Isolation Of mRNA Research Articles

Drug-Induced Differential Gene Expression Analysis on Nanoliter Droplet Microarrays: Enabling Tool for Functional Precision Oncology.

Drug-induced differential gene expression analysis (DGEA) is essential for uncovering the molecular basis of cell phenotypic changes and understanding individual tumor responses to anticancer drugs. Performing high throughput DGEA is challenging due to the high cost and labor-intensive multi-step sample preparation protocols. In particular, performing drug-induced DGEA on cancer cells derived from patient biopsies is even more challenging due to the scarcity of available cells. A novel, miniaturized, nanoliter-scale method for drug-induced DGEA is introduced, enabling high-throughput and parallel analysis of patient-derived cell drug responses, overcoming the limitations and laborious nature of traditional protocols. The method is based on the Droplet Microarray (DMA), a microscope glass slide with hydrophilic spots on a superhydrophobic background, facilitating droplet formation for cell testing. DMA allows microscopy-based phenotypic analysis, cDNA extraction, and DGEA. The procedure includes cell lysis for mRNA isolation and cDNA conversion followed by droplet pooling for qPCR analysis. In this study, the drug-induced DGEA protocol on the DMA platform is demonstrated using patient-derived chronic lymphocytic leukemia (CLL) cells. This methodology is critical for DGEA with limited cell numbers and promise for applications in functional precision oncology. This method enables molecular profiling of patient-derived samples after drug treatment, crucial for understanding individual tumor responses to anticancer drugs.

DMF-DM-seq: Digital-Microfluidics Enabled Dual-Modality Sequencing of Single-Cell mRNA and microRNA with High Integration, Sensitivity, and Automation.

Multimodal measurement of single cells provides deep insights into the intricate relationships between individual molecular layers and the regulatory mechanisms underlying intercellular variations. Here, we reported DMF-DM-seq, a highly integrated, sensitive, and automated platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics. This platform first integrates the processes of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enables complete separation of single-cell mRNA and miRNA via a magnetic field application, resulting in a higher miRNA detection ability. DMF-DM-seq revealed differential expression patterns of single cells of noncancerous breast cells and noninvasive and aggressive breast cancer cells at both mRNA and miRNA levels. The results demonstrated the anticorrelated relationship between miRNA and their mRNA targets. Further, we unravel the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, along with important miRNA-interaction networks involved in significant signaling pathways. We also deconstruct the miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool for a comprehensive study of the expression heterogeneity of single-cell mRNA and miRNA, which will be widely applied in basic and clinical research.

Combined amphiregulin and epiregulin expression as a prognostic and predictive biomarker of panitumumab (Pmab), fluorouracil, and folinic acid (FU/FA) or FU/FA maintenance therapy following Pmab+FOLFOX induction in RAS wildtype metastatic colorectal cancer (mCRC): PANAMA trial (AIO-KRK-0212).

3528 Background: mRNA expression of amphiregulin ( AREG) and epiregulin ( EREG) as ligands of the epidermal growth factor receptor (EGFR) were associated with response to anti-EGFR directed antibodies in metastatic colorectal cancer (mCRC). Their role for maintenance treatment remained unclear yet and was investigated in the PanaMa trial (panitumumab (Pmab) and fluorouracil/folinic acid (FU/FA) vs. FU/FA alone after Pmab+FOLFOX induction therapy in RAS wild-type mCRC). Methods: Gene expression was measured after mRNA isolation in 196 of 248 patients of the full analysis set. The analysis was conducted using a customized Nanostring PanCancer Progression Panel. AREG and EREG mRNA expression was dichotomized by median. High combined expression was defined as mRNA expression of AREG and EREGequal or above the median. Median progression-free (PFS) and overall survival (OS) since start of maintenance were estimated by Kaplan-Meier-method and Cox-regression, using the log rank test. Objective response rates (ORR) of maintenance therapy were compared by Fisher’s exact test. Treatment interaction was tested by Cox regression. Results: In 196 patients with available mRNA expression data, high AREG, EREG and combined AREG / EREG expression were observed in 103 (52.6%), 97 (49.5%) and 84 (42.9%), respectively. Regardless of treatment, high combined AREG / EREG expression was associated with prolonged PFS (7.7 vs. 5.8 months, HR 0.69 (95% CI 0.51 – 0.93), P=0.016), OS (30.1 vs. 24.4 months, HR 0.58 (95% CI 0.41 – 0.82), P=0.002) and increased ORR of maintenance treatment (39.3% vs. 28.6%, P=0.13), compared to low expression. Tumors with high combined AREG / EREG expression were associated with numerical longer PFS, OS and higher ORR when treated with Pmab+FU/FA maintenance (Table). Significant interaction of combined AREG / EREG expression with treatment was observed in terms of PFS ( P=0.003) and OS ( P=0.016). Conclusions: Combined AREG and EREG mRNA expression was confirmed as prognostic biomarker for mCRC patients receiving maintenance treatment. Efficacy of maintenance treatment might potentially be higher for Pmab+FUFA maintenance in mCRC with high combined AREG / EREG expression in the PanaMa trial. [Table: see text]

