Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

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.

The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate.

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.

Folding-upon-Repair DNA Nanoswitches for Monitoring the Activity of DNA Repair Enzymes.

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.

A journey down to hell: new thermostable protein-tags for biotechnology at high temperatures.

The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag® and the Halotag®. These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H5) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag® for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H5, proposing them as alternatives for in vivo studies in these extreme model organisms.

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability.

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.

Thermostability enhancement of the α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense by using the anchoring-and-self-labelling-protein-tag system (ASLtag).

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.

An AGT-based protein-tag system for the labelling and surface immobilization of enzymes on E. coli outer membrane.

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the ß-glycoside hydrolase (SsßGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.

First thermostable CLIP-tag by rational design applied to an archaeal O6 -alkyl-guanine-DNA-alkyl-transferase.

Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O6 -benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC8 . The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC8 was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O6 -alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag variant.

Breast Cancer Management During COVID-19 Pandemic in Madrid: Surgical Strategy.

BACKGROUND: From the first case of SARS-CoV-2 infection in Wuhan (China), the infection spread all around the world causing a pandemic of coronavirus disease-2019 (COVID-19). Spain has been one of the most severely affected countries, and Madrid has reported a high number of cases and deaths. We discuss our strategies for optimal breast cancer management during COVID-19 pandemic. PATIENTS AND METHODS: This was a retrospective observational study at Clínico San Carlos Hospital to analyze the management of patients with breast cancer during the pandemic outbreak and the surgical strategy after the pandemic outbreak. We created a practical and dynamic tool based on a "traffic light" system for prioritizing surgical time. Every patient was contacted by telephone with a preoperative COVID-19 protocol. After surgical procedures, patient satisfaction was assessed using the European Organisation for Research and Treatment of Cancer in-patient satisfaction with cancer care questionnaire (EORTC IN-PATSAT32). RESULTS: Patients with breast cancer actively treated with surgical procedures were put on a waiting list and received systemic therapy. Telemedicine was used to evaluate any side effects and to avoid unnecessary hospital visits. Surgery was only considered after the pandemic outbreak, and then, only those procedures designed to minimize surgical complications and, therefore, reduce hospital stay. We also measured patients' satisfaction with medical and nursing scales that resulted in a "very good" evaluation tending to "excellent". CONCLUSION: It is necessary to adapt management of oncology treatment and surgical strategy to optimize resources during the COVID-19 pandemic. Patients' perception of care quality and the degree of patients' satisfaction with health services has potential relevance in the absence of outcome data.

Detecciòn de anticuerpos para el virus del Herpes simple tipo 1 y 2 (VHS 1 y 2), por el mètodo de Elisa en trabajadoras del sexo / Annual reports 1996 / detection of antibodies for herpes simplex virus 1 and 2 by the Elisa method in sex workers

Las tècnicas màs comùnmente utilizadas para el diagnòstico de la infecciòn por el virus del herpes simple (VHS) son: aislamiento del virus en cultivo de tejidos, examinaciòn citològica, ensayos serològicos como inmunofluorescencia indirecta, radioinmunoanàlisis, fijaciòn de complemento, hemaglutinaciòn indirecta y el mètodo de ELISA. Los ensayos serològicos mencionados podrìan distinguir infecciones por VHS-1 del 2. El objetivo de este trabajo fue determinar la presencia de anticuerpos de tipo IgG e IgM para el VHS-1 y 2, utilizando una tècnica de ELISA, en trabajadoras del sexo. Para tal efecto fueron estudiadas 56 trabajadoras del sexo, provenientes de las ciudades de Encarnaciòn, Edelira y Ayolas. El 78,5 por ciento de la poblaciòn en estudio presentò anticuerpos contra el virus VHS-1 y el 91 por ciento para el VHS-2. El 71 por ciento presentò anticuerpos del tipo IGG y el 74,1 por ciento del tipo IgM para ambos serotipos virales, respectivamente. Nuestros hallazgos podrìan indicar o una especificidad del mètodo de ELISA utilizado para la discriminaciòn del VHS-1 del 2 o la existencia de co-infecciòn en las mujeres estudiadas. Debido al alto porcentaje de anticuerpos contra VHS-2 detectados en las trabajadoras del sexo del Paraguay, consideramos de suma importancia ampliar el numero de pacientes y comparar el ELISA con otras tècnicas serològicas.

Detecciòn de anticuerpos para el virus del Herpes simple tipo 1 y 2 (VHS 1 y 2), por el mètodo de Elisa en trabajadoras del sexo / Annual reports 1996 / Detection of antibodies for herpes simplex virus 1 and 2 by the Elisa method in sex workers

Las tècnicas màs comùnmente utilizadas para el diagnòstico de la infecciòn por el virus del herpes simple (VHS) son: aislamiento del virus en cultivo de tejidos, examinaciòn citològica, ensayos serològicos como inmunofluorescencia indirecta, radioinmunoanàlisis, fijaciòn de complemento, hemaglutinaciòn indirecta y el mètodo de ELISA. Los ensayos serològicos mencionados podrìan distinguir infecciones por VHS-1 del 2. El objetivo de este trabajo fue determinar la presencia de anticuerpos de tipo IgG e IgM para el VHS-1 y 2, utilizando una tècnica de ELISA, en trabajadoras del sexo. Para tal efecto fueron estudiadas 56 trabajadoras del sexo, provenientes de las ciudades de Encarnaciòn, Edelira y Ayolas. El 78,5 por ciento de la poblaciòn en estudio presentò anticuerpos contra el virus VHS-1 y el 91 por ciento para el VHS-2. El 71 por ciento presentò anticuerpos del tipo IGG y el 74,1 por ciento del tipo IgM para ambos serotipos virales, respectivamente. Nuestros hallazgos podrìan indicar o una especificidad del mètodo de ELISA utilizado para la discriminaciòn del VHS-1 del 2 o la existencia de co-infecciòn en las mujeres estudiadas. Debido al alto porcentaje de anticuerpos contra VHS-2 detectados en las trabajadoras del sexo del Paraguay, consideramos de suma importancia ampliar el numero de pacientes y comparar el ELISA con otras tècnicas serològicas