Presentation of Meibomian Acini Compared to Dermal Papillae of the Eyelid Margin, Using Confocal Laser Scanning Microscopy and Corresponding Histology.

BACKGROUND: Numerous studies have investigated the eyelid margin using confocal laser scanning microscopy (CLSM) and have presented morphological alterations of the examined structures, which were presumed to be Meibomian acini. However, recent data confirm that these structures are the cross-sections of dermal papillae of the dermoepidermal junction. This study aims to present the morphological appearance of Meibomian acini examined by confocal laser scanning microscopy in comparison to dermal papillae, and to reveal the corresponding patterns with specific histological sections. METHODS AND MATERIAL: Twenty healthy patients were examined with a CLSM device in vivo at the marginal edge of the eyelid. Twenty-two samples of full-thickness eyelid wedges from 22 patients treated surgically with ectropion were collected, of which 11 freshly excised samples were imaged on the incision surface with CLSM ex vivo and 11 eyelids underwent conventional histological preparation. The represented structures on CLSM images were compared to Meibomian acini on histological sections in terms of area, longest and shortest diameter, as well as depth and density. RESULTS: On in vivo CLSM images, Meibomian orifices, epidermal cells, and dermal connective tissue could be identified, the latter in a cross-sectional view of the dermal papillae surrounded by basal cells of the epidermis, forming reflective ring-like structures. All morphological parameters of these structures differed from Meibomian acini measured on histological sections. In contrast, the CLSM images of the incision surface showed acinar units with the same morphology as the Meibomian acini seen in the histological images and no statistically significant difference was found between the corresponding parameters. CONCLUSION: The morphological appearance of Meibomian acini differs from the structures that were previously presumed as Meibomian glands on CLSM images. In vivo imaging of Meibomian glands by commonly used in vivo CLSM cannot be performed.

Comparison of immune activation of the COVID vaccines: ChAdOx1, BNT162b2, mRNA-1273, BBIBP-CorV, and Gam-COVID-Vac from serological human samples in Hungary showed higher protection after mRNA-based immunization.

OBJECTIVE: To gain insight into the different protective mechanisms of approved vaccines, this study focuses on the comparison of humoral and cellular immune responses of five widely used vaccines including ChAdOx1 (AZD1222, AstraZeneca), BNT162b2 (Pfizer), mRNA-1273 (Moderna), BBIBP-CorV (Sinopharm), and Gam-COVID-Vac (Sputnik V). MATERIALS AND METHODS: Isolated plasma from 95 volunteers' blood samples was used to measure anti-SARS-CoV-2 humoral and cellular immune responses. Positive controls were recovered patients from COVID-19 (unvaccinated). Specific quantification kits for anti-nucleocapsid IgG, anti-Spike protein IgG, neutralizing antibodies as well as specific SARS-CoV-2 antigens for T-cell activation were used and Spearman correlation and matrix analyses were performed to compare overall immune responses. RESULTS: Nucleocapsid antibodies were significantly higher for the BBIBP-CorV and convalescent group when compared to other vaccines. In contrast, subjects vaccinated with BNT162b2 and mRNA-1273 presented significantly higher anti-spike IgG. In fact, 9.1% of convalescent, 4.5% of Gam-COVID-Vac, 28.6% of ChAdOx1, and 12.5% of BBIBP-CorV volunteers did not generate anti-spike IgG. Similarly, a positive correlation was observed after the neutralization assay. T-cell activation studies showed that mRNA-based vaccines induced a T-cell driven immune response in all cases, while 55% of convalescents,  8% of BNT162b1,  12,5% of mRNA-1273, 9% of Gam-COVID-Vac,  57% of ChAdOx1,  and  56% of BBIBP-CorV subjects presented no cellular response. Further correlation matrix analyses indicated that anti-spike IgG and neutralizing antibodies production, and T-cell activation follow the same trend after immunization. CONCLUSIONS: RNA-based vaccines induced the most robust adaptive immune activation against SARS-CoV-2 by promoting a significantly higher T-cell response, anti-spike IgG and neutralization levels. Vector-based vaccines protected against the virus at a comparable level to convalescent patients.

Biological properties of influenza A virus mutants with amino acid substitutions in the HA2 glycoprotein of the HA1/HA2 interaction region.

