Aggregation induced nucleic acids recognition by homodimeric asymmetric monomethyne cyanine fluorochromes in mesenchymal stem cells.

In the light of recent retrovirus pandemics, the issue of discovering new and diverse RNA-specific fluorochromes for research and diagnostics became of acute importance. The great majority of nucleic acid-specific probes either do not stain RNA or cannot distinguish between DNA and RNA. The versatility of polymethine dyes makes them suitable as stains for visualization, analysis, and detection of nucleic acids, proteins, and other biomolecules. We synthesized the asymmetric dicationic homodimeric monomethine cyanine dyes 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium) bromide (Т1) and 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium) bromide (M1) and tested their binding specificity, spectral characteristics, membrane penetration in living and fixed cells, cellular toxicity, and stability of fluorescent emission. Mesenchymal cells have diverse phenotypes and extensive proliferation and differentiation properties. We found dyes T1 and M1 to show high photochemical stability in living mesenchymal stem cells from apical papilla (SCAP) with a strong fluorescent signal when bound to nucleic acids. We found M1 to perform better than control fluorochrome (Hoechst 33342) for in vivo DNA visualization. T1, on the other hand, stains granular cellular structures resembling ribosomes in living cells and after permeabilization of the nuclear membrane stains the nucleoli and not the chromatin in the nucleus. This makes T1 suitable for the visualization of structures rich in RNA in living and fixed cells.

Novel Tetrahydroisoquinoline-Based Heterocyclic Compounds Efficiently Inhibit SARS-CoV-2 Infection In Vitro.

The ongoing COVID-19 pandemic has caused over six million deaths and huge economic burdens worldwide. Antivirals against its causative agent, SARS-CoV-2, are in urgent demand. Previously, we reported that heterocylic compounds, i.e., chloroquine (CQ) and hydroxychloroquine (HCQ), are potent in inhibiting SARS-CoV-2 replication in vitro. In this study, we discussed the syntheses of two novel heterocylic compounds: tert-butyl rel-4-(((3R,4S)-3-(1H-indol-3-yl)-1-oxo-2-propyl-1,2,3,4-tetrahydroisoquinolin-4-yl)methyl)piperazine-1-carboxylate (trans-1) and rel-(3R,4S)-3-(1H-indol-3-yl)-4-(piperazin-1-ylmethyl)-2-propyl-3,4-dihydroisoquinolin-1(2H)-one (trans-2), which effectively suppressed authentic SARS-CoV-2 replication in Vero E6 cells. Compound trans-1 showed higher anti-SARS-CoV-2 activity than trans-2, with a half maximal effective concentration (EC50) of 3.15 µM and a selective index (SI) exceeding 63.49, which demonstrated comparable potency to CQ or HCQ. Additional anti-SARS-CoV-2 tests on Calu-3 human lung cells showed that trans-1 efficiently inhibited viral replication (EC50 = 2.78 µM; SI: > 71.94) and performed better than CQ (EC50 = 44.90 µM; SI = 2.94). The time of an addition assay showed that the action mechanism of trans-1 differed from that of CQ, as it mainly inhibited the post-entry viral replication in both Vero E6 and Calu-3 cells. In addition, the differences between the antiviral mechanisms of these novel compounds and CQ were discussed.

Synthesis of Novel 1-Oxo-2,3,4-trisubstituted Tetrahydroisoquinoline Derivatives, Bearing Other Heterocyclic Moieties and Comparative Preliminary Study of Anti-Coronavirus Activity of Selected Compounds.

A series of novel 1-oxo-2,3,4-trisubstituted tetrahydroisoquinoline (THIQ) derivatives bearing other heterocyclic moieties in their structure were synthesized based on the reaction between homophthalic anhydride and imines. Initial studies were carried out to establish the anti-coronavirus activity of some of the newly obtained THIQ-derivatives against two strains of human coronavirus-229E and OC-43. Their antiviral activity was compared with that of their close analogues, piperidinones and thiomorpholinones, previously synthesized in our group, with aim to expand the range of the tested representative sample and to obtain valuable preliminary information about biological properties of a wider variety of compounds.

Time and Temperature Stability of TGF-ß1, EGF and IGF-1 in 20% and 100% Human Serum.

