.

A liquid biopsy model to detect eventual pollutants as “one health” tool

Abstract Environmental pollution (EP), defined as the unfavorable chemical and physical alterations of our surroundings, still represents a relevant issue of this century. In this scenario, several changes may directly or indirectly impact on human health by promoting neoplastic cell transformation due to DNA damage in human tissue. The identification of a specific molecular signature observed in people exposed to environmental contaminants could improve risk calculation of exposition. As regards, DNA methylation is considered an epigenetic modification that could play a pivotal role in the risk calculation of patients under EC exposure. This mechanism, based on the introduction of methyl groups on cytosine's in CpG islands, is carried out by methyltransferases enzymes able to activate this modification in promotor region by influencing regulation process. Several studies demonstrated that methylation pattern is drastically alterated in subjects under EC exposure. To date, several approaches for the early detection of contaminants in biological fluids are under investigation. Liquid biopsy consists in a peripheral blood withdrawn where several biomolecules [cell-free DNA (ccfDNA), circulating tumor cells (CTCs), circulating cell-free RNA (ccfRNA) and exosomes] may be isolated. Students participate to the validation of a LP assay for the evaluation of environmental determinants with the Lab Residents. They assessed the cost-efficacy and the minimally invasive approach in order to construct a reliable model to be exported for the identification of epigenetic changes in the early stage of contamination process in high risk people. The young students role was also to create an informative schedule in order to explain the role and the potential of Liquid biopsy as a non-invasive tool that could allow the analysis of nucleic biomarkers as circulating cell-free DNA (ccfDNA), a potential stable biomarkers of diverse disease conditions in humans and animals.

The utility of plasma circulating cell-free messenger RNA as a biomarker of glioma: a pilot study

BackgroundResearch into the potential utility of plasma-derived circulating cell-free nucleic acids as non-invasive adjuncts to radiological imaging have been occasioned by the invasive nature of brain tumour biopsy. The objective of this study was to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and TCGA glioma-derived RNA.MethodsBlood samples were collected from twenty glioma patients prior to tumour resection. Plasma ccfmRNAs and glioma-derived RNA were extracted and profiled.ResultsBCL2L1, GZMB, HLA-A, IRF1, MYD88, TLR2, and TP53 genes were significantly over-expressed in glioma patients (p < 0.001, versus control). GZMB and HLA-A genes were significantly over-expressed in high-grade glioma patients (p < 0.001, versus low-grade glioma patients). Moreover, the fold change of the BCL2L1 gene was observed to be higher in patients with high-grade glioma (p = 0.022, versus low-grade glioma patients). There was positive correlation between the magnitude of fold change of differentially expressed genes in plasma- and glioma-derived RNA (Spearman r = 0.6344, n = 14, p = 0.017), and with the mean FPKM in TCGA glioma-derived RNA samples (Spearman r = 0.4614, n = 19, p < 0.05). There was positive correlation between glioma radiographic tumour burden and the magnitude of fold change of the CSF3 gene (r = 0.9813, n = 20, p < 0.001).ConclusionWe identified significant differential expression of genes involved in cancer inflammation and immunity crosstalk among patients with different glioma grades, and there was positive correlation between their transcriptomic profile in plasma and tumour samples, and with TCGA glioma-derived RNA.

Non-Invasive Characterization of the Pancreas During Bariatric Surgery via Circulating Pancreatic Specific Cell-free Messenger RNA.

Bariatric surgery results in sustained weight loss and improvement in glucose homeostasis. However, the lack of accessible non-invasive tools to examine molecular alterations occurring in the pancreas limits our understanding of the causes and recovery of glucose homeostasis. Here, we describe the use of a circulating cell free mRNA (cfmRNA) based multiplex qPCR assay to selectively amplify and quantify circulating pancreatic specific transcripts levels within plasma. We applied this assay to a cohort of 58 plasma samples consisting of 10 patients that tracks multiple time points including pre and post-bariatric surgery. In our targeted multiplex screen of 14 selected pancreatic specific circulating transcripts, we identified 13 pancreatic specific transcripts that can be amplified from plasma. Furthermore, when quantifying the amplicons obtained in the short-term post-surgery (2 weeks–1 month) and long-term (3–12 months), we observed a consistent reduction of circulating GCG transcripts during short term post-surgery. Across the cohort, GCG cfmRNA levels correlated significantly with common metrics of improvement following bariatric surgery such as: haemoglobin A1c levels (R: −0.41, p-value: 0.0039) and percentage of excess weight loss (R: 0.29, p-value: 0.046).