Influenza A viruses (IAVs) enter into cells by receptor-dependent endocytosis. Subsequently, conformational changes of haemagglutinin are triggered by low environmental pH and the N terminus of HA2 glycoprotein (gp) is inserted into the endosomal membrane, resulting in fusion pore formation and genomic vRNA release into the cytoplasm. However, the pH optimum of membrane fusion is host- and virus-specific and can have an impact on virus pathogenicity. We prepared mutants of neurotropic IAV A/WSN/33 (H1N1) with aa substitutions in HA2 gp at the site of HA1/HA2 interaction, namely T642H (HA2 numbering position 64, H1 numbering position HA407; referred to as mutant '64'), V662H ('66') (HA409); and a double mutant ('D') with two aa substitutions (T642H, V662H). These substitutions were hypothesized to influence the pH optimum of fusion. The pH optimum of fusion activity was measured by a luciferase assay and biological properties of viruses were monitored. The in vitro and in vivo replication ability and pathogenicity of mutants were comparable (64) or lower (66, D) than those of the wild-type virus. However, the HA2 mutation V662H and double mutation T642H, V662H shifted the fusion pH maximum to lower values (ranging from 5.1 to 5.3) compared to pH from 5.4 to 5.6 for the wild-type and 64 mutant. The decreased replication ability and pathogenicity of 66 and D mutants was accompanied by higher titres in late intervals post-infection in lungs, and viral RNA in brains compared to wild-type virus-infected mice. These results have implications for understanding the pathogenicity of influenza viruses.

Methods to Study Autophagy in Zebrafish.

Autophagy (cellular self-eating) is a highly regulated degradation process of the eukaryotic cell during which parts of the cytoplasm are delivered into, and broken down within, the lysosomal compartment. The process serves as a main route for the elimination of superfluous and damaged cellular constituents, thereby mediating macromolecular and organellar turnover. In addition to maintaining cellular homeostasis, autophagy is involved in various other cellular and developmental processes by degrading specific regulatory proteins, and contributing to the clearance of intracellular pathogens. The physiological roles and pathological involvement of autophagy can be effectively studied in divergent eukaryotic model systems ranging from yeast to mice. Such a tractable animal modelapplied only recently for autophagy researchis the zebrafish Danio rerio, which also facilitates the analysis of more specific biological processes such as tissue regeneration. In this chapter, we overview the main methods and tools that are used to monitor autophagic structures and to assay autophagic responses in this vertebrate organism. We place emphasis on genetic (functional) approaches applied for exploring novel cellular and developmental roles of the autophagic process.

Single-cell studies of IFN-ß promoter activation by wild-type and NS1-defective influenza A viruses.

Deletion or truncation of NS1, the principal IFN antagonist of influenza viruses, leads to increased IFN induction during influenza virus infection. We have studied activation of the IFN induction cascade by both wild-type and NS1-defective viruses at the single-cell level using a cell line expressing GFP under the control of the IFN-ß promoter and by examining MxA expression. The IFN-ß promoter was not activated in all infected cells even during NS1-defective virus infections. Loss of NS1 expression is therefore insufficient per se to induce IFN in an infected cell, and factors besides NS1 expression status must dictate whether the IFN response is activated. The IFN response was efficiently stimulated in these cells following infection with other viruses; the differential IFN response we observe with influenza viruses is therefore not cell specific but is likely due to differences in the nature of the infecting virus particles and their subsequent replication.

The significance of micro- and macrovascular biomarkers on cardiovascular outcome in chronic kidney disease: a prospective cohort study.

Measures of small and large artery dysfunction have not been investigated in a single cohort for the prediction of cardiovascular (CV) events in patients with nondialysed (ND) chronic kidney disease (CKD). This prospective cohort study aimed to determine whether central pulse wave velocity (cPWV), central pulse pressure (CPP) or microvascular post-occlusive reactive hyperaemia area (PORHHA) independently predict CV events and mortality in CKD-ND. A total of 94 stage 1-5 CKD-ND (65.3±13.1 years; estimated glomerular filtration rate 35.3 (22.8-49.4) ml min(-1) per 1.73 m(2)) patients were followed-up for a median of 52 (36-65) months and had baseline cPWV and CPP measured by applanation tonometry and PORHHA by laser Doppler flowmetry. Multiple failure time Cox regression models were used to determine the predictive role of vascular parameters on CV mortality and events. Based on multiple linear regressions, baseline age, diabetes, CV disease, and systolic blood pressure (SBP) were independently related to cPWV (R(2)=0.3), SBP and PORHHA to CPP (R(2)=0.45), whereas CPP was the only parameter independently related to PORHHA (R(2)=0.16, all P<0.05). During follow-up, 41 CV events occurred (14 CV deaths). In univariate analyses, cPWV (1.07 (1.02-1.13) per m s(-1)), CPP (1.04 (1.01-1.07) per mm Hg) and lnPORHHA (0.70 (0.58-0.85) per ln(PU × s)) were all related to the outcome. Baseline diabetes (HR 3.07 (1.65-5.68)), lnFGF23 (fibroblast growth factor-23; 1.86 (1.13-3.06) per RU ml(-1)) and CPP (1.04 (1.01-1.07) per mm Hg) were independent predictors of CV events. The impaired pulsatile component of large arteries (CPP) independently of other vascular markers (cPWV, PORHHA) predicted CV outcomes in CKD-ND. CPP may integrate the information provided by cPWV and PORHHA.