#1493 Upregulated miR-205 as a biomarker of diet-induced obesity-related glomerulopathy

Abstract Background and Aims Obesity is a modifiable risk factor for the development and progression of chronic kidney disease (CKD). Pathways through which obesity might cause renal disease are not completely understood, as not all obese subjects develop CKD. In terms of deepening the knowledge of the preliminary steps of obesity-related renal damage, our group has developed a diet-induced obese murine model with obesity related glomerulopathy (ORG). The objective of this study was to investigate ORG at a cellular, structural and transcriptomic level as a way to discover the triggering pathogenic phenomena of ORG and evaluate potential biomarkers. Method Thirty Wistar rats were randomized into two groups: control rats (n = 15), which were fed with standard diet (SD) and study rats (n = 15), which were fed with high-fat diet (HFD). After 10 weeks, weight, parameters of kidney function, renal histological features, transcriptomic changes, miRNA and mRNA isolation were compared. Results HFD gained significantly more weight (55.8%) than SD(29.2%), p = 0.001. Albuminuria was also significantly higher in HFD (10,384.04) compared with SD (5,845.45), p = 0.001. HFD rats showed typical lesions of early-stages of ORG, with a predominance of mesangial matrix increase (MMI) and podocyte hypertrophy (PH). All histologic lesions correlated with genes differentially expressed (DE) in kidneys of HFD group and, both MMI and PH, also correlated with specific miRNAs DE in the urine of HFD group. The functional analysis showed 4 miRNAs DE in the kidneys of HFD group that negatively regulates PTEN gene, which promotes podocyte endocytosis of lipids in ORG. The electronic microscope confirmed the spaces of lipid vacuoles in the podocytes of HFD rats. Between those 4 miRNAs DE, miR-205 was also found to be upregulated in the urine of HFD group. Conclusion Wistar rats fed with HFD for 10 weeks developed early-stages of ORG, with a specific targetome of miRNAs and gene expression. The upregulation of miR-205 in kidney and its isolation in urine is associated with lipid endocytosis of podocytes, which takes place in the early-stages of ORG and could become a plausible biomarker of early-stages of ORG and open new avenues for future therapeutics research.

Rapid and high recovery isolation of mRNA from in vitro transcription system by ammonium sulphate precipitation at room temperature

Messenger RNA (mRNA) technologies have shown great potential in prophylactic vaccines and therapeutic medicines. Rapidly and effectively separating mRNA from in vitro transcription (IVT) products is important to reduce the accumulation of byproducts and avoid degradation of the mRNA. In this study, the salting out of mRNA encoding enhanced green fluorescent protein (EGFP) by ammonium sulfate (AS) that was widely employed in protein precipitation was examined. Effects of precipitation conditions, including AS concentration, pH, temperature, and time, on the precipitation rate and re-dissolution yield of mRNA were first investigated by the design of experiments (DOE) method, using LiCl-purified mRNA. The optimal condition was determined to be 2 M AS, pH 7.0, 20 °C, and 2 h of precipitation time, under which almost 100 % precipitation rate and nearly 100 % dissolution rate could be reached. This optimal condition was then applied to separate mRNA from IVT and co-transcriptional capping system, overall recovery of about 80 % of mRNA was obtained; meanwhile, more than 80 % DNA template and 99.0 % residual NTP were removed. In co-transcriptional capping system, >90 % dsRNA was removed. The mRNA purified by AS precipitation was transfected into HEK293T cells to successfully express EGFP with efficiency of 45.65 % which was similar to that by LiCl-purified mRNA (43.85 %). Though the removal rate for T7 polymerase was low (about 30 %) due to protein-mRNA interaction induced co-precipitation in 2 M AS, our results demonstrated significant advantages of AS precipitation over conventional LiCl precipitation in terms of biosafety and energy cost. Thus, the AS precipitation provides a cost-effective method to rapidly separate, concentrate, and store the mRNA from the IVT system, as well as during subsequent downstream processes.

