Recently, Type III-A CRISPR-Cas systems were found to catalyze the synthesis of cyclic oligoadenylates (cOAs), a second messenger that specifically activates Csm6, a Cas accessory RNase and confers antiviral defense in bacteria. To test if III-B CRISPR-Cas systems could mediate a similar CRISPR signaling pathway, the Sulfolobus islandicus Cmr-α ribonucleoprotein complex (Cmr-α-RNP) was purified from the native host and tested for cOA synthesis. We found that the system showed a robust production of cyclic tetra-adenylate (c-A4), and that c-A4 functions as a second messenger to activate the III-B-associated RNase Csx1 by binding to its CRISPR-associated Rossmann Fold domain. Investigation of the kinetics of cOA synthesis revealed that Cmr-α-RNP displayed positively cooperative binding to the adenosine triphosphate (ATP) substrate. Furthermore, mutagenesis of conserved domains in Cmr2α confirmed that, while Palm 2 hosts the active site of cOA synthesis, Palm 1 domain serves as the primary site in the enzyme-substrate interaction. Together, our data suggest that the two Palm domains cooperatively interact with ATP molecules to achieve a robust cOA synthesis by the III-B CRISPR-Cas system.

In this study, a facile synthesis of the titanium (IV) ion immobilized poly-glycidyl methacrylate microparticles functionalized with polyethylenimine and adenosine triphosphate was developed for efficient enrichment of intact phosphoproteins. The titanium (IV) ion immobilized microparticles had higher saturated adsorption capacity for phosphoproteins (1217.6 mg/g for β-casein) than non-phosphoproteins (97.1 mg/g for bovine serum albumin) and the average particle diameter was about 1.4 μm with good dispersibility. In application, as demonstrated by SDS-PAGE, titanium (IV) ion immobilized microparticles exhibited good performance in enriching intact phosphoproteins from standard protein mixtures of β-casein and bovine serum albumin with high specificity and selectivity; in addition, titanium (IV) ion immobilized microparticles were also successfully applied in intact phosphoprotein enrichment from complex biological samples including non-fat milk, chicken egg white and mouse heart tissue extract. This article is protected by copyright. All rights reserved.

This study aimed to elucidate anticoagulant/antiplatelet mechanisms of two previously purified PLA2 s from Cerastes cerastes venom, here, termed Cc1 -PLA2 and Cc2 -PLA2 . Both PLA2 s present close molecular weights of 13,534 and 13,430 Da and Isoectric pH (pI) 7.38 and 7.86 respectively, for Cc1 -PLA2 and Cc2 -PLA2 . These Ca2+ -dependent enzymes showed a high catalytic activity upon phospholipids, inducing indirect hemolysis, since they conserve the catalytic domain of PLA2 s 26 CYCGWGGKG34 . They exhibited dual inhibition of platelet aggregation by targeting P2 Y12 and TPα receptors preventing Adenosine diphosphate/arachidonate binding and blood clotting. These effects are due to the interaction of Cc1 -PLA2 s/Cc2 -PLA2 s with factor FXa through a noncatalytic PL-independent mechanism leading to nonreleased thrombin. Both proteins consist of 120 amino acid residues and share similar three-dimensional structures close to other SV-PLA2 s. Structural data of PLA2 s allowed the relevant residues involved in binding to FXa and platelet receptors. These findings may lead to the design of novel noncompetitive FXa inhibitors.

Human polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the immune response to bacterial and fungal infections and eliminate pathogens through phagocytosis. During phagocytosis of microorganisms, the 5-lipoxygenase (5-LOX) pathway is activated resulting in generation of leukotrienes, which mediate host defense. In this study, a library of oligodeoxyribonucleotides (ODNs) with varying numbers of human telomeric repeats (d(TTAGGG)n) and their analogues with phosphorothioate internucleotide linkages and single-nucleotide substitutions was designed. These ODNs with the potential to fold into G-quadruplex structures were studied from structural and functional perspectives. We showed that exogenous G-quadruplex-forming ODNs significantly enhanced 5-LOX metabolite formation in human neutrophils exposed to Salmonella Typhimurium bacteria. However, the activation of leukotriene synthesis was completely lost when G-quadruplex formation was prevented by substitution of guanosine with 7-deazaguanosine or adenosine residues at several positions. To our knowledge, this study is the first to demonstrate that G-quadruplex structures are potent regulators of 5-LOX product synthesis in human neutrophils in the presence of targets of phagocytosis.

A series of clickable bile acid-nucleosides conjugates linked directly or via amino acid linker were synthesized, and characterized by spectroscopic techniques such as 1H NMR, 13C NMR, FT-IR, HRMS and HPLC. The synthesized compounds 6a-p were screened for their in vitro anticancer property against a panel of three cancer cell lines (PC-3, MCF-7, IMR-32). In addition, the synthesized derivatives were also tested for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC 27294 strain). Among the screened compounds, cholic acid-uridine clicked conjugate (6c), and cholic acid-uridine clicked conjugate liked via phenylalanine moiety (6m) were found to be most active against MCF-7 and IMR-32 exhibiting an IC50 value of 8.084 and 8.71 µM, respectively. The antimycobacterial study of the synthesized conjugates revealed all the conjugates to be active with MIC values in the range of 4.09-15.41 µM. Deoxycholic acid-adenosine clicked conjugate (6b) showed most promising antituberculosis property with MIC value of 4.09 µM. Most of the synthesized conjugates were found to be safe at 50 µM against normal human embryonic kidney (HEK 293T) cell line.

Beyond thromboembolic events, peri-procedural bleeding remains one of the most frequent complications after transcatheter aortic valve implantation (TAVI). The majority of TAVI patients receive a dual anti-platelet treatment (DAPT) regimen. This analysis from the EVERY-TAVI register database aimed to analyse whether the level of on-treatment adenosine diphosphate-induced platelet reactivity predicts early outcomes at 30 days after TAVI. A total of 146 consecutive TAVI patients on DAPT who underwent platelet function testing with the Multiplate analyser were included here. Definition of bleeding events was done according to the Valve Academic Research Consortium-2 (VARC-2) classification. In our cohort, a status of low platelet reactivity (LPR, ≤ 18 units) was observed in 79 patients (54%), while high platelet reactivity (HPR, ≥ 46 units) was present in 18 patients (12%). At 30-day follow-up, the incidence of VARC-2 bleeds was 45.6% (n = 36) in LPR patients and 23.9% (n = 16) in patients without LPR (hazard ratio [HR] 2.10, 95% confidence interval [CI], 1.17-3.79; p = 0.01). In age-adjusted multivariate analysis, a status of LPR was independently associated with VARC-2 bleeding events (HRadj, 2.06, 95% CI, 1.14-3.71; p = 0.02). HPR was not associated with the 30-day risk of death, stroke, or myocardial infarction (p ≥ 0.43). In summary, presence of LPR was associated with bleeding events in patients undergoing TAVI while presence of HPR was not associated with ischaemic outcomes at 30 days. The value of platelet function testing for bleeding risk prediction and for a possible guidance of anti-thrombotic treatment in the elderly TAVI population warrants further investigation.