Modeling the dynamics of a tracer particle in an elastic active gel.

The internal dynamics of active gels both in artificial (in vitro) model systems and inside the cytoskeleton of living cells has been extensively studied with experiments of recent years. These dynamics are probed using tracer particles embedded in the network of biopolymers together with molecular motors, and distinct nonthermal behavior is observed. We present a theoretical model of the dynamics of a trapped active particle, which allows us to quantify the deviations from equilibrium behavior, using both analytic and numerical calculations. We map the different regimes of dynamics in this system and highlight the different manifestations of activity: breakdown of the virial theorem and equipartition, different elasticity-dependent "effective temperatures," and distinct non-Gaussian distributions. Our results shed light on puzzling observations in active gel experiments and provide physical interpretation of existing observations, as well as predictions for future studies.

Energetics of active fluctuations in living cells.

The nonequilibrium activity taking place in a living cell can be monitored with a tracer embedded in the medium. While microrheology experiments based on optical manipulation of such probes have become increasingly standard, we put forward a number of experiments with alternative protocols that, we claim, will provide insight into the energetics of active fluctuations. These are based on either performing thermodynamiclike cycles in control-parameter space or determining response to external perturbations of the confining trap beyond simple translation. We illustrate our proposals on an active itinerant Brownian oscillator modeling the dynamics of a probe embedded in a living medium.

The RNA polymerase of influenza a virus: mechanisms of viral transcription and replication.

The influenza A virus RNA genome segments are packaged in ribonucleoprotein complexes containing RNA polymerase and nucleoprotein. The ribonucleoprotein is involved in the transcription of viral genes and replication of the viral RNA genome in the nucleus of the infected cells, and represents the minimal transcriptional and replicative machinery of an influenza virus. During transcription, the viral RNA polymerase synthesizes capped and polyadenylated mRNA using 5΄ capped RNA primers. During replication, the viral RNA polymerase generates a complementary RNA (cRNA) replication intermediate, a full-length complement of the vRNA that serves as a template for the synthesis of new copies of vRNA. The nucleoprotein is also an essential component of the viral transcriptional machinery. The molecular determinants of the transcriptional and replicative activities of the viral RNA polymerase are not fully understood, but recent data suggest that transcription is performed by a cis-acting RNA polymerase, forming part of the ribonucleoprotein complex, while replication might be carried out by a trans-acting RNA polymerase. Viral as well as cellular factors are known to be involved in the regulation of the activities of the RNA polymerase, e.g. the viral nuclear export protein has been shown to regulate the accumulation of viral transcription and replication products. The viral transcriptional machinery represents an attractive target for the development of antiviral drugs and lead compounds targeting nucleoprotein and the PA endonuclease domain of the RNA polymerase have already been identified.

Modulation of the pro-inflammatory cytokines and matrix metalloproteinases production in co-cultivated human keratinocytes and melanocytes.

The human epidermis exerts immunoregulatory functions through the variety of cytokines and other molecules elaborated by keratinocytes and melanocytes. Their constitutive production is very low; however, considerably increased upon stimulation. In vivo, keratinocytes and melanocytes have a typical exposure in the skin, referred as melanocyte epidermal unit. In the present study we co-cultivated these cells in vitro proposing to elucidate some communication links in close cell-to-cell association. We assessed the amounts of IL-6, IL-8, and matrix metalloproteinases (MMP-2 and MMP-9) in individually and co-cultured cells, exposed or not to UVB radiation. Normal human epidermal keratinocytes and melanocytes were grown in specific media and supplements. Cells were exposed to UVB radiation (100 mJ/cm(2)) to create comparable stress to the environmental one. Cytokines were determined with ELISA and confirmed with Western blot and metalloproteinases with gel zimography. Pure cultures of keratinocytes and melanocytes released low amounts of cytokines and metalloproteinases, these secretions being enhanced by UVB irradiation. In co-cultures, the cell-to-cell proximity triggered signals which markedly augmented the cytokines' secretions, whereas metalloproteinases were down-regulated. UVB irradiation did not influence either of these secretions in co-cultures. Concurrently with the highest levels of the pro-inflammatory cytokines, MMP-9 was up-regulated creating pro-inflammatory conditions and premises for changes in cellular survival, differentiation and phenotype. A complex network of interactions occurred between keratinocytes and melanocytes in co-cultures, resulting in modulated pro-inflammatory cytokines and metalloproteinases productions. Therefore, any disturbances in the microenvironmental signaling system and its molecular constituents may result in inflammation or even tumorigenesis in the epidermis.