Simultaneous Isolation and Amplification of mRNA and Genomic DNA of a Single Cell.

Many biological or pathological processes are driven by cells difficult to identify or isolate, i.e., rare cells. Very often, these cells have elusive biology. Therefore, their detailed characterization is of utmost importance. There are many approaches that allow analysis of few or even many targets within one class of biomacromolecules/analytes (e.g., DNA, RNA, proteins, etc.) in single cells. However, due to rarity of the cells of interest, there is a great need to comprehensively analyze multiple analytes within these cells, in other words to perform multi-omics analysis. In this chapter, I describe a method to isolate, separate, and amplify total mRNA and genomic DNA of a single cells, using whole transcriptome (WTA) and whole genome amplification (WGA). These WTA and WGA products enable simultaneous analysis of transcriptome and genome of a single cell using various downstream high-throughput approaches.

Updated Pseudo-seq Protocol for Transcriptome-Wide Detection of Pseudouridines.

Pseudouridine (Ψ), the most prevalent modified base in cellular RNAs, has been mapped to numerous sites not only in rRNAs, tRNAs, and snRNAs but also mRNAs. Although there have been multiple techniques to identify Ψs, due to the recent development of sequencing technologies some reagents are not compatible with the current sequencer. Here, we show the updated Pseudo-seq, a technique enabling the genome-wide identification of pseudouridylation sites with single-nucleotide precision. We provide a comprehensive description of Pseudo-seq, covering protocols for RNA isolation from human cells, library preparation, and detailed data analysis procedures. The methodology presented is easily adaptable to any cell or tissue type with high-quality mRNA isolation. It can be used for discovering novel pseudouridylation sites, thus constituting a crucial initial step toward understanding the regulation and function of this modification. Key features • Identification of Ψ sites on mRNAs. • Updated Pseudo-seq provides precise positional and quantitative information of Ψ. • Uses a more efficient library preparation with the latest, currently available materials.

An experimental workflow for identifying RNA m6A alterations in cellular senescence by methylated RNA immunoprecipitation sequencing.

N6-methyladenosine (m6A), the most prevalent mRNA modification in eukaryotic cells, is known to play regulatory roles in a wide array of biological processes, including aging and cellular senescence. To investigate such roles, the m6A modification can be identified across the entire transcriptome by immunoprecipitation of methylated RNA with an anti-m6A antibody, followed by high-throughput sequencing (meRIP-seq or m6A-seq). Presented here is a protocol for employing meRIP-seq to profile the RNA m6A landscape in senescent human cells. We described, in detail, sample preparation, mRNA isolation, immunoprecipitation, library preparation, sequencing, bioinformatic analysis and validation. We also provided tips and considerations for the optimization and interpretation of the results. Our protocol serves as a methodological resource for investigating transcriptomic m6A alterations in cellular senescence as well as a valuable paradigm for the validation of genes of interest.

Ketorolac Tromethamine Loaded Nano-Spray Dried Nanoparticles: Preparation, Characterization, Cell Viability, COL1A1 Gene Simulation and Determination of Anti-inflammatory Activity by In vivo HET-CAM Assay.