1104 Plasma Circulating Cell-Free Messenger RNA as a Potential Biomarker of Melanoma

Abstract Introduction Blood-borne cell-free nucleic acids are increasingly emerging as significant non-invasive adjuncts to current methods of disease status evaluation in cancer patients. In this study, we sought to examine whether significant differences exist in the plasma transcriptomic profile of advanced melanoma patients with a high disease burden compared to patients with therapeutic response. Method Plasma circulating cell-free messenger ribonucleic acid (ccfmRNA) was extracted from twenty patients with stage IV melanoma receiving immunotherapy. Pathway focused gene expression analysis was performed. Patients were assessed with paired blood sample collection and CT scan assessments at baseline and at 3 months follow up. Response Evaluation Criteria in Solid Tumours (RECIST 1.1) was used for tumour burden estimation. Results In stage IV melanoma patients, CCL5, GZMB and MYD88 genes were significantly over-expressed (p &amp;lt; 0.05 versus healthy controls). In patients with high disease burden or progressive disease, CCL18, CCR1, CCR4, CD274, CSF2, and GBP1 genes were significantly over-expressed (p &amp;lt; 0.05 versus patients with therapeutic response). Finally, in stage IV melanoma patients with brain metastases, CCL18, CCR1, CCR4, CD274, CSF2, EGF, GBP1, and PTGS2 genes were significantly over-expressed (p &amp;lt; 0.05, versus patients without brain metastasis). Conclusions Significant differences were observed in the plasma transcriptomic profile between the various melanoma patient groups, and we postulate that these differences may be exploited to identify novel therapeutic targets or biomarkers relevant to melanoma. CCL4 and CCL5 are prognostic in melanoma, both genes had significantly higher expressions in low disease burden patients compared to patients with a high disease burden.

1095 The Utility of Plasma Circulating Cell-Free Messenger RNA as A Biomarker of Glioma: A Pilot Study

Abstract Introduction Research into the potential utility of plasma-derived circulating-cell-free nucleic acids as non-invasive adjuncts to radiological imaging has been occasioned by the invasive nature of brain tumour biopsy. Circulating-cell-free messenger RNAs are short fragments of RNA present in blood. The objective of this study was to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and TCGA glioma derived RNA. Method Blood samples were collected from twenty-nine patients prior to tumour resection. Plasma-ccfmRNA and glioma derived RNA were extracted and profiled. Results BCL2L1, CXCL5, GZMB, HLA-A, HLA-C, IRF1, MYD88, TGFB1, TLR2, and TP53 genes were significantly over-expressed in glioma (high-grade-glioma-HGG and low-grade-glioma-LGG) patients (p &amp;lt; 0.05, versus control). BCL2L1, GZMB and HLA-A genes were significantly over-expressed in HGG patients (p &amp;lt; 0.05, versus LGG patients). There was positive correlation between the magnitude of fold change of differentially expressed genes in plasma and glioma derived RNA (Spearman r = 0.6344, n = 14, p = 0.017), and with the mean FPKM of TCGA glioma derived RNA samples (Spearman r = 0.4614, n = 19, p = 0.047). There was positive correlation between glioma radiographic tumour burden and the magnitude of fold change of CSF3 gene (r = 0.9813, n = 20, p &amp;lt; 0.001). Conclusions We identified significant differential expression of genes involved in cancer inflammation and immunity among patients with different glioma grades, and we identified positive correlation between the plasma transcriptomic profile and tumour samples, and with TCGA glioma derived RNA.

Nanostructures in non-invasive prenatal genetic screening.