The highly conserved, N-terminal (RXXX)8 motif of mouse Shadoo mediates nuclear accumulation.

The prion protein (PrP)-known for its central role in transmissible spongiform encephalopathies-has been reported to possess two nuclear localization signals and localize in the nuclei of certain cells in various forms. Although these data are superficially contradictory, it is apparent that nuclear forms of the prion protein can be found in cells in either the healthy or the diseased state. Here we report that Shadoo (Sho)-a member of the prion protein superfamily-is also found in the nucleus of several neural and non-neural cell lines as visualized by using an YFP-Sho construct. This nuclear localization is mediated by the (25-61) fragment of mouse Sho encompassing an (RXXX)8 motif. Bioinformatic analysis shows that the (RXXX)n motif (n=7-8) is a highly conserved and characteristic part of mammalian Shadoo proteins. Experiments to assess if Sho enters the nucleus by facilitated transport gave no decisive results: the inhibition of active processes that require energy in the cell, abolishes nuclear but not nucleolar accumulation. However, the (RXXX)8 motif is not able to mediate the nuclear transport of large fusion constructs exceeding the size limit of the nuclear pore for passive entry. Tracing the journey of various forms of Sho from translation to the nucleus and discerning the potential nuclear function of PrP and Sho requires further studies.

Nuclear import of the influenza A virus transcriptional machinery.

Unusually for an RNA virus, influenza A viruses transcribe and replicate their genomes in the nuclei of infected cells. As a result the viral ribonucleoprotein complexes (RNPs), and their newly synthesised protein subunits, must interact with the host nuclear import machinery. In this review we discuss how the virus exploits nuclear import pathways to allow regulated and chaperoned assembly of RNPs in the nucleus, and describe how the import machinery itself can be a determinant of host tropism.

GITR-expressing regulatory T-cell subsets are increased in tumor-positive lymph nodes from advanced breast cancer patients as compared to tumor-negative lymph nodes.

Lymph node (LN) infiltration by neoplastic process involves important changes in lymph node immune microenvironment. In particular, regulatory T cells (Treg) seem to have a key role in altering the immunoediting function of the immune system which leads to the elusion of the tumor from immune surveillance. In this study, we evaluated the expression of T-cell markers in CD4+ and CD8+ subsets from tumor-positive and tumor-negative lymph nodes from the same, advanced stage breast cancer patient. The study was carried out on 3 patients and similar results were obtained. Flow cytometric analysis of CD8+ cells demonstrated a significant difference in the expression of CD25, CD45RA, CD45RO, and GITRL (Glucocorticoid-Induced TNF receptor-Related ligand). Flowcytometric analysis of CD4+ cells demonstrated a significant difference in the expression of GITR (Glucocorticoid-Induced TNF receptor-Related), CD25, FoxP3 (Forkhead box P3), CD28, and CD45RA. Multiple staining allowed the identification of two Treg subpopulations, CD4+ CD25 highGITR+ CD127-/low and CD4+ CD25 low GITR+ CD127+ cells, proving that both are increased in the positive nodes in comparison with the negative nodes from the same patient. We identified for the first time the CD4+ CD25 low GITR+ CD127+ Treg subpopulation in cancer, and the 2.6 fold increase in positive LN suggests that this Treg subpopulation could be a key player in metastasis. We also found GITRL expression in the CD8 lymphocytes, which may also contribute to the changes of metastatic lymph node microenvironment. These findings make both GITR and GITRL good possible co-candidates for future therapeutical intervention against metastasis and perhaps also as disease evolution biomarkers.

Arsenic trioxide sensitizes cancer stem cells to chemoradiotherapy. A new approach in the treatment of inoperable glioblastoma multiforme.