Autologous serum eye drops (ASEDs) are used as a treatment for severe dry eye disease. The concentration and stability of various growth factors in ASEDs is determinative for their efficiency. We therefore assessed the concentrations of transforming growth factor beta 1 (TGF-ß1), epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) in ASEDs following storage at 4-8, -20, -80 and -156 °C. Twenty % and 100% sera from eight healthy volunteers were analysed by the sandwich enzyme immunoassay at different time intervals up to seven months. The mean levels of TGF-ß1 and EGF in undiluted and 20% serum did not differ significantly from the baseline levels in fresh serum for any storage conditions after 7 days at 4-8 °C, as well as after 4- and 7-month preservation at sub-zero temperatures. In 20% serum, no IGF-1 concentration decrease was found following 7 days of preservation at 4-8 °C. However, a decrease to 78 % and 81 % (P < 0.01) of baseline values was found in 20% serum after 4-month storage at -20 °C and 7-month storage at -156 °C, respectively. A more pronounced decrease in IGF-1 was observed in undiluted serum. All assessed growth factors present in 20% frozen serum remained stable for up to 7 months. The highest stability was achieved at -80 °C. At -20 and -156 °C, some decrease in IGF-1 occurred. Our results indicate that 20% ASEDs can be stored frozen up to 7 months under proper conditions.

The interplay between total mercury, methylmercury and dissolved organic matter in fluvial systems: A latitudinal study across Europe.

Large-scale studies are needed to identify the drivers of total mercury (THg) and monomethyl-mercury (MeHg) concentrations in aquatic ecosystems. Studies attempting to link dissolved organic matter (DOM) to levels of THg or MeHg are few and geographically constrained. Additionally, stream and river systems have been understudied as compared to lakes. Hence, the aim of this study was to examine the influence of DOM concentration and composition, morphological descriptors, land uses and water chemistry on THg and MeHg concentrations and the percentage of THg as MeHg (%MeHg) in 29 streams across Europe spanning from 41°N to 64 °N. THg concentrations (0.06-2.78 ng L-1) were highest in streams characterized by DOM with a high terrestrial soil signature and low nutrient content. MeHg concentrations (7.8-159 pg L-1) varied non-systematically across systems. Relationships between DOM bulk characteristics and THg and MeHg suggest that while soil derived DOM inputs control THg concentrations, autochthonous DOM (aquatically produced) and the availability of electron acceptors for Hg methylating microorganisms (e.g. sulfate) drive %MeHg and potentially MeHg concentration. Overall, these results highlight the large spatial variability in THg and MeHg concentrations at the European scale, and underscore the importance of DOM composition on mercury cycling in fluvial systems.

[Tear Osmolarity in Patients with Severe Dry Eye Syndrome Before and After Autologous Serum Treatment: a Comparison with Tear Osmolarity in Healthy Volunteers]. / Osmolarita slz u pacientu s tezkým syndromem suchého oka pred a po aplikaci autologního séra. Porovnání s hodnotami zdravých dobrovolníku.

INTRODUCTION: Dry eye syndrome (DES) is a multifactorial disease of the tears and ocular surface. Recently, treatment with autologous serum eye drops (AS-ed) has been frequently used in these patients. Significant improvement correlates well with clinical, laboratory and subjective findings. It is assumed that one of the key factors in the development of the disease is increased tear osmolarity. The aims of our study were to test tear osmolarity measurements in clinical practice, to examine if osmolarity values differ before and after a 3-month application of 20% AS-ed, and to determine if the values differ between patients with severe DES and healthy individuals. METHODS: The study included 35 patients with severe DES (Schirmer test<5 mm/5 min) and 23 healthy volunteers. Tear osmolarity values (TearLab Osmolarity System), the Schirmer test (ST1), vital ocular surface staining and subjective feelings (the OSDI questionnaire) were assessed in patients with DES before and after treatment with 20% AS-ed. Further, the tear osmolarity values were compared between healthy subjects and patients with DES before and after treatment with AS-ed. RESULTS: The values of OSDI and vital staining significantly decreased in patients with DES after the treatment (p<0.0001). ST1 and tear osmolarity did not change significantly after the treatment. ST1 values in healthy individuals were significantly higher (p<0.0001) and the OSDI values significantly lower (p<0.0001) than the results obtained in patients before and after treatment. Tear osmolarity was not statistically different between healthy subjects (306 mosmol/l) and patients with DES (302 and 301 mosmol/l before and after treatment respectively). CONCLUSION: We demonstrated a positive effect of AS treatment on the ocular surface in patients with DES. However, the osmolarity values did not differ before and after treatment with AS, and they also did not differ significantly between DES patients and healthy individuals. In accordance with other recent studies, our results raise questions concerning the value of the TearLab Osmolarity System for evaluating therapeutic effect and also as a tool for DES diagnosis.

[Prospective, multicentric, non-interventional study to assess the existing treatment of type 2 diabetes mellitus patients inadequately controlled with metformin monotherapy - KOMETA CZ]. / Prospektivní, multicentrická, neintervencní studie na zhodnocení soucasné lécby pacientu s diabetes mellitus 2. typu, nedostatecne kompenzovaných monoterapií metforminem - KOMETA CZ.

