Characterization of the TCRß repertoire of peripheral MR1-restricted MAIT cells in psoriasis vulgaris patients.

Psoriasis vulgaris (PV) is an inflammatory skin disease largely driven by aberrant αßT cells. Mucosal-associated invariant T (MAIT) cells, which constitute the largest circulating innate-like αßT cell community in human adults, are characterized by a semi-invariant TCRVα7.2 receptor and MR1-restricted affinity toward microbial metabolites. Limited MAIT TCRα diversity is complemented by a more variable TCRß repertoire, but its footprint in the MAIT repertoire of PV patients has never been tested. Here, we used bulk TCRSeq, MiXCR, VDJTools, and Immunarch pipelines to decipher and compare TCRß clonotypes from flow-sorted, peripheral TCRVα7.2+MR1-5-OP-RU-tet+MAIT cells from 10 PV patients and 10 healthy, matched controls. The resulting TCRß collections were highly private and individually unique, with small public clonotype content and high CDR3ß amino acid length variability in both groups. The age-related increase in the 'hyperexpanded' clonotype compartment was observed in PV, but not in healthy MAIT repertoires. The TCRß repertoires of PV patients were also marked by skewed TRBV/TRBJ pairing, and the emergence of PV-specific, public CDR3ß peptide sequences closely matching the published CDR3ß record from psoriatic skin. Overall, our study provides preliminary insight into the peripheral MAIT TCRß repertoire in psoriasis and warrants further evaluation of its diagnostic and clinical significance.

The Mechanism of Anti-Tumor Activity of 6-Morpholino- and 6-Amino-9-Sulfonylpurine Derivatives on Human Leukemia Cells.

The aim of this study was to explore the mechanism of antitumor effect of (E)-6-morpholino-9-(styrylsulfonyl)-9H-purine (6-Morpholino-SPD) and (E)-6-amino-9-(styrylsulfonyl)-9H-purine (6-Amino-SPD). The effects on apoptosis induction, mitochondrial potential, and accumulation of ROS in treated K562 cells were determined by flow cytometry. The RT-PCR method was used to measure the expression of Akt, CA IX, caspase 3, and cytochrome c genes, as well as selected miRNAs. Western blot analysis was used to determine the expression of Akt, cytochrome c, and caspase 3. The results demonstrate the potential of the tested derivatives as effective antitumor agents with apoptotic-inducing properties. In leukemic cells treated with 6-Amino-SPD, increased expression of caspase 3 and cytochrome c genes was observed, indicating involvement of the intrinsic mitochondrial pathway in the induction of apoptosis. Conversely, leukemic cells treated with 6-Morpholino-SPD showed reduced expression of these genes. The observed downregulation of miR-21 by 6-Morpholino-SPD may contribute to the induction of apoptosis and disruption of mitochondrial function. In addition, both derivatives exhibited increased expression of Akt and CA IX genes, suggesting activation of the Akt/HIF pathway. However, the exact mechanism and its relations to the observed overexpression of miR-210 need further investigation. The acceptable absorption and distribution properties predicted by ADMET analysis suggest favorable pharmacokinetic properties for these derivatives.

Rhodanine Derivatives as Anticancer Agents: QSAR and Molecular Docking Studies.

BACKGROUND: Rhodanine derivatives have a proven wide range of biological activities. OBJECTIVE: The aim of this study was to evaluate the cytotoxic effect of a series of rhodanine derivatives and investigate the quantitative structure-activity relationships, as well as binding modes to tyrosine kinase. METHODS: Cytotoxic effect on cell proliferation (CaCo-2, HeLa, MDCK-1, Hut-78, K562) in vitro was evaluated by the MTT viability assay. QSAR analysis was performed with Dragon descriptors using QSARINS software. Molecular docking was performed on the tyrosin kinase (c-Src) (PDB ID: 3G6H) using iGEMDOCK. RESULTS: Compounds with the best inhibiting activity toward all cell lines were the ones possessing only one group in the C2 of the phenyl ring. QSAR study on the cytotoxic activity against Human T cell lymphoma achieved the model that satisfies the fitting and internal cross-validation criteria (R2 = 0.75; Q2 LOO = 0.64). Descriptors included in the model (MATS2e, MATs7e, RDF060p) revealed the importance of the presence of atoms with higher polarizability in the outer region of molecules. The findings of the molecular docking study performed on the c-Src are in accordance with the results of the QSAR study. The key interactions with binding site residues were achieved through oxygen atoms from phenoxy and rhodanine groups and rhodanine sulphur atoms. CONCLUSION: Rhodanine derivatives could be developed as novel tyrosine kinase inhibitors in the treatment of leukemia.

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression.

6-Morpholino- and 6-amino-9-sulfonylpurine derivatives. Synthesis, computational analysis, and biological activity.

