Background: Autosomal dominant polycystic kidney disease (ADPKD) is responsible for 10% of cases of the end stage renal disease. Early diagnosis, especially of potential fast progressors would be of benefit for efficient planning of therapy. Urine excreted proteome has become a promising field of the search for marker patterns of renal diseases including ADPKD. Up to now however, only the low molecular weight fraction of ADPKD proteomic fingerprint was studied. The aim of our study was to characterize the higher molecular weight fraction of urinary proteome of ADPKD population in comparison to healthy controls as a part of a general effort aiming at exhaustive characterization of human urine proteome in health and disease, preceding establishment of clinically useful disease marker panel. Results: We have analyzed the protein composition of urine retentate (>10 kDa cutoff) from 30 ADPKD patients and an appropriate healthy control group by means of a gel-free relative quantitation of a set of more than 1400 proteins. We have identified an ADPKD-characteristic footprint of 155 proteins significantly up- or downrepresented in the urine of ADPKD patients. We have found changes in proteins of complement system, apolipoproteins, serpins, several growth factors in addition to known collagens and extracellular matrix components. For a subset of these proteins we have confirmed the results using an alternative analytical technique. Conclusions: Obtained results provide basis for further characterization of pathomechanism underlying the observed differences and establishing the proteomic prognostic marker panel.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis. The first symptoms of IgAN are erytrocyturia or hematuria, proteinuria, and decline in renal function, or any combination of the above. One of the promising diagnostic methods is urine proteomics. We studied urine proteomics in patients with IgAN and age- and sex‑matched healthy controls. To minimize the risk of protein degradation, we proposed a new protocol for urine collection and preparation. A total of 30 patients with IgAN and 30 controls were enrolled into the study. Thirty urine samples of the IgAN group were divided into 3 disease pooled samples (DPS I, II, and III) and 30 urine samples of the control group were divided into 3 control pooled samples (CPS I, II, and III). We used isoelectric focusing/liquid chromatography-mass spectrometry/mass spectrometry (IEF/LC‑MS/MS) to detect all proteins larger than 10 kDa. Using qualitative analysis, we identified 761, 951, and 956 proteins in each of the 3 IEF/LC‑MS/MS experiments. The results were combined, yielding a dataset with 1238 proteins identified by at least 2 peptides. The statistical analysis of the quantitative results revealed 18 proteins that were differently populated in the urine of IgAN patients compared with healthy controls. We found increased urinary concentrations of complement components, coagulation factors, extracellular matrix, intracellular, transmembrane, and other proteins in patients with IgAN. Some of them have never been linked to IgAN before. We demonstrated that urine proteomics is a promising tool for diagnosing and monitoring patients with IgAN.

There is a huge disparity between the number of bacterial and yeast probiotics in favor of the former. The latest reports indicate that extracellular vehicles (EVs) play a significant role in probiotic mechanisms. In the present work, we compared the probiotic properties of Saccharomyces cerevisiae strains (WUT3 and WUT151), which have never been previously characterized in this context, with commercial probiotic yeast—Saccharomyces cerevisiae var. boulardii CNCM-745. Notably, WUT3 and WUT151 reacted more mildly to the unfavorable simulated environment of saliva, stomach, small, and large intestines. As a result, we confirmed that WUT3 and WUT151 were superior to S. boulardii in terms of probiotic properties. Then, we performed a complex analysis of their EVs, isolated by a multistep filtration process. The nanoparticle tracing analysis showed no significant difference in the diameter of the vesicles between the strains. MTT studies confirmed that EVs are not toxic against normal human colorectal cell lines CCD-18 Co and CCD 841 CoN. However, toxicity was observed against the HT-29 cancer line. By staining EVs with Nile Red, we successfully visualized EVs–cell interactions. Finally, we explored the profile of proteins transported with the EVs, identifying a significant overrepresentation of extracellular proteins. Based on comparison with other proteomic data, we selected marker proteins for S. cerevisiae EVs. This knowledge will be helpful for further studies on tracking the transfer of the protein cargo of yeast EVs to human cells using, for instance, specific antibodies to these marker proteins.

