For what factors should we normalize urinary extracellular mRNA biomarkers?

Background and Aims Blood and urinary mRNA biomarkers are non-invasive tools with potential for detecting kidney allograft rejection. However, their ability to provide added diagnostic value beyond current standard-of-care (SOC) patient monitoring remains unclear. Method In this prospective study, adult kidney transplant recipients were enrolled across seven French referral centres between July 2018 and December 2019 (ClinicalTrials.gov, NCT03582436). During the first-year post-transplantation, we quantified 19 blood and 12 urinary mRNA biomarkers during kidney allograft biopsies, including both protocol-specified and clinically indicated procedures. Primary outcome was the occurrence of allograft rejection, including antibody-mediated rejection (AMR), T cell-mediated rejection (TCMR), and mixed rejection, classified according to the Banff 2019 criteria. Results Overall, 733 kidney transplant patients (64.1% male, 35.9% female) were included, with 1,549 biopsies paired with the mRNA biomarkers. The cumulative incidence of rejection was 9.7% (95% CI; 7.6%–12.1%). None of the blood biomarkers tested (AKR1C3, CD3E, CD4, CD40, CD8A, CD9, CTLA4, ENTPD1, FOXP3, GZMB, ID3, IL7R, MS4A1, MZB1, POU2AF1, POU2F1, TCL1A, TLR4, and TRIB1) had a significant association with allograft rejection (Fig. 1A). However, among the 12 urinary biomarkers (CXCL9, CD25, CD3, Perf, NKG2∼4, FN1, PSMB10, PSMB9, αSMA, IP10, GZB, ECadh), eight-out-of-twelve, CXCL9 (p = 0.001), CD3 (p = 0.001), Perforin (p < 0.001), NKG2∼4 (p < 0.001), PSMB10 (p ≤ 0.001), PSMB9 (p = 0.005), aSMA (p = 0.043), IP10 (p = 0.006) were significantly associated with allograft rejection (Fig. 1B). Moreover, PSMB9 was the only urinary biomarker to show a discriminatory ability between TCMR and AMR (p = 0.022, higher in TCMR). Despite these findings, integrating urinary biomarkers with SOC monitoring parameters failed to improve predictive accuracy compared to SOC monitoring. Conclusion Urinary mRNA—but not blood—biomarkers showed significant associations with kidney allograft rejection during the first-year post-transplantation. Nonetheless, these biomarkers did not improve predictive performance when added to existing SOC practices, underscoring the need for further research to optimise non-invasive diagnostic strategies.

Calcineurin inhibitor (CNI) toxicity is a significant cause of graft dysfunction in kidney transplant recipients, yet distinguishing it from acute rejection (AR) and acute tubular necrosis (ATN) remains challenging. This study investigated the use of urinary mRNA biomarkers as a noninvasive tool for identifying CNI toxicity. We retrospectively enrolled 110 kidney transplant recipients and classified them into four groups based on pathological findings: stable graft function (n=35), CNI toxicity (n=25), AR (n=30), and ATN (n=20). Candidate biomarkers were selected using the GEO database. Urinary mRNA was extracted from cell pellets, reverse-transcribed, and quantified by real-time polymerase chain reaction. Estimated glomerular filtration rates were comparable among the CNI toxicity, AR, and ATN groups. Four transcripts (LTF, NNMT, WFDC2, and HIF1A) were identified as candidate biomarkers. Urinary mRNA levels of LTF, NNMT, and HIF1A were significantly lower in the CNI toxicity group than the AR group. NNMT and HIF1A levels were also significantly lower than those observed in the ATN group. In contrast, WFDC2 levels did not differ significantly across groups. A three-gene signature (LTF, NNMT, and HIF1A) effectively differentiated CNI toxicity from AR and ATN (area under the curve [AUC], 0.867; 95% confidence interval [CI], 0.787-0.947) and significantly enhanced the diagnostic performance of the clinical variable-based model (AUC increased from 0.776; 95% CI, 0.660-0.892, to 0.934; 95% CI, 0.881-0.986). Urinary mRNA levels of LTF, NNMT, and HIF1A may serve as useful biomarkers for identifying CNI toxicity in kidney transplant recipients with graft dysfunction.