The synthesis of novel 6-chloro/morpholino/amino/-9-sulfonylpurine derivatives was accomplished in two ways, either (i) involving the condensation reaction of 6-chloropurine with commercially available arylsulfonyl chlorides in acetone and the presence of aqueous KOH at 0 °C, followed by the substitution of C6-chlorine with morpholine, or (ii) employing a reversed synthetic approach where 6-morpholinopurine and commercially available adenine bases were reacted with the corresponding alkyl, 2-arylethene and arylsulfonyl chlorides giving the N9 sulfonylated products, the latter particularly used where prior nonselective sulfonylation was observed. In both approaches, the sulfonylation reaction occurred regioselectively at the purine N9 position lacking any concurrent N7 derivatives, except in the case of a smaller methyl substituent on SO2 and the free amino group at C6 of the purine ring. The tautomeric features of initial N9 unsubstituted purines, as well as stability trends among the prepared N-9-sulfonylpurine derivates, were investigated using DFT calculations with an important conclusion that electron-donating C6 substituents are beneficial for the synthesis as they both promote the predominance of the desired N9 tautomers and help to assure the stability of the final products. The newly synthesized 6-morpholino and 6-amino-9-sulfonylpurine derivatives showed antiproliferative activity on human carcinoma, lymphoma, and leukemia cells. Among the tested compounds, 6-morpholino 17 and 6-amino 22 derivatives, with trans-ß-styrenesulfonyl group attached at the N9 position of purine, proved to be the most effective antiproliferative agents, causing accumulation of leukemia cells in subG0 cell cycle phase.

Adamantyl pyran-4-one derivatives and their in vitro antiproliferative activity.

Pyran-4-one (maltol, kojic acid and chlorokojic acid 1) esters of adamantan-1-ylacetic acid were prepared through efficient synthetic routes in good yields and evaluated for their in vitro antiproliferative activity on four cancer cell lines: K562 (chronic myelogenous leukemia), HeLa (cervical cancer), Caco-2 (colorectal adenocarcinoma) and NCI-H358 (bronchioalveolar carcinoma). The results indicate that the presence and the position of the adamantyl acyl group or chlorine atom are the necessary requirement for antitumor activity of pyranone systems. Derivatives of kojic acid with either free (compounds 1 and 8) or acylated 5-OH group (compounds 2 and 9) have shown good-to-moderate activity (IC50 values ranging from 13.1 to 43.0 µM) on all cell lines. Adamantyl kojic acid derivative 5 with a free OH group on the position 2 showed activity only on the K562 cell line. It seems that removal of halogen or adamantyl unit from position 2 elicits antileukemic activity, as observed in compound 5. The positive influence of the adamantyl unit was also observed on a 3-OH acylated derivative of maltol I which was also selectively active on the same cell line. 5-O-benzylated adamantyl compounds 6 and 7 and unmodified starting pyranones were found to be inactive. Antibacterial activity of compounds was also evaluated on S. aureus ATCC 13709, M. catarrhalis ATCC 23246, E. faecalis ATCC29212 and E. coli TolC-Tn10, but no activity was observed (MIC values 128-256 µg/mL).

LIFESTYLE RISK FACTORS AND BONE MASS IN RECURRENT STONE-FORMING PATIENTS: A CROSS-SECTIONAL STUDY IN 144 SUBJECTS.

Patients with urolithiasis, particularly hypercalciuria, may have reduced bone mineral density (BMD). There are numerous risk factors contributing to reduction of BMD such as advanced age, sedentary lifestyle, smoking, low calcium intake, etc. The aim of our study was to investigate the association of lifestyle risk factors and daily intake of milk and dairy products with determinants of BMD in a group of recurrent calcium stone formers (RSF) compared with healthy subjects (HS). The study was carried out at the Department of Mineral Research, Faculty of Medicine in Osijek, Croatia. The study included 144 subjects, i.e. 56 RSF and 78 HS. BMD was assessed by dual-energy x-ray absorptiometry. A standard self-reported questionnaire was used to collect data on lifestyle risk factors. Current dietary intake was assessed by personal interview that included questions about milk and dairy product intake. Low BMD was observed in 44.64% of RSF and 35.90% of HS. RSF consumed significantly less milk and dairy products than HS. Calcium restriction in dietary recommendations might be unnecessary due to the impact on bone mineral loss in RSF and dual-energy x-ray absorptiometry should be included in the routine evaluation of RSF.

New quinoline-arylamidine hybrids: Synthesis, DNA/RNA binding and antitumor activity.