Ketorolac tromethamine (KT) is a non-steroidal anti-inflammatory drug from the heteroaryl acetic acid derivatives family. The most widely used new nanotechnological approaches for topical drug delivery are polymeric nanoparticles (NPs). Successful results have been obtained with low doses in many treatments, such as cancer, antimicrobial, pain, made with nanoparticle formulations of drug active ingredients. NPs were prepared using Nano Spray-Dryer. The cytotoxicity of the optimum formulation in BJ (ATCC® CRL-2522™) human fibroblast cells was determined by the WST- 1 method and the gene activity was elucidated by mRNA isolation and real-time polymerase chain reaction (RT-PCR). The in vivo HET- CAM assay was performed for anti-inflammatory activity. NPs presented PDI values lower than 0.5, and therefore particle size distribution was decided to be monodisperse. Positive zeta potential values of NPs highlighted the presence of the cationic ammonium group of Eudragit® RS 100. The release rates observed from KT-NP coded formulations after 24 hours were 78.4% ± 2.9, demonstrating extended release from all formulations, relative to pure KT. The lowest concentration of KT-NP increased fibroblast cell proliferation higher than the highest concentration of KT. The 5-fold increased effect of KT-NP formulation on collagen gene expression compared to KT is also related to the enhanced anti-inflammatory effect in line with the in vivo HET-CAM assay results. With the obtained cell viability, gene expression, and HET-CAM results, it has the hope of a successful nano-topical formulation, especially in both wound healing and anti-inflammatory treatment.

Normalization methods for RT‐qPCR assessment of circulating microRNAs in Alzheimer’s and other aging‐related diseases

AbstractBackgroundCirculating miRNAs in blood plasma present significant potential for use as minimally‐invasive biomarkers for diagnosis of aging‐related diseases, such as cancer or Alzheimer’s disease (AD). The most sensitive and inexpensive analysis method of circulating miRNA is RT‐qPCR. However, lack of RT‐qPCR standardisation is one of the major causes of data inconsistency across different studies and it hinders progress towards widespread implementation of miRNA biomarkers in clinical use. Normalisation of RT‐qPCR data to stably expressed miRNAs is a key step in data analysis. Current methods of identifying optimal normalisers are lacking in their evaluation of the stability of normalisers and their combinations in aging populations. To address these needs, we created a novel, transparent, method for selecting optimal normalisers in aging populations, using the Python programming language (v. 3.7.3).MethodOur method uses the Python packages: pandas (v. 1.2.3), numpy (v. 1.17.1), scipy (v. 1.3.1). The method uses three measures to evaluate the miRNA Cq values for potential normalisers: Kolmogorov‐Smirnov score (KS‐score), average displacement from a mean of zero and mean standard deviation. In each case, the comparators are two distinct biological groups of interest (such as AD and cognitively normal controls). Before comparison, the group of potential normalisers were normalised according to the log2(2−ΔΔCq) method. The miRNA data were collected from Polish subjects including 44 AD patients and 70 similar‐aged, cognitively normal, controls). Circulating miRNA molecules were isolated from plasma, followed by RT‐qPCR with a selection of 7 miRNAs tested as potential normalisers, along with suitable controls for mRNA isolation and reverse transcription.ResultWe provide a novel method for identification of optimal normalisers with the advantages of: assessments of a greater number of combined potential normalisers (7 in this study), transparency of decision making and the ability for end users to weight that decision making according to their judgment of relative importance.ConclusionWe recommend assessment of plasma miRNA levels in an aging population employing a novel set of normalisers.Work supported by the Polish National Science Centre grant OPUS 2018/29/B/NZ7/02757and by the EU Horizon2020 FETOPEN grant no 737390 (ArrestAD).

NSUN2-mediated m5 C RNA methylation dictates retinoblastoma progression through promoting PFAS mRNA stability and expression.

The precise temporal and spatial regulation of N5 -methylcytosine (m5 C) RNA modification plays essential roles in RNA metabolism, and is necessary for the maintenance of epigenome homeostasis. Howbeit, the mechanism underlying the m5 C modification in carcinogenesis remains to be fully addressed. Global and mRNA m5 C levels were determined by mRNA isolation and anti-m5 C dot blot in both retinoblastoma (RB) cells and clinical samples. Orthotopic intraocular xenografts were established to examine the oncogenic behaviours of RB. Genome-wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m5 C-methylated RNA immunoprecipitation sequencing (m5 C-meRIP-seq). Organoid-based single-cell analysis and gene-correlation analysis were performed to verify the NSUN2/ALYREF/m5 C-PFAS oncogenic cascade. Herein, we report that NSUN2-mediated m5 C RNA methylation fuels purine biosynthesis during the oncogenic progression of RB. First, we discovered that global and mRNA m5 C levels were significantly enriched in RBs compared to normal retinas. In addition, tumour-specific NSUN2 expression was noted in RB samples and cell lines. Therapeutically, targeted correction of NSUN2 exhibited efficient therapeutic efficacy in RB both in vitro and in vivo. Through multiomics analyses, we subsequently identified phosphoribosylformylglycinamidine synthase (PFAS), a vital enzyme in purine biosynthesis, as a downstream candidate target of NSUN2. The reintroduction of PFAS largely reversed the inhibitory phenotypes in NSUN2-deficient RB cells, indicating that PFAS was a functional downstream target of NSUN2. Mechanistically, we found that the m5 C reader protein ALYREF was responsible for the recognition of the m5 C modification of PFAS, increasing its expression by enhancing its RNA stability. Conclusively, we initially demonstrated that NSUN2 is necessary for oncogenic gene activation in RB, expanding the current understanding of dynamic m5 C function during tumour progression. As the NSUN2/ALYREF/m5 C-PFAS oncogenic cascade is an important RB trigger, our study suggests that a targeted m5 C reprogramming therapeutic strategy may be a novel and efficient anti-tumour therapy approach.