PURPOSE: glioblastoma multiforme (GBM) still bears a very dismal prognosis even with complete resection followed by adjuvant chemoradiation. The aim of the current study was to evaluate in vitro the antitumor efficacy of arsenic trioxide (ATO) in combination with ionizing radiation plus temozolomide and bevacizumab against cultured glioblastoma stem-like cells, as possible way to increase the therapeutic index in patients diagnosed with recurrent, therapy-refractory GBM. METHODS: stem-like tumor cells isolated from a GBM biopsy were established by cell proliferation assays and upregulation of stem cell markers, as proven by reverse transcription - polymerase chain reaction (RT-PCR). Low concentrations of ATO were added prior to temozolomide, bevacizumab and ionizing irradiation. RESULTS: molecular analysis showed that cells expressed CXCR4, Oct-3/4 and GAPDH when compared to placental mesenchymal stem cells, as well as nestin, GFAP and neurofilament protein. Low concentrations of ATO led to morphologic differentiation, with fewer stem cells in Go state and differentiation-associated cytochemical features, like increased sensitivity to cytostatic drugs and radiotherapy. CONCLUSION: ATO exposure before conventional postoperative chemoradiotherapy for GBM might increase treatment efficacy. Further in vivo experiments on laboratory animals and analysis of absorption rate and side effects are required.

Comparative study of two evaluation methods for the genotoxic effects of environmental heavy metals on normal cells.

Genetic modifications caused by chronic exposure to low levels of toxic metals may activate stress-signaling pathways, thus increasing cancer incidence among affected individuals. The aim of this study was to evaluate the relationship between exposure to heavy metals and the incidence of chromosomal aberrations and DNA lesions in a chronically exposed population by using specific biomarkers. The study included 156 subjects divided into two major groups: exposed individuals (in a heavy metal contaminated region, Maramures, Romania) and non-exposed population, as control group (Cluj, Romania). We compared the results of two cytogenetic methods for the detection and quantification of DNA lesions and chromosomal aberrations in normal human cells: Single Cell Gel Electrophoresis or Comet assay and Cytokinesis Block Micronucleus assay. The methods were performed on lymphocytes isolated from whole blood in density gradient. The basal DNA lesions and chromosomal aberrations were evaluated, as well as the repair capacity of the supplementary lesions induced by genotoxic agents such as ionizing radiations. Our results showed a great interindividual variability in the basal level of the DNA lesions and chromosomal aberrations, between and within the groups, the most affected being the heavy metals-exposed groups. Non-exposed subjects from rural area Cluj appeared to be more susceptible to the induction of supplementary DNA lesions and chromosomal aberrations by irradiation. The most efficient repair capacity of the radio-induced DNA lesions was observed in the non-exposed Cluj urban group. Both cytogenetic assays (as tools for detection of DNA lesions and chromosomal aberrations) may be used in human biomonitoring studies as indicators of early biological effects induced by exposure to heavy metals.

Interfacial water structure controls protein conformation.

A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacteriorhodopsin and myoglobin. The experimental results support the ability of the new formalism to account for the diverse manifestations of salt effects on protein conformation, dynamics, and stability, and to resolve the puzzle of chaotropes stabilizing certain proteins (and other anomalies). It is also shown that the relation between interfacial tension and protein structural stability is straightforwardly linked to protein conformational fluctuations, providing a keystone for the microscopic interpretation of Hofmeister effects. Implications of the results concerning the use of Hofmeister effects in the experimental study of protein function are discussed.

Alteration of serum semicarbazide-sensitive amine oxidase activity in chronic renal failure.

Despite recent intensive investigations, physiological and pathological role of semicarbazide-sensitive amine oxidase (SSAO) is far from clear. In this study, serum SSAO activity was determined, radiochemically, in various groups of uremic patients: haemodialysed (HD), peritoneally dialysed (PD) and those receiving conservative treatment but still not dialysed (ND), as well as in controls. Reduced enzyme activity was found in HD uremic patients before and after dialysis treatment, compared to controls (5260 +/- 862 and 6011 +/- 958 pmol/h/ml vs. 8601 +/- 283 pmol/h/ml, p < 0.01 and p < 0.05, respectively). The activity was slightly lower in PD, and normal in ND patients. In HD patients SSAO activity was also determined by an assay based on the formation of hydrogen peroxide, and was found to be elevated compared to controls (2384 +/- 323 pmol/h/ml vs. 1437 +/- 72 pmol/h/ml, p < 0.05). The elevated serum SSAO activity measured through the detection of the enzyme-generated hydrogen peroxide in HD patients might indicate its contribution to the accelerated atherosclerotic disease observed in uremia.

Orthomyxovirus replication, transcription, and polyadenylation.

Efficient in vitro and in vivo systems are now in place to study the role of viral proteins in replication and/or transcription, the regulation of these processes, polyadenylation of viral mRNAs, the viral promoter structures, or the significance of noncoding regions for virus replication. In this chapter, we review the status of current knowledge of the orthomyxovirus RNA synthesis.

Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene.

The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U-A replacing G-C at nucleotide positions 12'-11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.