Nobel for RNAi

Epigenetics in evolution and disease

When one is better than two: RNA with dual functions

As stated by the central dogma of molecular biology, in living organisms the sequence information of DNA is trans- ferred to RNA and then to protein, but such information cannot be transferred back from protein to nucleic acids. In this article, it is proposed that the sequence in- formation encoded by protein can be arti- ficially transferred back to DNA and RNA, respectively, based on transcription activa- tor-like effectors (TALE) and Pumilio/fem-3 mRNA-binding factors (PUF). Specifically, mono- and/or dinucleotides are assumed to be arranged along the characteristic amino acids of TALE and PUF, and then assembled as oligonucleotides by ligase or condensation agents. This hypothesis suggests a new protein-based strategy for synthesizing DNA and RNA molecules. INTRODUCTION The central dogma of molecular biology, as proposed by Francis Crick in 1958 1 5,6,7,8 . In this article, I suggest the possibility that the sequence information encoded by protein can be artificially transferred resi - due by residue directly to DNA and RNA, respectively, based on transcription ac- tivator-like effectors (TALE) and Pumilio/ fem-3 mRNA-binding factors (PUF). This hypothesis, if proved experimentally, sug- gests a new strategy for the synthesis of short DNA/RNA molecules.

The central dogma of biology, that DNA makes RNA makes protein, was again shown to be outdated with the recent discovery of RNAs that have essential regulatory functions (e.g., metabolite-binding RNAs, transcription regulation) [2, 15]. These finding have stimulated a large effort to search for small, functional RNA motifs (either embedded inside larger messenger RNA molecules or as separate molecules in the cell). For example, it is known that cells make use of a variety of small non-coding RNAs (such as microRNAs) as a mechanism for gene regulation. The very existence of these small motifs in Nature suggests that the functional, artificial RNA molecules developed through (experimental) in vitro selection technology may shed some light on the scope and functional diversity of these small RNA molecules in vivo. The binding and catalytic properties of nucleic acid molecules are conferred by specific sequence and structural motifs. Indeed, recent discoveries show that metabolite-induced RNA conformational changes constitute another form of bacterial gene regulation [12, 20, 21]. Since in vitro selection is a process that simulates evolution, it is reasonable that novel nucleic acid motifs discovered through in vitro selection experiments may have also evolved in the cell, especially since such motifs often target molecules (e.g., ATP, cAMP, antibiotics, etc.) that are prevalent in Nature. The structure of RNA is hierarchical in nature. The primary (1D) structure of an RNA molecule is the oriented, linear ordering of the nucleotides A, C, G, and U. The secondary (2D) structure of the molecule is described by ∗To whom correspondence should be addressed 1In vitro selection is a process that mimics evolution in which a large pool of small, random-sequence RNA molecules is subjected to an iterative process that selects for a specific physical or chemical property; see [19] for a review.

The clinical manifestations of diabetic kidney disease (DKD) are more heterogeneous than those previously reported, and these observations mandate the need for the recruitment of patients with biopsy-proven DKD in biomarker research. In this study, using the public gene expression omnibus (GEO) repository, we aimed to identify urinary mRNA biomarkers that can predict histological severity and disease progression in patients with DKD in whom the diagnosis and histologic grade has been confirmed by kidney biopsy. We identified 30 DKD-specific mRNA candidates based on the analysis of the GEO datasets. Among these, there were significant alterations in the urinary levels of 17 mRNAs in patients with DKD, compared with healthy controls. Four urinary mRNAs—LYZ, C3, FKBP5, and G6PC—reflected tubulointerstitial inflammation and fibrosis in kidney biopsy and could predict rapid progression to end-stage kidney disease independently of the baseline eGFR (tertile 1 vs. tertile 3; adjusted hazard ratio of 9.68 and 95% confidence interval of 2.85–32.87, p < 0.001). In conclusion, we demonstrated that urinary mRNA signatures have a potential to indicate the pathologic status and predict adverse renal outcomes in patients with DKD.

The central dogma of molecular biology is that DNA makes RNA and RNA makes protein. Thus the genetic information in the DNA is first converted into an RNA molecule and in turn the information in the RNA is used to make a protein which exerts a particular biological function. A central role in this process is evidently played by the process of transcription in which the DNA is converted into an RNA equivalent. This is true both in prokaryotes where the information in this RNA molecule is rapidly translated to produce a protein and in eukaryotes where a number of processes such as RNA splicing and RNA transport must intervene before the initial RNA transcript is in a form which can be translated into protein.