Prenatal screening is an important issue during pregnancy to ensure fetal and maternal health, as well as preventing the birth of a defective fetus and further problems such as extra costs for the family and society. The methods for the screening have progressed to non-invasive approaches over the recent years. Limitations of common standard screening tests, including invasive sampling, high risk of abortion and a big delay in result preparation have led to the introduction of new rapid and non-invasive approaches for screening. Non-invasive prenatal screening includes a wide range of procedures, including fetal cell-free DNA analysis, proteome, RNAs and other fetal biomarkers in maternal serum. These biomarkers require less invasive sampling than usual methods such as chorionic villus sampling, amniocentesis or cordocentesis. Advanced strategies including the development of nanobiosensors and the use of special nanoparticles have provided optimization and development of NIPS tests, which leads to more accurate, specific and sensitive screening tests, rapid and more reliable results and low cost, as well. This review discusses the specifications and limitations of current non-invasive prenatal screening tests and introduces a novel collection of detection methods reported studies on nanoparticles' aided detection. It can open a new prospect for further studies and effective investigations in prenatal screening field.

Comprehensive RNA analysis of CSF reveals a role for CEACAM6 in lung cancer leptomeningeal metastases

Non-small cell lung cancer (NSCLC) metastatic to the brain leptomeninges is rapidly fatal, cannot be biopsied, and cancer cells in the cerebrospinal fluid (CSF) are few; therefore, available tissue samples to develop effective treatments are severely limited. This study aimed to converge single-cell RNA-seq and cell-free RNA (cfRNA) analyses to both diagnose NSCLC leptomeningeal metastases (LM), and to use gene expression profiles to understand progression mechanisms of NSCLC in the brain leptomeninges. NSCLC patients with suspected LM underwent withdrawal of CSF via lumbar puncture. Four cytology-positive CSF samples underwent single-cell capture (n = 197 cells) by microfluidic chip. Using robust principal component analyses, NSCLC LM cell gene expression was compared to immune cells. Massively parallel qPCR (9216 simultaneous reactions) on human CSF cfRNA samples compared the relative gene expression of patients with NSCLC LM (n = 14) to non-tumor controls (n = 7). The NSCLC-associated gene, CEACAM6, underwent in vitro validation in NSCLC cell lines for involvement in pathologic behaviors characteristic of LM. NSCLC LM gene expression revealed by single-cell RNA-seq was also reflected in CSF cfRNA of cytology-positive patients. Tumor-associated cfRNA (e.g., CEACAM6, MUC1) was present in NSCLC LM patients’ CSF, but not in controls (CEACAM6 detection sensitivity 88.24% and specificity 100%). Cell migration in NSCLC cell lines was directly proportional to CEACAM6 expression, suggesting a role in disease progression. NSCLC-associated cfRNA is detectable in the CSF of patients with LM, and corresponds to the gene expression profile of NSCLC LM cells. CEACAM6 contributes significantly to NSCLC migration, a hallmark of LM pathophysiology.

P-055: Extracellular RNA: an emerging biomarker for therapeutic monitoring in multiple myeloma

P-055: Extracellular RNA: an emerging biomarker for therapeutic monitoring in multiple myeloma

RNA Origami: Packaging a Segmented Genome in Orbivirus Assembly and Replication.

Understanding how viruses with multi-segmented genomes incorporate one copy of each segment into their capsids remains an intriguing question. Here, we review our recent progress and describe the advancements made in understanding the genome packaging mechanism of a model nonenveloped virus, Bluetongue virus (BTV), with a 10-segment (S1–S10) double-strand RNA (dsRNA) genome. BTV (multiple serotypes), a member of the Orbivirus genus in the Reoviridae family, is a notable pathogen for livestock and is responsible for significant economic losses worldwide. This has enabled the creation of an extensive set of reagents and assays, including reverse genetics, cell-free RNA packaging, and bespoke bioinformatics approaches, which can be directed to address the packaging question. Our studies have shown that (i) UTRs enable the conformation of each segment necessary for the next level of RNA–RNA interaction; (ii) a specific order of intersegment interactions leads to a complex RNA network containing all the active components in sorting and packaging; (iii) networked segments are recruited into nascent assembling capsids; and (iv) select capsid proteins might be involved in the packaging process. The key features of genome packaging mechanisms for BTV and related dsRNA viruses are novel and open up new avenues of potential intervention.

Recent advances in blood-based and artificial intelligence-enhanced approaches for gastrointestinal cancer diagnosis.

