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The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

We present a new class of DNA-based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding-upon-repair DNA nanoswitches are synthetic DNA sequences containing O6 -methyl-guanine (O6 -MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The nanoswitches are rationally designed so that only upon enzymatic demethylation of the O6 -MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNA structure. We have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations. We then demonstrated that the folding-upon-repair DNA nanoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors.

We present the case of a 36-year-old primigravida who gave birth to a 3200 g baby by vacuum-assisted (Kiwi OmniCup™) operative vaginal delivery with mediolateral episiotomy. A "y"-shaped perineal tear with a grade IIIC obstetric anal sphincter injury (OASI) was diagnosed and repaired. Two days after delivery, in the absence of suture dehiscence, she started experiencing complete anal incontinence. A decision was made in association with a proctologic surgeon for an early secondary repair. Before surgery, a Three-dimensional transperineal ultrasound (TPUS) was performed. The exam revealed a major defect of the external anal sphincter at the 11 o'clock position. This allowed for the reopening of only a circumscribed area of the perineal suture and repair of the sphincters using the end-to-end technique. The symptoms regressed completely, and follow-up TPUS demonstrated the gradual wound healing process. Anal incontinence, secondary to obstetric anal sphincter injury (OASI), has a severe negative impact on women's quality of life. TPUS is an effective method to detect sphincter defects and monitor the healing process. This report investigates the feasibility of identifying the sphincter tear in an incontinent puerperal patient without suture dehiscence in order to target early secondary repair while minimizing its extent. TPUS has proven a safe and effective tool to guide early secondary repair of symptomatic OASI complications while minimizing the invasiveness of the procedure. Multidisciplinary management is crucial to ensure the adequate standard of care. [ABSTRACT FROM AUTHOR]

SNAP-tag® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag® and its relative thermostable version (SsOGT-H5) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag. [ABSTRACT FROM AUTHOR]

The mechanism by which the activity of the active-site mutants Glu387Ala/Gly of the beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus is restored through addition of external nucleophiles was investigated. Experimental results showed that the beta-glycosidase hydrolyzes beta-glycosides following a retaining mechanism which is dependent on the action of the amino acids Glu387 and Glu206. The type of nucleophile used influences the reaction mechanism behind the restoration process.

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H5-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions. [ABSTRACT FROM AUTHOR]

The structural, kinetic and thermodynamic dissection of binding of two glucoimidazole-derived compounds, which are among the most potent glycosidase inhibitors reported to date, with two family 1 [beta]-glycosidases is described. Inhibition of glycosidases has great potential in the quest for highly potent and specific drugs to treat diseases such as diabetes, cancer, and viral infections.