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BackgroundColonic epithelial cells form a frontline intestinal barrier and maintain its function which deteriorates early in ulcerative colitis (UC). MicroRNAs (miRNAs) participate in regulation of intestinal epithelial integrity and barrier permeability. However, there is a lack of understanding about cell type-specific expression of miRNAs in UC.MethodsTwo independent cohorts composed of active and quiescent UC patients (n=74), and healthy controls (HC; n=50) were studied. Crypt-bottom (CD44+) and crypt-top (CD66a+) colonic epithelial cell populations were enriched using FACS. Small RNA-sequencing was performed on colon biopsy and colonic epithelial cell population samples. Data processing encompassed differential expression, gene-set enrichment analysis (GSEA) and clinical correlation analysis.ResultsWe describe differentially expressed miRNAs among active and quiescent UC compared to HC colon tissue and propose their involvement in intestinal barrier integrity regulation. We further focus on crypt-bottom and crypt-top colonic epithelial cells and characterise common and cell population-specific miRNA expression in response to UC-caused inflammation. We suggest that differentially expressed miRNAs are commonly involved in inflammation- and intestinal barrier integrity-related processes (such as signalling of interleukin-4 and interleukin-13), while differences between cell populations might reflect their function, i.e., crypt-bottom cell miRNA target genes are enriched in regulation of cell differentiation. Moreover, we show cell population-specific miRNA expression correlations with endoscopic disease activity, i.e., let-7b-5p and let-7e-5p negatively correlates with activity score only in the crypt-bottom cells, while miR-24-3p and miR-27a-3p positively correlates only in the crypt-top cells.ConclusionsChanges in miRNA expression during UC are epithelial cell type- and UC activity-specific (including correlations with endoscopic Mayo score). Further, irrespective of the UC stage and colonic cell population, deregulated miRNAs are potentially involved in signalling pathways responsible for regulation of intestinal barrier integrity and permeability.Key MessagesWhat is already known?Colonic epithelium plays an important role in pathogenesis of ulcerative colitis (UC), while microRNAs (miRNA) have been implicated in modulation of intestinal homeostasis.What is new here?Cell type-specific miRNA expression of colonic epithelial cells during UC was unknown. Here, we show cell population-specific miRNA expression in response to UC-caused inflammation in crypt-top and crypt-bottom colonic cells.How can this study help patient care?The identified cell type-specific correlations of miRNA expression and endoscopic Mayo score might be further evaluated in UC monitoring and diagnostics as well as selected as targets for further therapeutics development.Graphical abstractSummaryWe present microRNA transcriptome analysis on different levels - colon tissue and epithelial cell population - in ulcerative colitis. Results indicate cell type- and disease stage-dependent microRNA deregulation, unveil associations with disease activity, and putative biological role of deregulated microRNAs.

Graphical abstract, During the pandemic, different methods for SARS-CoV-2 detection and COVID-19 diagnostics were developed, including antibody and antigen tests. For a better understanding of the interaction mechanism between SARS-CoV-2 virus proteins and specific antibodies, total internal reflection ellipsometry based evaluation of the interaction between SARS-CoV-2 nucleoprotein (SCoV2-rN) and anti-SCoV2-rN antibodies was performed. Results show that the appropriate mathematical model, which takes into account the formation of an intermediate complex, can be applied for the evaluation of SCoV2-rN/anti-SCoV2-rN complex formation kinetics. The calculated steric factor indicated that SCoV2-rN/anti-SCoV2-rN complex formation has very strict steric requirements. Estimated Gibbs free energy (ΔGAssoc) for SCoV-rN and anti-SCoV-rN binding was determined as −34 kJ/mol. The reported findings are useful for the design of new analytical systems for the determination of anti-SCoV2-rN antibodies and for the development of new anti-SARS-CoV-2 medications.

Zoonotic transmission of Hendra virus (HeV) from primary hosts (pteropid bats) to horses, and, occasionally, onward adventitious spread to humans, is associated with high mortality rates in both affected secondary species. The introduction of an effective recombinant G protein vaccine for use in horses has been a major advance for the suppression of disease risk. However, equine HeV vaccination induces neutralising antibody that is indistinguishable from a post infection immune response when using most first line serology assays (eg. VNT and some ELISAs). We have constructed and evaluated an IgM antibody capture (MAC) ELISA which employs yeast expressed HeV nucleoprotein (N). All other serology tests use the G protein which does not detect early infection and is present in the current Hendra virus vaccine and may cause ambiguity in interpretation of results. Thus, this is the first test developed using a N protein which can successfully detect a recent (primarily within the last four weeks) infection of horses with HeV and is not affected by vaccination induced antibody. Testing a limited panel (21 samples) of post infection sera, a normal serum panel (288 samples) and a post vaccination panel (163 samples), we have estimated DSe to be 100 % (95 % CI, 83.9–100.0 %) and DSp to be 98.4 % (95 % CI, 96.8–99.4 %) relative to assigned serology results (VNT, ELISA and Luminex) for the test panels. The HeV IgM MAC ELISA is intended to supplement other molecular and serology test results, with selective use, and is the only serology test which can provide an indication for recent infection which is otherwise not available.

The serologic diagnosis of coronavirus disease 2019 (COVID-19) and the evaluation of vaccination effectiveness are identified by the presence of antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we present the electrochemical-based biosensing technique for the detection of antibodies specific to the SARS-CoV-2 proteins. Recombinant SARS-CoV-2 spike proteins (rSpike) were immobilised on the surface of a gold electrode modified by a self-assembled monolayer (SAM). This modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against the rSpike (anti-rSpike). Electrochemical impedance spectroscopy (EIS) was used to observe the formation of immunocomplexes while cyclic voltammetry (CV) was used for additional analysis of the surface modifications. It was revealed that the impedimetric method and the elaborate experimental conditions are appropriate for the further development of electrochemical biosensors for the serological diagnosis of COVID-19 and/or the confirmation of successful vaccination against SARS-CoV-2.