Gastrointestinal (GI) cancers are among the most common cancer types and leading causes of cancer-related deaths worldwide. There is a tremendous clinical need for effective early diagnosis for better healthcare of GI cancer patients. In this article, we provide a short overview of the recent advances in GI cancer diagnosis. In the first part, we discuss the applications of blood-based biomarkers, such as plasma circulating cell-free DNA, circulating tumor cells, extracellular vesicles, and circulating cell-free RNA, for cancer liquid biopsies. In the second part, we review the current trends of artificial intelligence (AI) for pathology image and tissue biopsy analysis for GI cancer, as well as deep learning-based approaches for purity assessment of tissue biopsies. We further provide our opinions on the future directions in blood-based and AI-enhanced approaches for GI cancer diagnosis, and we think that these fields will have more intensive integrations with clinical needs in the near future.

A Multiplex Thyroid-Specific Assay for Quantification of Circulating Thyroid Cell-Free RNA in Plasma of Thyroid Cancer Patients.

BackgroundThe standard of care for thyroid cancer management is thyroidectomy and adjuvant radioactive iodine (RAI). There is a paucity of clinical tool that quantifies residual thyroid volume reliably for precise adjuvant RAI dosing. Serum thyroglobulin (TG), tumour marker for thyroid cancer, takes 4 weeks for complete clearance due to its long half-life, and might be undetectable in 12% of structural disease patients. It detects recurrence with a sensitivity of 19–40%, mainly attributed to issue of TG antibody interference with TG immunometric assay. We hypothesise that the quantity of thyroid-specific cell-free RNA (cfRNA) is indicative of amount of thyroid tissues, and that during thyroid surgery, cfRNA levels decrease accordingly.MethodsWe identified 11 biologically significant and highly expressed thyroid-specific targets from Human Protein Atlas and literature. To assess for a fall in thyroid-specific cfRNA level, we recruited 16 patients undergoing thyroid surgery or RAI for malignant or benign thyroid disease, and tracked longitudinal trend of cfRNA. To assess the utility of cfRNA in detecting metastatic thyroid cancer, cfRNA of 11 patients at intermediate to high risk of recurrence was measured during surveillance and at time of clinical recurrence.ResultsThe multiplex assay was capable of amplifying and quantifying multiple thyroid-specific genes in a single reaction. The selected targets were amplified successfully from RNA extracted directly from the thyroid (positive control), indicating that they were highly expressed within thyroid tissue. These cfRNAs were present in plasma, in amounts quantifiable using qRT-PCR. Four cfRNA transcripts (TPO, GFRA2, IVD, TG) fell post-treatment in more than 50% of cohort. The thyroid peroxidase (TPO) cfRNA fell post-therapy in 63% of cohort by 80%, as early as 1 day post-treatment, supporting the potential role as early indicator of remnant thyroid tissue volume. We demonstrated the clinical relevance of circulating TPO cfRNA by tracking temporal changes in setting of peri-treatment, recurrence, and TG Ab positive state.ConclusionUsing a multiplex pre-amplification approach, the TPO cfRNA was a potential biomarker that can track residual thyroid mass. It can be further optimised for quantification of thyroid volume to guide RAI doses and for detection of thyroid cancer recurrence.

Comparative Analyses of Liquid-Biopsy MicroRNA371a-3p Isolation Protocols for Serum and Plasma.

Simple SummaryThe active disease status of patients with a malignant germ cell tumor can be evaluated using detection of specific body-circulating microRNAs. However, various methods are reported to isolate and detect microRNAs from blood, possibly influencing the score as positive or negative. Here, we investigated two frequently used techniques for microRNA isolation from blood, either serum or plasma, to evaluate possible differences. These data are required to compare published studies and to select the best methods in the future. No effect of either starting with plasma or serum was found, indicating that both blood products can be used. The bead-based method was more stable and applicable on small blood volumes, whereas the total RNA method exhibited a higher sensitivity due to a larger starting volume. These results are important to develop the optimal method for the detection of microRNAs in blood to monitor malignant germ cell tumor patients in clinic practice.MicroRNAs (miRNAs) are short, non-coding RNAs involved in translation regulation. Dysregulation has been identified in cancer cells. miRNAs can be secreted and detectable in body fluids; therefore, they are potential liquid-biopsy biomarkers. The miR-371a-3 cluster members are an example, monitoring the presence of malignant germ cell tumors based on patient serum/plasma analyses. However, a large variety of isolation techniques on sample types (serum vs. plasma) are reported, hampering interstudy comparisons. Therefore, we analyzed the impact of using the miRNeasy Serum/Plasma Kit (cell-free total RNA purification) Qiagen extraction kit and the TaqMan anti-miRNA bead-capture procedure of ThermoFisher for miRNA isolation. Ten normal male matched serum and plasma samples and seventeen testicular germ cell tumor patient serum samples were investigated. The Qiagen kit requires a higher input volume (200 µL vs. 50 µL), resulting in higher sensitivity. Serum and plasma comparison demonstrated high similarity in miRNA levels. Titration experiments showed that the bead-capture procedure is superior in cases of lower starting volumes (<100 µL). This study highlights the strengths and limitations of two different isolation protocols, relevant for in vivo analysis with small starting volumes. In summary, miRNA detection levels results varied little between plasma and serum, whereas for low volumes the bead capture isolation method is preferable.