Abstract 6221: Support for targeted therapy selection in triple negative breast cancer patients using aberrant signal transduction pathway activity profiles

Abstract Introduction: Success of therapeutic interventions largely rely on the pathological and biological characteristics of the tumor and varies due to the heterogeneous nature of breast cancers. Triple negative breast cancers (TNBC) stain IHC negative for both hormone receptors (ER, PR) and HER-2. TNBC is usually treated with chemotherapy because of absence of any molecular target. This study is initiated to characterize TNBC based on a new methodology that measures and quantifies signal transduction pathways (STPs) activities to reveal potential tumor-driving STPs in TNBC and create new options for targeted therapy. Method: Using the mRNA-based OncoSIGNal pathway activity profiling PCR test (InnoSIGN) STP activities of 7 pathways (AR, ER, PI3K, MAPK, HH, TGFβ and Notch) in 39 samples from normal breast tissue and 12 TNBC patients were quantified and expressed on a scale from 0-100. Normal healthy breast tissue was obtained from breast reduction surgery and areas where epithelial cells content was at least 50% were annotated and harvested for mRNA isolation and STP analyses. The normal STP activity is representing the normal physiological activity and is used as reference value. Aberrant high pathway activity in a patient tumor sample was concluded when this score was higher than twice the standard deviation above the mean of normal breast tissue STP activity. Results: STP thresholds for interpretation of high aberrant activity were determined from 39 normal breast samples. Using these thresholds, STP activities in 12 TNBC samples were analyzed. In these TNBC samples, MAPK was in 75%, HH in 67%, PI3K in 58%, and AR, Notch and TGFβ were in 17% of the cases above the thresholds. In 50% of the cases a combination of MAPK and HH or a combination of PI3K and HH were found above thresholds. In 42% of the cases MAPK in combination with PI3K were above thresholds. Increased activity of AR was never observed in combination with increased HH activity. Conclusion: By using the OncoSIGNal test high aberrant STP activities could be determined in TNBC patient samples. Results show that different pathways are aberrantly active, depending on the patient sample, which opens the opportunity for personalized targeted treatment of individual TNBC patients. Next steps: The TNBC data set will be extended to further profile the STP activity in relation to different clinical outcomes and to investigate new opportunities for targeted therapies. Citation Format: Diederick M. Keizer, Yvonne J. Wesseling, Dianne A. van Strijp, Saskia M. Vermeer, Eveline C. den Biezen. Support for targeted therapy selection in triple negative breast cancer patients using aberrant signal transduction pathway activity profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6221.

ISOLATION AND IDENTIFICATION OF PARTIAL GROWTH HORMONE (GH) mRNA OF TIGER SHOVELNOSE CATFISH Pseudoplatystoma fasciatum (LINNAEUS, 1766)