As part of the 'Central Dogma' of molecular biology, the function of proteins and nucleic acids within a cell is determined by their primary sequence. Recent work, however, has shown that within living cells the role of many proteins and RNA molecules can be influenced by the physical state in which the molecule is found. Within living cells, both protein and RNA molecules are observed to condense into non-membrane-bound yet distinct structures such as liquid droplets, hydrogels and insoluble aggregates. These unique intracellular organizations, collectively termed biomolecular condensates, have been found to be vital in both normal and pathological conditions. Here, we review the latest studies that have developed molecular tools attempting to recreate artificial biomolecular condensates in living cells. We will describe their design principles, implementation and unique characteristics, along with limitations. We will also introduce how these tools can be used to probe and perturb normal and pathological cell functions, which will then be complemented with discussions of remaining areas for technological advance under this exciting theme.

Introduction: Urine proteomics plays an important role in the screening of biomarkers for infant diseases. However, there is no unified standard for the selection of urine samples for urine proteomics. It is also unclear whether there are differences in proteomics between whole urine and urine supernatant. Therefore, the urine of preterm infants was used as the research sample to explore the differences in protein profiles between the whole urine and urine supernatant of preterm infants by proteomics. Methods: Urine samples were collected from five preterm infants with a gestational age of <28 weeks at their corrected gestational age of 37 weeks. Each preterm urine was divided into whole urine and supernatant. Urine protein was extracted and analyzed by liquid chromatography-tandem mass spectrometry. Results: The two groups of urine samples did not show significant clustering in the principal component analysis. A total of 2,607 proteins were detected in the two groups of urine samples, of which 82 proteins were unique to whole urine samples and 56 proteins were unique to urine supernatant samples. The molecular functions, the main biological processes, and subcellular localization of the differential proteins were analyzed. In other neonatal-related diseases, there was no significant difference in protein enrichment between whole urine and urine supernatant. Conclusions: This study analyzed the differences between whole urine and urine supernatant in urine proteomics of preterm infants. In neonatal-related diseases, there is no significant difference in urinary protein biomarkers between whole urine and urine supernatant.

Identifying noninvasive biomarkers of kidney disease is valuable for diagnostic and therapeutic purposes. Hypoxia inducible factor 1 (HIF‐1) expression is known to be elevated in the kidneys in several renal disease pathologies. We hypothesized that the urinary HIF‐1a mRNA level may be a suitable biomarker for expression of this protein in chronic kidney disease (CKD). We compared HIF‐1a mRNA levels from urine pellets of CKD and healthy subjects. To ensure that urinary HIF‐1a mRNA is of kidney origin, we examined colocalization of HIF‐1a mRNA with two kidney specific markers in urine cells. We found that HIF‐1a mRNA is readily quantifiable in urine pellets and its expression was significantly higher in CKD patients compared with healthy adults. We also showed that the urinary HIF‐1a mRNA comes primarily from cells of renal origin. Our data suggest that urinary HIF‐1a mRNA is a potential biomarker in CKD and can be noninvasively assessed in patients.

Pancreatic cancer is a solid tumour that is often fatal. Hence, there is an urgent need to identify new drug targets for this disease. Highly proliferating cancer cells have an increased demand for nutrients and, therefore, need to up-regulate selective amino acid transporters. Here, we investigated which amino acid transporters are up-regulated in pancreatic cancer and whether any of these transporters has potential as a drug target for this fatal disease. The expression of amino acid transporters in pancreatic cancer was analysed using publicly available microarray datasets, and the findings with the transporter SLC6A14 were validated by mRNA and protein analysis. The potential of SLC6A14 as a drug target was evaluated using a pharmacological blocker in vitro and in vivo. SLC6A14 was up-regulated several fold in patient-derived xenografts, primary tumour tissues and pancreatic cancer cells lines compared to normal pancreatic tissue or normal pancreatic epithelial cells. The magnitude of the up-regulation of SLC6A14 was the highest among the amino acid transporters examined. A pharmacological blocker of SLC6A14, α-methyltryptophan, induced amino acid starvation in pancreatic cancer cells and reduced the growth and proliferation of these cells, both in vitro and in vivo. The salient features of this study are that SLC6A14 is markedly up-regulated in pancreatic cancer and that pharmacological blockade of this transporter interferes with amino acid nutrition and reduces growth and proliferation of pancreatic cancer cells. These findings identify SLC6A14 as a novel druggable target for pancreatic cancer.

The Medawar Prize Acceptance Speech 2022.

Long non-coding RNA: Multiple effects on the differentiation, maturity and cell function of dendritic cells