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

INFLUENCE OF LITTER SIZE ON ULTRASOUND ESTIMATED FETAL GROWTH CURVE, MATERNAL STEROIDS, OXIDATIVE STRESS AND SERUM FREE RNA IN GOATS

This study aimed at evaluating the litter size influence on fetal growth (marked by biparietal diameter), steroid hormones (estradiol and progesterone), oxidative stress markers [Total antioxidant capacity (TAC), superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx), and malondialdehyde (MDA)], total proteins, and serum-free RNA. Goats (n=150) were blood sampled and assessed ultrasonographically during the mid-stage of pregnancy (6th to 14th week) and were classified into non-pregnant (n=64), single (n= 55) twine (n= 25), and triple (n= 6) pregnancy according to a number of feti. The correlation coefficient of caprine fetal growth was R² = 0.9609, 0.9418, and, 0.928 in single, twin, and triple feti, respectively. The area under the curve of the fetal growth was 286.2, 282.1, and 263.4 for single, twin, and triple caprine fetuses. The mean reduction rate in fetal growth compared to singleton pregnancy was 1.65±1.03 and 8.32±2.41 % in twine and triple feti, respectively. Estradiol significantly (P&lt; 0.05) decreased, while progesterone (P&lt; 0.01) and serum-free RNA (P&lt; 0.001) increased in pregnant animals compared to non-pregnant. TAC and MDA increased in multiple pregnancies compared to non-pregnancy in association with the decrease of SOD and catalase activities. GPx activity and total proteins substantially decreased in triple pregnancy than non-pregnancy. Cell-free RNA negatively correlated with estradiol, CAT, GPx, and total proteins, and positively correlated with P4, TAC, and MDA. In conclusion, litter size greatly impacted fetal growth, maternal steroids, and serum-free RNA, and preload to oxidative stressmediated health disorders in pregnant goats.

Validation of Cell-Free RNA and Circulating Tumor Cells for Molecular Marker Analysis in Metastatic Prostate Cancer.

Since tissue material is often lacking in metastatic prostate cancer (mPCa), there is increasing interest in using liquid biopsies for treatment decision and monitoring therapy responses. The purpose of this study was to validate the usefulness of circulating tumor cells (CTCs) and plasma-derived cell-free (cf) RNA as starting material for gene expression analysis through qPCR. CTCs were identified upon prostate-specific membrane antigen and/or cytokeratin positivity after enrichment with ScreenCell (Westford, Massachusetts, USA) filters or the microfluidic ParsortixTM (Guildford, Surrey, United Kingdom) system. Overall, 50% (28/56) of the patients had ≥5 CTCs/7.5 mL of blood. However, CTC count did not correlate with Gleason score, serum PSA, or gene expression. Notably, we observed high expression of CD45 in CTC samples after enrichment, which could be successfully eliminated through picking of single cells. Gene expression in picked CTCs was, however, rather low. In cfRNA from plasma, on the other hand, gene expression levels were higher compared to those found in CTCs. Moreover, we found that PSA was significantly increased in plasma-derived cfRNA of mPCa patients compared to healthy controls. High PSA expression was also associated with poor overall survival, indicating that using cfRNA from plasma could be used as a valuable tool for molecular expression analysis.