After several years of diabetes, the first line anti-diabetic therapy of metformin usually starts failing. The KOMETA study was a prospective, multicentric, non-interventional epidemiological study in patients with type 2 diabetes mellitus inadequately controlled with metformin therapy. The study was conducted between 2010 and 2011. The study demonstrated a significant improvement in diabetes control, reduction of waist circumference, reduction in blood pressure and adjustment of dyslipidemia compared to baseline. Based on this study, our doctors select anti-diabetic drugs responsibly and they often use medicines without the hypoglycaemia risk. Nevertheless, more than two thirds of patients achieve the HbA1c control of under 6% according to IFCC. The main conclusion of this study is that under standard conditions it is possible to achieve full metabolic control of type

[Efficacy and safety of vildagliptin as a second-line therapy vs other oral antidiabetic agents in patients with type 2 diabetes: Czech results within the worldwide prospective cohort EDGE study]. / Úcinnost a bezpecnost vildagliptinu jako léku druhé volby ve srovnání s jinými perorálními antidiabetiky u pacientu s diabetes mellitus 2. typu: ceské výsledky v rámci celosvetové prospektivní kohortové studie EDGE.

INTRODUCTION: Metformin monotherapy is recommended as initial treatment of type 2 diabetes. The selection of optimal second-line therapy that is often necessary due to the progressive nature of the disease is still a subject of ongoing discussions. AIM OF THE STUDY: The aim of the international EDGE (Effectiveness of Diabetes control with vildaGliptin and vildagliptin/mEtformin) study was to prospectively compare the efficacy and safety of vildagliptin vs other oral antidiabetic agents in patients with type 2 diabetes not adequately controlled on monotherapy in a real-life clinical setting. In this paper, we present the data of patients participating in the EDGE study in the Czech Republic. MATERIAL AND METHODS: Patients with type 2 diabetes not adequately controlled on monotherapy were enrolled into the study, and randomised into either the vildagliptin arm or control arm with another OAD at the discretion of the treating physician. Patients with the addition of other incretin-based medications were not enrolled into the study. The efficiency was evaluated as a proportion of patients reaching the combined endpoint of decreasing HbA1c> 3 mmol/mol without hypoglycaemia, peripheral oedema or treatment termination due to gastrointestinal side effects during the 12 months of treatment. RESULTS: 654 patients were enrolled into the study in the Czech Republic. The mean age of the patients when enrolled into the study (vildagliptin group vs control group) was 59.5 ± 10.6 vs 63.7 ± 8.5 years, mean body mass index was 32.4 ± 5.7 vs 31.7 ± 6.5 kg/m2, mean HbA1c was 62 ± 12 vs 64 ± 11 mmol/mol. The probability of reaching the combined primary endpoint (calculated using a binary logistic regression model to calculate the odds ratios with 95% confidence intervals) was higher for vildagliptin regardless of baseline HbA1c or type of medication added in the control group. Primary endpoint was reached by 60.6 % of patients in the vildagliptin group vs 51.3 % of patients in the control group, odds ratio 1.46 (1.06, 1.99); p< 0.019. The proportion of patients reaching secondary endpoint (HbA1c< 54 mmol/mol without hypoglycemic event or weight gain 3 % with baseline glycated hemoglobin > 54 mmol/mol was higher for vildagliptin 45.7 % vs 31.4 % in the control arm, odds ratio 1.84 (1.26, 2.68), p< 0.001. The rate of adverse events was comparable in both groups. CONCLUSION: In a real-life clinical set-ting, the percentage of patients reaching the combined endpoint of decreasing HbA1c> 3 mmol/mol, without hypoglycaemia, peripheral oedema or treatment termination due to gastrointestinal side effects was higher after the addition of vildagliptin as compared to other antidiabetic agents with comparable rate of side effects.

[The application of the autologous serum eye drops results in significant improvement of the conjunctival status in patients with the dry eye syndrome]. / Aplikace autologního séra-ocních kapek vede ke statisticky významnému zlepsení stavu spojivky pacientu se syndromem suchého oka--pilotní studie.