Four series of new hybrid molecules with 7-chloroquinoline and arylamidine moieties joined through the rigid -O- (groups I (2a-g) and II (5a-g)) or flexible -NH-CH2-CH2-O- (groups III (8a-g) and IV (10a-g)) linker were synthesized, and their DNA/RNA binding properties and cytotoxic activity were tested, against several human cancer lines. The compounds and their interaction with DNA and RNA were studied by UV-Vis and CD spectroscopy. The obtained results showed that the binding affinity of the investigated compounds increases proportionally with the increase of the length and number of groups able to form hydrogen bonds with ds-polynucleotides. Improvement of binding was additionally achieved by reduction of the structural rigidity of the investigated compounds, new hybrid compounds preferentially bind to ctDNA. For most of them the DNA/RNA grooves are dominant binding sites, except for the compounds from group II for which intercalation in polyA-polyU was the dominant binding mode. The antiproliferative effects were tested by the MTT test on normal (MDCK1), carcinoma (HeLa and CaCo2) and leukemia cell lines (Raji and K462). The GI50 values for all investigated compounds ranged from 5 to more than 100 × 10-6 mol dm-3. Carcinoma cells were more resistant to the investigated compounds than leukemia cells. The most effective compounds against leukemia cell lines were from group IV (10a-g), with GI50 values ranging from of 5 and 35 × 10-6 mol dm-3. The cell cycle arrest was investigated by flow cytometry and the obtained results indicate that the selected compounds, 2d, 2e, 8a, 10d, 10e, and 10f, induce changes in the cell cycle of treated cells, but the cycle phase distribution varies between them. A significant decrease in the number of cells in S phase (p < 0.001) was observed in all treated cells, but only 10d and 10f induce cell cycle arrest at G0/G1 phase, dominantly.

Effects of cell penetrating Notch inhibitory peptide conjugated to elastin-like polypeptide on glioblastoma cells.

Notch pathway was found to be activated in most glioblastomas (GBMs), underlining the importance of Notch in formation and recurrence of GBM. In this study, a Notch inhibitory peptide, dominant negative MAML (dnMAML), was conjugated to elastin-like polypeptide (ELP) for tumor targeted delivery. ELP is a thermally responsive polypeptide that can be actively and passively targeted to the tumor site by localized application of hyperthermia. This complex was further modified with the addition of a cell penetrating peptide, SynB1, for improved cellular uptake and blood-brain barrier penetration. The SynB1-ELP1-dnMAML was examined for its cellular uptake, cytotoxicity, apoptosis, cell cycle inhibition and the inhibition of target genes' expression. SynB1-ELP1-dnMAML inhibited the growth of D54 and U251 cells by inducing apoptosis and cell cycle arrest, especially in the presence of hyperthermia. Hyperthermia increased overall uptake of the polypeptide by the cells and enhanced the resulting pharmacological effects of dnMAML, showing the inhibition of targets of Notch pathway such as Hes-1 and Hey-L. These results confirm that dnMAML is an effective Notch inhibitor and combination with ELP may allow thermal targeting of the SynB1-ELP1-dnMAML complex in cancer cells while avoiding the dangers of systemic Notch inhibition.

Evaluation of methods for urine inhibitory potential for precipitation of calcium oxalate.

Renal lithiasis is a significant medical and social problem. Worldwide recurrence is anywhere from 3% to 5%. Objective of this paper is to evaluate two methods for distinguishing between stone formers and non-stone formers. Urine samples were titrated with calcium and seed crystals were added to facilitate precipitation. Ionic calcium levels were monitored and compared between the two groups. Stone formers showed impaired tolerance to the calcium added and increased precipitation on seed crystals. Both methods discriminated between stone formers and non-stone formers. Further evaluations are needed to establish the better of the two for wider clinical use.

Influence of pH, temperature and common ion on magnesium hydrogenurate octahydrate solubility.

Physico-chemical investigation of urolithiasis today is mostly focused on applying physico-chemical description of precipitation processes to the stone formation with the aim to distinguish between stone formers and nonstone formers. This is done by calculating supersaturation for different solid phases which can be formed in urine using data obtained by urine analysis and existing solubility product, dissociation and complexation constants. In order for this approach to succeed it is of utmost importance that system is described as detailed as possible, i.e., that all species that can be formed are taken into account. Magnesium hydrogenurate octahydrate, Mg(C5H3N4O3)28H2O (Mg(HU)2.8H2O), is among species which can precipitate in the urine and for which solubility data doesn't exist. In order to fill this void crystals of Mg(C5H3N4O)2.8H2O phase I and phase II have been prepared and characterized. Solubility product constant of Mg(C5H3N4O3)2.8H2O phase I in water at 37 degrees C and phase II at different temperatures, pH and in different solvents have been determined by measuring total concentration of uric acid and magnesium ions in solutions at different time periods. Results show that in water at 37 degrees C thermodynamically less stable phase I is more soluble (Ksp = (5.64 +/- 0.20).10(-9) mol3 dm(-9)) than phase II (Ksp = (1.66 +/- 0.13).10(-9) mol3 dm(-9)). Solubility of Mg(HU)2.8H2O phase II increases with temperature. At equilibrium the solubility of phase II is the lowest in the presence of excess of magnesium ions, while solubility in the presence of uric acid is comparable with the one obtained in water.