The growth of fish is regulated by growth hormones secreted in a limited amount by its pituitary glands. Tiger shovelnose catfish has a relatively faster growth indicating that some growth hormones suspectedly play roles in the process and could be used in improving other farmed fish species. This study aimed to isolate and identify the growth hormone (GH) mRNA gene in Tiger shovelnose catfish (Pseudoplatystoma fasciatum Linnaeus, 1766). The sample was isolated from the pituitary, the fish measuring 602 g and 43 cm body length; Total RNA was extracted using the Tri Reagent Kit, followed with cDNA synthesis. The success of the extraction was confirmed by quantification and PCR using the β-actin gene as an internal control. GH mRNA gene was isolated by RT-PCR method, with degenerated primers from seven catfish species sequence data in the NCBI gene bank. The single band from GH gene was cloned and sequenced. Total RNA quantification with a concentration of 227 ng/µL and purity of 1.821. The successful isolation of mRNA from the pituitary gland was confirmed by amplifying the β-actin gene generated at 300 bp. This isolation of the GH mRNA gene had a sequence length of 234 bp. Tiger shovelnose catfish GH gene consists of 17 amino acid residues. The GH gene of Tiger shovelnose catfish was close to that of striped catfish (P. hypophthalmus) and Indian catfish (Clarias batrachus) with almost similar homology value of 90.6 %. Partial GH gene from tiger shovelnose catfish can be used a molecular marker in revealing the role of growth hormone on fish development, fish biology, growth gene expression, selective breeding and other mechanisms related to the aquaculture.

Combination of Small Extracellular Vesicle-Derived Annexin A2 Protein and mRNA as a Potential Predictive Biomarker for Chemotherapy Responsiveness in Aggressive Triple-Negative Breast Cancer

Small extracellular vesicles (sEVs), mainly exosomes, are nanovesicles that shed from the membrane as intraluminal vesicles of the multivesicular bodies, serve as vehicles that carry cargo influential in modulating the tumor microenvironment for the multi-step process of cancer metastasis. Annexin A2 (AnxA2), a calcium(Ca2+)-dependent phospholipid-binding protein, is among sEV cargoes. sEV-derived AnxA2 (sEV-AnxA2) protein is involved in the process of metastasis in triple-negative breast cancer (TNBC). The objective of the current study is to determine whether sEV-AnxA2 protein and/or mRNA could be a useful biomarkers to predict the responsiveness of chemotherapy in TNBC. Removal of Immunoglobulin G (IgG) from the serum as well as using the System Bioscience's ExoQuick Ultra kit resulted in efficient sEV isolation and detection of sEV-AnxA2 protein and mRNA compared to the ultracentrifugation method. The standardized method was applied to the twenty TNBC patient sera for sEV isolation. High levels of sEV-AnxA2 protein and/or mRNA were associated with stage 3 and above in TNBC. Four patients who responded to neoadjuvant chemotherapy had high expression of AnxA2 protein and/or mRNA in sEVs, while other four who did not respond to chemotherapy had low levels of AnxA2 protein and mRNA in sEVs. Our data suggest that the sEV-AnxA2 protein and mRNA could be a combined predictive biomarker for responsiveness to chemotherapy in aggressive TNBC.

Urinary cell mRNA profiling of kidney allograft recipients: Development of a portable protocol for noninvasive diagnosis of T cell mediated rejection and BK virus nephropathy

