The objective of this review is to focus on putative modified epithelial functions related to allergy. The dysregulation of the epithelial barrier might result in the allergen uptake, which could be the primary defect in the pathogenesis of allergic reaction. We review the literature of the role of respiratory epithelium as an active barrier, how allergens are transported through it and how it senses the hostile environmental allergens and other dangerous stimuli.

RNA sequencing (RNA-seq) has become an indispensable tool to identify disease associated transcriptional profiles and determine the molecular underpinnings of diseases. However, the broad adaptation of the methodology into the clinic is still hampered by inconsistent results from different RNA-seq protocols and involves further evaluation of its analytical reliability using patient samples. Here, we applied two commonly used RNA-seq library preparation protocols to samples from acute leukemia patients to understand how poly-A-tailed mRNA selection (PA) and ribo-depletion (RD) based RNA-seq library preparation protocols affect gene fusion detection, variant calling, and gene expression profiling. Overall, the protocols produced similar results with consistent outcomes. Nevertheless, the PA protocol was more efficient in quantifying expression of leukemia marker genes and showed better performance in the expression-based classification of leukemia. Independent qRT-PCR experiments verified that the PA protocol better represented total RNA compared to the RD protocol. In contrast, the RD protocol detected a higher number of non-coding RNA features and had better alignment efficiency. The RD protocol also recovered more known fusion-gene events, although variability was seen in fusion gene predictions. The overall findings provide a framework for the use of RNA-seq in a precision medicine setting with limited number of samples and suggest that selection of the library preparation protocol should be based on the objectives of the analysis.

Being enucleated, RBCs lack typical transcriptomes, but are known to contain small amounts of diverse long transcripts and microRNAs. However, the exact role and importance of these RNAs are lacking. Shedding of extracellular vesicles (EVs) from the plasma membrane constitutes an integral mechanism of RBC homeostasis, by which RBCs remove unnecessary cytoplasmic content and cell membrane. To study this further, we explored the transcriptomes of RBCs and extracellular vesicles (EVs) of RBCs using next-generation sequencing. Furthermore, we performed single-cell RNA sequencing on RBCs, which revealed that approximately 10% of the cells contained detectable levels of mRNA and cells formed three subpopulations based on their transcriptomes. A decrease in the mRNA quantity was observed across the populations. Qualitative changes included the differences in the globin transcripts and changes in the expression of ribosomal genes. A specific splice form of a long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), was the most enriched marker in one subpopulation of RBCs, co-expressing with ribosomal structural transcripts. MALAT1 expression was confirmed by qPCR in CD71-enriched reticulocytes, which were also characterized with imaging flow cytometry at the single cell level. Analysis of the RBC transcriptome shows enrichment of pathways and functional categories required for the maturation of reticulocytes and erythrocyte functions. The RBC transcriptome was detected in their EVs, making these transcripts available for intercellular communication in blood.

In the present experiments the cDNA coding for a truncated form of the β1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67–74-kDa fusion protein. Typically a yield of 750 μg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, converting the acceptor to the branched pentasaccharide Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and ¹H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc. Accordingly, we call the recombinant β1,6-GlcNAc transferase cIGnT6 to emphasize its action atcentral rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, β1,4galactosyltransferase and β1,3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.

Background: Transcriptomic and proteomic profiling of human brain tissue is hindered by the availability of fresh samples from living patients. Postmortem samples usually represent the advanced disease stage of the patient. Furthermore, the postmortem interval can affect the transcriptomic and proteomic profiles. Therefore, fresh brain tissue samples from living patients represent a valuable resource of metabolically intact tissue. Implantation of deep brain stimulation (DBS) electrodes into the human brain is a neurosurgical treatment for, e.g., movement disorders. Here, we describe an improved approach to collecting brain tissues from surgical instruments used in implantation of DBS device for transcriptomics and proteomics analyses. Methods: Samples were extracted from guide tubes and recording electrodes used in routine DBS implantation procedure to treat patients with Parkinson’s disease, genetic dystonia and tremor. RNA sequencing was performed in tissues extracted from the recording microelectrodes and liquid chromatography-mass spectrometry (LC-MS) performed in tissues from guide tubes. To assess the performance of the current approach, the obtained datasets were compared with previously published datasets representing brain tissues. Results: Altogether, 32,034 RNA transcripts representing the unique Ensembl gene identifiers were detected from eight samples representing both hemispheres of four patients. By using LC-MS, we identified 734 unique proteins from 31 samples collected from 14 patients. The datasets are available in the BioStudies database (accession number S-BSST667). Our results indicate that surgical instruments used in DBS installation retain brain material sufficient for protein and gene expression studies. Comparison with previously published datasets obtained with similar approach proved the robustness and reproducibility of the protocol. Conclusions: The instruments used during routine DBS surgery are a useful source for obtaining fresh brain tissues from living patients. This approach overcomes the issues that arise from using postmortem tissues, such as the effect of postmortem interval on transcriptomic and proteomic landscape of the brain, and can be used for studying molecular aspects of DBS-treatable diseases.

Background and Objectives: There is increasing evidence that frequent blood donation depletes the iron stores of some blood donors. The FinDonor 10 000 study was set up to study iron status and factors affecting iron stores in Finnish blood donors. In Finland, iron supplementation for at-risk groups has been in place since the 1980’s. Material and Methods: 2584 blood donors (N= 8003 samples) were recruited into the study alongside the standard donation at three donation sites in the capital region of Finland between 5/2015 and 12/2017. All participants were asked to fill out a questionnaire about their health and lifestyle. Blood samples were collected from the sample pouch of whole blood collection set, kept in cool temperature and processed centrally. Whole blood count, CRP, ferritin and sTFR were measured from the samples and DNA was isolated for GWAS studies. Results: Participant demographics, albeit in general similar to the general blood donor population in Finland, indicated some bias toward older and more frequent donors. Participation in the study increased median donation frequency of the donors. Analysis of the effect of time lag from the sampling to the analysis and the time of day when sample was drawn revealed small but significant time-dependent changes. Conclusion: The FinDonor cohort now provides us with tools to identify potential donor groups at increased risk of iron deficiency as factors explaining this risk. The increase in donation frequency during the study suggests that scientific projects can be used to increase the commitment of blood donors.