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a &lsquo, wound healing&rsquo, assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.

Introduction.: There is no effective and reliable biomarker to distinguish benign thyroid nodules (BTN) from papillary thyroid carcinomas (PTC). In this study we analyzed a set of four miRNA molecules in plasma of patients with papillary thyroid cancer, benign nodules and healthy controls to identify miRNA molecules that may be markers of PTC.Aim.: We aimed to investigate the dysregulation of plasma miRNAs in PTC and evaluate the diagnostic value for differentiation of PTC from BTN. Methods.: The expression levels of 4 miRNAs (miR-221, miR-222, miR-146b, miR-21) were measured in 48 PTC patients before thyroidectomy and again after thyroidectomy in a subgroup of 36 patients. Preoperative and postoperative plasma miRNA expression levels were compared with baseline levels established in plasma from the heathy controls group (N=57) and patients with BTN (N=22). MicroRNA-222 and miR-146b, miR-221, miR-21 were included in a panel because they all reportedly were overexpressed in PTC compared to benign nodules or normal thyroid tissue.Results.: Compared with baseline levels in the healthy controls group, miR-221, miR-222, miR-146b, miR-21 levels were significantly higher in the preoperative PTC group (P

Many miRNAs and cancer-related mutations have been proposed as promising molecular markers of papillary thyroid carcinoma (PTC). However, there are limited data on the correlation between miRNA expression, BRAFV600E mutation, and PTC recurrence. Therefore, to evaluate the potential of BRAFV600E mutation and five selected miRNAs (-146b, -222, -21, -221, -181b) in predicting PTC recurrence, these molecular markers were analyzed in 400 formalin-fixed, paraffin-embedded PTC tissue specimens. The expression levels of miRNAs were measured using qRT-PCR. It was demonstrated that expression levels of all analyzed miRNAs are significantly higher in recurrent PTC than in non-recurrent PTC (p &lt, 0.05). Moreover, higher expression levels of miR-146b, miR-222, miR-21, and miR-221 were associated with other clinicopathologic features of PTC, such as tumor size and lymph node metastases at initial surgery (p &lt, 0.05). No significant differences in the frequency of BRAFV600E mutation in recurrent PTC and non-recurrent PTC were determined. Our results suggest that miRNA expression profile differs in PTC that is prone to recurrence when compared to PTC that does not reoccur after the initial surgery while BRAFV600E mutation frequency does not reflect the PTC recurrence status. However, the prognostic value of the analyzed miRNAs is rather limited in individual cases as the pattern of miRNA expression is highly overlapping between recurrent and non-recurrent PTC.

Many miRNAs and cancer-related mutations have been proposed as promising molecular markers of papillary thyroid carcinoma (PTC). However, there are limited data on the correlation between miRNA expression, BRAFV600E mutation, and PTC recurrence. Therefore, to evaluate the potential of BRAFV600E mutation and five selected miRNAs (-146b, -222, -21, -221, -181b) in predicting PTC recurrence, these molecular markers were analyzed in 400 formalin-fixed, paraffin-embedded PTC tissue specimens. The expression levels of miRNAs were measured using qRT-PCR. It was demonstrated that expression levels of all analyzed miRNAs are significantly higher in recurrent PTC than in non-recurrent PTC (p < 0.05). Moreover, higher expression levels of miR-146b, miR-222, miR-21, and miR-221 were associated with other clinicopathologic features of PTC, such as tumor size and lymph node metastases at initial surgery (p < 0.05). No significant differences in the frequency of BRAFV600E mutation in recurrent PTC and non-recurrent PTC were determined. Our results suggest that miRNA expression profile differs in PTC that is prone to recurrence when compared to PTC that does not reoccur after the initial surgery while BRAFV600E mutation frequency does not reflect the PTC recurrence status. However, the prognostic value of the analyzed miRNAs is rather limited in individual cases as the pattern of miRNA expression is highly overlapping between recurrent and non-recurrent PTC.

Rat hepatitis E virus (HEV) is an orthohepevirus which is related to other HEV found in humans and other mammals. It was first identified in Norway rats (Rattus norvegicus) from Germany in 2010, and later it has been detected in Black rats (Rattus rattus) and Norway rats from USA, China, Indonesia, Vietnam and many European countries. In this study, we describe molecular and serological investigations of Black and Norway rats trapped in Lithuania, Eastern Europe, for infections with rat HEV and human HEV genotypes 1-4. Rat HEV-specific real-time reverse transcription-PCR (RT-qPCR) analysis of rat liver samples revealed the presence of rat HEV in 9 of 109 (8.3%) samples. In contrast, a RT-qPCR specific for HEV genotypes 1-4 did not reveal any positive samples. A nested broad spectrum RT-PCR was used for a confirmation of rat HEV infection with a subsequent sequencing of the amplified rat HEV genome fragment. Phylogenetic analysis revealed a clustering of all newly identified rat HEV sequences with Norway rat-derived rat HEV sequences from Germany within the species Orthohepevirus C. An indirect ELISA using a yeast-expressed truncated rat HEV capsid protein variant revealed 31.2% seropositive samples indicating a high rate of rat HEV circulation in the rat population examined. In conclusion, the current investigation confirms rat HEV infections in Norway and Black rats in Lithuania, Eastern Europe, and the non-persistent nature of HEV infection.