BackgroundWe developed urinary cell mRNA profiling for noninvasive diagnosis of acute T cell mediated rejection (TCMR) and BK virus nephropathy (BKVN), two significant post-transplant complications. Our profiling protocol for the multicenter Clinical Trial of Transplantation-04 (CTOT-04) study consisted of centrifugation of urine to prepare cell pellets, washes, addition of an RNA preservative, storage at 800C and shipment in cold containers to our Gene Expression Monitoring (GEM) Core for RNA isolation and quantification of mRNA in RT-qPCR assays. To simplify profiling, we developed a filter-based protocol (ZFBP) that eliminated the need for centrifugation, RNA preservative, storage at 800C, and shipment in cold containers for mRNA profiling. Furthermore, we trained kidney allograft recipients to perform the filtration of urine at home using the filter and post the urinary cell lysate containing the RNA at ambient temperature to our GEM Core for profiling. Here, we report our refinement of ZFBP and investigation of its diagnostic performance characteristics. MethodsTotal RNA was isolated from kidney allograft biopsy-matched urines using a filter-based protocol complemented by a silica-membrane-based cartridge for mRNA enrichment, the Weill Cornell Hybrid Protocol (WCHP). Absolute copy numbers of CD3ε mRNA, CXCL10 mRNA, and 18S rRNA, components of the CTOT-04 three-gene TCMR diagnostic signature, and urinary cell BKV VP 1 mRNA copy number were measured using RT-qPCR assays. Mann-Whitney test, Fischer exact test, and receiver operating characteristic (ROC) curve analysis were used for data analyses. ResultsUrinary cell three-gene TCMR diagnostic signature scores in urines processed using the WCHP discriminated kidney allograft recipients with TCMR (12 TCMR biopsies from 11 patients) from those without TCMR or BKVN (29 No TCMR/No BKVN biopsies from 29 patients). The median (25th and 75th percentiles) score of the CTOT-04 three-gene TCMR diagnostic signature was −0.448 (−1.664, 0.204) in the TCMR group and − 2.542 (−3.267, −1.365) in the No TCMR/ No BKVN group (P = 0.0005, Mann-Whitney test). ROC curve analysis discriminated the TCMR group from the No TCMR/ No BKVN group; the area under the ROC curve (AUROC) was 0.84 (95% Confidence Intervals [CI], 0.69 to 0.98) (P < 0.001), and TCMR was diagnosed with a sensitivity of 67% (95% CI, 35 to 89) at a specificity of 86% (95% CI, 67 to 95) using the CTOT-04 validated cutpoint of −1.213 (P = 0.0016, Fisher exact test). BKV VP1 mRNA copy number in urines processed using the WCHP discriminated patients with BKVN (n = 7) from patients without TCMR or BKVN (n = 29) and the AUROC was 1.0 (95% CI, 1.00 to 1.00) (P < 0.0001) and BKVN was diagnosed with a sensitivity of 86% (95% CI, 42 to 99) at a specificity of 100% (95% CI, 85 to 100) with the previously validated cutpoint of 6.5 × 108 BKV-VP1 mRNA copies per microgram of RNA (P < 0.0001, Fisher exact test). ConclusionUrine processed using the WCHP predicted TCMR and BKVN in kidney allograft recipients. WCHP represents not only a significant advance toward the portability of urinary cell mRNA profiling but also improved patient management by minimizing their visits for urine collection.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

Synbiotic Supplementation Modulates Gut Microbiota, Regulates β-Catenin Expression and Prevents Weight Gain in ob/ob Mice: Preliminary Findings.

Background: Obesity is one of the main health problems in the world today, and dysbiosis seems to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation on obesity and the microbiota in ob/ob mice. Twenty animals were divided into four groups: obese treated (OT), obese control (OC), lean treated (LT) and lean control (LC). All animals received a standard diet for 8 weeks. The treated groups received a synbiotic (Simbioflora-Invictus Farmanutrição Ltd., Sao Paulo, Brazil) in water, while the nontreated groups received only water. After 8 weeks, all animals were sacrificed, and gut tissue and stool samples were collected for mRNA isolation and microbiota analysis, respectively. β-Catenin, occludin, cadherin and zonulin in the gut tissue were analyzed via RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using an Ion PGM Torrent platform. Results: Synbiotic supplementation reduced body weight gain in the OT group compared with the OC group (p = 0.0398) and was associated with an increase in Enterobacteriaceae (p = 0.005) and a decrease in Cyanobacteria (p = 0.047), Clostridiaceae (p = 0.026), Turicibacterales (p = 0.005) and Coprococcus (p = 0.047). On the other hand, a significant reduction in Sutterella (p = 0.009) and Turicibacter (p = 0.005) bacteria was observed in the LT group compared to the LC group. Alpha and beta diversities were different among all treated groups. β-Catenin gene expression was significantly decreased in the gut tissue of the OT group (p ≤ 0.0001) compared to the other groups. No changes were observed in occludin, cadherin or zonulin gene expression in the gut tissue. Conclusions: Synbiotic supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces β-catenin expression in ob/ob mice.

Synthesis of point-modified mRNA.

Synthetic mRNA has recently moved into the focus of therapeutic and vaccination efforts. Incorporation of modified nucleotides during in vitro transcription can improve translation and attenuate immunogenicity, but is limited to triphosphate nucleotides which are accepted by RNA polymerases, and their incorporation is either random or complete. In contrast, site-specific modification, herein termed 'point modification' in analogy to point mutations, holds significant technical challenge. We developed fundamental techniques for isolation of long, translatableand internally point-modified mRNAs. Enabling concepts include three-way-one-pot splint ligations, and isolation of mRNA by real-time elution from agarose gels. The use of blue light permitted visualization of mRNA in pre-stained gels without the photochemical damage associated with the use of hard UV-radiation. This allowed visualization of the mRNA through its migration in the agarose gel, which in turn, was a prerequisite for its recovery by electroelution into precast troughs. Co-eluting agarose particles were quantified and found to not be detrimental to mRNA translation in vitro. Translation of EGFP-coding mRNA into functional protein was quantified by incorporation of 35S-labelled methionine and by in-gel EGFP fluorescence. This enabled the functional analysis of point modifications, specifically of ribose methylations in the middle of a 1371 nt long mRNA.