Introduction: The receptor SorCS2 is involved in the trafficking of membrane receptors and transporters. It has been implicated in brain disorders and has previously been reported to be indispensable for ionotropic glutamatergic neurotransmission in the hippocampus.Aim: We aimed to study the role of SorCS2 in the control of astrocyte‐neuron communication, critical for neurovascular coupling.Methods: Brain slices from P8 and 2‐month‐old wild‐type and SorCS2 knockout (Sorcs2^−/−) mice were immunostained for SorCS2, GFAP, AQP4, IB4, and CD31. Neurovascular coupling was assessed in vivo using laser speckle contrast imaging and ex vivo in live brain slices loaded with calcium‐sensitive dye. Bulk and cell surface fraction proteomics was analyzed on freshly isolated and cultured astrocytes, respectively, and validated with Western blot and qPCR.Results: SorCS2 was strongly expressed in astrocytes, primarily in their endfeet, of P8 mice; however, it was sparsely represented in 2‐month‐old mice. Sorcs2^−/− mice demonstrated reduced neurovascular coupling associated with a reduced astrocytic calcium response to neuronal excitation. No differences in vascularization or endothelium‐dependent relaxation ex vivo between the 2‐month‐old groups were observed. Proteomics suggested changes in glutamatergic signaling and suppressed calcium signaling in Sorcs2^−/− brains from both P8 and 2‐month‐old mice. The increased abundance of glutamate metabotropic receptor 3 in Sorcs2^−/− astrocytes was validated by PCR and Western blot. In cultured Sorcs2^−/− astrocytes, AQP4 abundance was increased in the bulk lysate but reduced in the cell surface fraction, suggesting impaired trafficking.Conclusion: The results suggest that SorCS2 expression is important for the development of neurovascular coupling, at least in part by modulating glutamatergic and calcium signaling in astrocytes.

Acute myeloid leukaemia (AML) is a therapeutically challenging malignancy driven by the self-renewal, quiescence, and therapy resistance of leukaemic stem cells (LSCs). CDK8, a kinase component of the Mediator complex, regulates oncogenic transcription, and the selective CDK8/CDK19 inhibitor RVU120 (Romaciclib) targets AML cells with CD34⁺/pSTAT5-high LSC-like characteristics; however, the epigenetic and transcriptional consequences of CDK8 blockade and actionable combinatorial strategies remain incompletely defined. Using the TEX cell line, an LSC-enriched surrogate model, we performed time-resolved RNA-seq, whole-proteome and phosphoproteomics mass spectrometry (MS), and CUT&Tag chromatin profiling following treatment with RVU120 and CCT251921. CDK8 protein–protein interactions were mapped by co-immunoprecipitation MS across five AML models, and synergy with Pelabresib (BET inhibitor) or CB6644 (RUVBL1/2 inhibitor) was assessed by high-content screening in three cell lines and three patient-derived xenograft (PDX) models. Both inhibitors suppressed STAT5 phosphorylation, induced loss of the CD34⁺/CD38⁻ LSC-enriched phenotype, and drove erythromegakaryocytic differentiation. Transcriptomic and proteomic responses were concordant, and CDK8 inhibition triggered widespread enhancer activation with redistribution of RNAP2, BRD3, and NFRKB. CDK8 combined with Pelabresib acted synergistically in MOLM-16 cells and two of three PDX models. These findings identify CDK8 as a transcriptional node of LSC-associated programs and provide a mechanistic rationale for combined CDK8-BET inhibition in molecularly defined AML subsets, which will require validation in functional LSC assays and primary specimens.

Transposable element-derived long intergenic noncoding RNAs are increasingly recognized as context-dependent regulators of gene expression, but the functional consequences of their ectopic activation in somatic cells remain poorly understood. We previously showed that U7 snRNA represses a subset of LTR12-associated lincRNAs, including lnc-ARRDC4-1 and lnc-ADCYAP1-2 , two testis-enriched lincRNAs with minimal expression in somatic cells. Here, we examined the consequences of their increased expression in somatic cells. We showed that overexpression of either lincRNA led to overlapping transcriptomic and proteomic changes, impaired migration, altered adhesion and proliferation, and a ∼50% reduction in protein synthesis. Furthermore, we identified lnc-ARRDC4-1 as an upstream regulator of lnc-ADCYAP1-2 transcription. Downstream of this event, lnc-ADCYAP1-2 interacts with the RNA helicase DHX36, a regulator of G-quadruplex-containing mRNAs. lnc-ADCYAP1-2 activation reduces DHX36 protein levels which is accompanied by decreased protein output from a subset of DHX36 mRNA targets. At the cellular level, these effects correlate with altered cell proliferation, migration, adhesion, and global translation. Our results suggest a lnc-ARRDC4-1: lnc-ADCYAP1-2 : DHX36 regulatory cascade linking de-repression of LTR12-containing lincRNAs to reduced protein synthesis and altered cellular processes in somatic cells.