AIM: To detect the changes on the conjunctiva surface before and after the application of the autologous serum (AS) eye drops in patients with dry eye syndrome, using both clinical and laboratory approaches, supplemented with subjective assessing the discomfort status. MATERIALS AND METHODS: The AS eye drops were applied during the period of 3 months in 8 patients with dry eye syndrome (Schirmer test < 5 mm and break-up time < 5 seconds), with the highest (maximum) frequency 8 times a day. The clinical (Schirmer test, break-up time, rose Bengal staining, examination of the tear meniscus, detritus and superficial punctate keratitis) and laboratory examinations (morphological assessment of the conjunctiva, detection of apoptotic cells) were performed at the start and at the end of the 3 months treatment period. Each day, patients reported their ocular status (dryness, discomfort, foreign body sensation, light sensitivity). RESULTS: The AS eye drops application improved significantly the values of the Schirmer test, detritus and superficial punctate keratitis as well. The goblet cells density on the conjunctival surface increased and the number of apoptotic cells decreased. The intensity of unpleasant feelings reported by the patients decreased significantly in all of the assessed categories. CONCLUSION: Because the application of AS eye drops caused the improvement of conjunctival status as well as the decrease of the severity of difficulties reported by the patients, the AS eye drops application should become common therapeutic practice in patients with dry eye syndrome.

Morphological and immunocytochemical characterization of snake-like chromatin cells.

Snake-like chromatin (SLC) is a nuclear alteration occurring under various pathological conditions and in different tissues. The aim of this study was the morphological and immunocytochemical characterization of SLC-positive conjunctival epithelial cells from keratoconjunctivitis sicca (KCS) patients. Impression cytology specimens from the upper bulbar conjunctiva of 10 controls and 10 KCS patients with a high incidence of SLC cells were assessed, the morphology of SLC nuclei evaluated by light microscopy, and proliferation markers, nucleolar proteins, lamins and cytokeratin filaments detected immunocytochemically. In KCS patients, SLC cells with a normal nuclear shape, with nuclear membrane notching (2.3% of cells) and with binuclear dumb-bell structures (4.4% of cells) were observed. The most striking features of SLC cells were the absence of an A/C lamin signal, the redistribution of fibrillarin into two spots adjacent to SLC structures and cytokeratin 14 positivity in the strangulation belt of the dumb-bell structures. The deficiency of lamin A/C is the probable reason for the disintegration of chromatin from the nuclear lamina in SLC cells. The occurrence of SLC-positive cells, SLC-positive dumb-bell shaped nuclei and SLC-positive binucleated cells, together with the absence of mitotic markers, leads to the conclusion that the SLC phenomenon might be a form of nuclear segregation.

Kinetics of leukocyte and myeloid cell traffic in the murine corneal allograft response.

BACKGROUND: Little information exists on the trafficking of myeloid and lymphoid cells between the transplanted cornea and the secondary lymphoid tissue. This study reports on changes in the cornea and the draining lymph node (DLN) from the time of graft emplacement. METHODS: Using a mouse corneal graft model (C57BL/10Sn to BALB/c), eyes and submandibular DLN were examined by immunohistochemistry and three-color flow cytometry for evidence of T cell activation and dendritic cell (DC) conditioning (up-regulation of costimulatory molecules) at various times (15 min to 24 days; n=4 for each time). RESULTS: In the DLN, early (2 hr) DC conditioning was sustained throughout allograft rejection whereas a remarkable drop in percentage of activated CD4+ and CD8+ T cells (P <0.001) was followed by a biphasic rise in activated CD4+ and, to a lesser extent, CD8+ T cells (24 hr, P <0.001 and 6 days, P <0.01). CD11b+ and MOMA-2+ macrophages, MHC Class II+ cells, CD86+ DC, and neutrophils were the earliest cells infiltrating the cornea (at 24 hr), whereas T cells appeared after 2 days, with CD4+ T cells being confined largely to the graft recipient border. CONCLUSIONS: Immediate and rapid changes in T cell and DC populations in the DLN correlate with the type of cellular infiltration in the corneal graft. The data are consistent with a model in which CD4+ T cell help for CD8+ cytotoxic T cells could be provided by sequential two-way activation of T cells and DC in the DLN. The majority of cells infiltrating the graft were macrophages and neutrophils, with fewer DC and T cells.

Effect of amino acid replacement on the stability of the tobacco mosaic virus protein structure.

A comparative polarographic study on the alkaline degradation of tobacco mosaic virus (TMV) strain vulgare and its mutant TMV 483, having histidine instead of glutamine at position 9 in the polypeptide chain, was performed. In the course of alkaline degradation and subsequent incubation in the supporting electrolyte at 0 degrees C TMV 483, unlike TMV vulgare, showed a polarographic effect indicating the unfolding of the TMV polypeptide. It was concluded that the replacement of glutamine-9 by histidine causes a decrease in the stability of the three-dimensional structure of the TMV protein subunit. A polarographic study of untreated virions as well as denatured proteins of both TMV strains showed that histidine, when incorporated into the polypeptide chain, is not active polarographically at the conditions used.