Expression of the Growth Hormone Gene during Early Development of Tiger Shovelnose Catfish (Pseudoplatystoma Fasciatum Linnaeus, 1766)

The early development stage of fish is crucial in fish farming, especially catfish species with cannibal characteristics, such as the Tiger Shovelnose Catfish (Pseudoplatystoma fasciatum Linnaeus 1766). Growth plays an essential role in fish’s life, which is influenced by the mechanism of growth hormone (GH). This study analyzed the level of growth hormone (GH) gene expression at several early developmental stages of Tiger Shovelnose Catfish. Samples were taken from an embryo, three days post hatched (dph) larvae to 60 dph juvenile. Isolation of mRNA was carried from the pituitary gland located at the head of the fish. Gene expression analysis was performed using RT-PCR method that compared GH/b-actin genes from band thickness. The results of the analysis showed the highest expression at 60 dph juvenile. The most significant increase in GH gene expression occurred from the egg to three dph larvae (P &lt; 0.05), then decreased to 20 dph juvenile. The increased phenotypic growth was seen from the increased length and weight at 45 dph juveniles. This indicates the amount of growth hormone released at the juvenile stage of 20 dph, thus triggering rapid growth in the next stage at 45 dph juvenile. Growth hormones can act as growth biomarkers in Tiger Shovelnose Catfish. The novelty of this study was to determine the level of GH gene expression in Pseudoplatystoma fasciatum ornamental fish as the object of research. Suggestions for further research to explore genes related to growth mechanisms through transcriptomic and proteomic technologies.

Consensus molecular subtypes (CMS) as prognostic and predictive biomarkers of panitumumab (Pmab), fluorouracil and folinic acid (FU/FA) or FU/FA maintenance therapy following Pmab-FOLFOX induction in RAS wildtype metastatic colorectal cancer (mCRC): PANAMA trial (AIO-KRK-0212).

3537 Background: Consensus molecular subtypes (CMS1-4) of colorectal cancer were evaluated as prognostic and predictive biomarkers in the PANAMA trial. PANAMA compared maintenance therapy with panitumumab (Pmab) and fluorouracil/folinic acid (FU/FA) vs. FU/FA alone after Pmab-FOLFOX induction therapy in RAS wildtype mCRC. Methods: Gene expression was measured after mRNA isolation in 179 of 248 patients of the full analysis set. The analysis was conducted using a customized Nanostring PanCancer Progression Panel. The original CMS classifier was re-derived for Nanostring data using a multinomial regression analysis.Median progression-free (PFS) and overall survival (OS) since start of maintenance were estimated by Kaplan-Meier-method and Cox-regression, using the log rank test. Objective response rates (ORR) of maintenance therapy were compared by Chi-square-test. Results: Prevalence of CMS was: CMS1, n = 15 (8.4 %); CMS2, n = 82 (45.8 %); CMS3, n = 20 (11.2 %) and CMS4, n = 62 (34.6 %). A prognostic impact of CMS regardless of treatment was not evident for PFS (p = 0.245) and OS (p = 0.169), but for ORR (p = 0.022), with CMS1 and CMS3 being associated with unfavourable efficacy during maintenance therapy. Potential predictive effects of CMS were observed in patients with CMS2 and CMS4 tumours. In CMS2 and CMS4 tumours, ORR was significantly higher when treated with Pmab-FU/FA in maintenance therapy (CMS2: 56.5% vs 30.6%, p = 0.026; CMS4: 55.6% vs. 28.6%, p = 0.040). In patients with CMS2 mCRC, this translated into a significant effect on PFS (Hazard ratio: 0.61 (95% CI 0.38 – 0.99) p = 0.046 (Table). Conclusions: CMS have limited prognostic impact for pmab-based maintenance therapy. However, CMS2 and CMS4 are positively associated with Pmab efficacy during maintenance therapy in the PANAMA trial. Further trials are necessary to confirm these results. [Table: see text]