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Deaminases, ubiquitous enzymes found in all living organisms from bacteria to humans, serve diverse and crucial functions. Notably, purine and pyrimidine deaminases, while biologically essential for regulating nucleotide pools, exhibit exceptional versatility in biotechnology. This review systematically consolidates current knowledge on deaminases, showcasing their potential uses and relevance in the field of biotechnology. Thus, their transformative impact on pharmaceutical manufacturing is highlighted as catalysts for the synthesis of nucleic acid derivatives. Additionally, the role of deaminases in food bioprocessing and production is also explored, particularly in purine content reduction and caffeine production, showcasing their versatility in this field. The review also delves into most promising biomedical applications including deaminase-based GDEPT and genome and transcriptome editing by deaminase-based systems. All in all, illustrated with practical examples, we underscore the role of purine and pyrimidine deaminases in advancing sustainable and efficient biotechnological practices. Finally, the review highlights future challenges and prospects in deaminase-based biotechnological processes, encompassing both industrial and medical perspectives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus . These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.

Muscle testing is an integral component in assessing musculoskeletal function and tailoring rehabilitation efforts. This study aimed i. to identify an objective evaluation system sensitive to analyze changes in different muscular conditions in different neuromuscular tests across a spectrum of professional experience levels; and ii. to analyze differences in objective parameters and clinical judgment between participants of different levels of expertise in different muscular conditions in different neuromuscular tests. Participants included 60 subjects with Level I to III expertise who performed blinded neuromuscular tests on the middle deltoid and rectus femoris muscles of 40 volunteer subjects. The methodology centered on standardizing test protocols to minimize variability, employing EMG to quantify muscle activity, thermography to capture thermographic muscular response, and digital dynamometry to measure muscular resistance. The findings revealed that while traditional methods like thermography and electromyography provide valuable insights, digital dynamometry stands out for its sensitivity in detecting muscle condition changes in neuromuscular test. Moreover, the data underscored the pivotal role of advanced training and expertise in enhancing the precision and accuracy of neuromuscular diagnostics, since there were significant differences in objective parameters and clinical judgment between participants of different levels of expertise in the different muscular conditions in Middle deltoid and Rectus femoris neuromuscular tests analyzed, presenting higher expertise participant clinical judgment like objective validated instrument., Competing Interests: Declaration of competing interest Guillermo Escribano-Colmena and Jorge Rey-Mota are affiliated with ®NeuroReEvolution., (Copyright © 2024. Published by Elsevier Inc.)

Directed enzyme prodrug therapy (DEPT) strategies show promise in mitigating chemotherapy side effects during cancer treatment. Among these, the use of immobilized enzymes on solid matrices as prodrug activating agents (IDEPT) presents a compelling delivery strategy, offering enhanced tumor targeting and reduced toxicity. Herein, we report a novel IDEPT strategy by employing a His-tagged Leishmania mexicana type I 2'-deoxyribosyltransferase (His- Lm PDT) covalently attached to glutaraldehyde-activated magnetic iron oxide nanoparticles (MIONPs). Among the resulting derivatives, PDT-MIONP3 displayed the most favorable catalyst load/retained activity ratio, prompting its selection for further investigation. Substrate specificity studies demonstrated that PDT-MIONP3 effectively hydrolyzed a diverse array of 6-oxo and/or 6-amino purine 2'-deoxynucleosides, including 2-fluoro-2'-deoxyadenosine (dFAdo) and 6-methylpurine-2'-deoxyribose (d6MetPRib), both well-known prodrugs commonly used in DEPT. The biophysical characterization of both MIONPs and PDT-MIONPs was conducted by TEM, DLS, and single particle ICPMS techniques, showing an ideal nanosized range and a zeta potential value of -47.9 mV and -78.2 mV for MIONPs and PDT-MIONPs, respectively. The intracellular uptake of MIONPs and PDT-MIONPs was also determined by TEM and single particle ICPMS on HeLa cancer cell lines and NIH3T3 normal cell lines, showing a higher intracellular uptake in tumor cells. Finally, the selectivity of the PDT-MIONP/dFAdo IDEPT system was tested on HeLa cells (24 h, 10 µM dFAdo), resulting in a significant reduction in tumoral cell survival (11% of viability). Based on the experimental results, PDT-MIONP/dFAdo presents a novel and alternative IDEPT strategy, providing a promising avenue for cancer treatment.

Lignin modifying enzymes (LMEs) have gained widespread recognition in depolymerization of lignin polymers by oxidative cleavage. LMEs are a robust class of biocatalysts that include lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP), laccase (LAC), and dye-decolorizing peroxidase (DyP). Members of the LMEs family act on phenolic, non-phenolic substrates and have been widely researched for valorization of lignin, oxidative cleavage of xenobiotics and phenolics. LMEs implementation in the biotechnological and industrial sectors has sparked significant attention, although its potential future applications remain underexploited. To understand the mechanism of LMEs in sustainable pollution mitigation, several studies have been undertaken to assess the feasibility of LMEs in correlating to diverse pollutants for binding and intermolecular interactions at the molecular level. However, further investigation is required to fully comprehend the underlying mechanism. In this review we presented the key structural and functional features of LMEs, including the computational aspects, as well as the advanced applications in biotechnology and industrial research. Furthermore, concluding remarks and a look ahead, the use of LMEs coupled with computational framework, built upon artificial intelligence (AI) and machine learning (ML), has been emphasized as a recent milestone in environmental research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

In recent decades, enzyme-based biocatalytic systems have garnered increasing interest in industrial and applied research for catalysis and organic chemistry. Many enzymatic reactions have been applied to sustainable and environmentally friendly production processes, particularly in the pharmaceutical, fine chemicals, and flavor/fragrance industries. However, only a fraction of the enzymes available has been stepped up towards industrial-scale manufacturing due to low enzyme stability and challenging separation, recovery, and reusability. In this context, immobilization and co-immobilization in robust support materials have emerged as valuable strategies to overcome these inadequacies by facilitating repeated or continuous batch operations and downstream processes. To further reduce separations, it can be advantageous to use multiple enzymes at once in one pot. Enzyme co-immobilization enables biocatalytic synergism and reusability, boosting process efficiency and cost-effectiveness. Several studies on multi-enzyme immobilization and co-localization propose kinetic advantages of the enhanced turnover number for multiple enzymes. This review spotlights recent progress in developing versatile biocatalytic cascade systems by multi-enzyme co-immobilization on environmentally friendly biopolymers and nanostructured materials and their application scope in the chemical and biotechnological industries. After a succinct overview of carrier-based and carrier-free immobilization/co-immobilizations, co-immobilization of enzymes on a range of biopolymer and nanomaterials-based supports is thoroughly compiled with contemporary and state-of-the-art examples. This study provides a new horizon in developing effective and innovative multi-enzymatic systems with new possibilities to fully harness the adventure of biocatalytic systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri ( Lk CPR). Experimental results revealed that Lk CPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6-9) and temperatures (40-60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol -1 K -1 . The K M values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, and the k cat values were 206.79 s -1 and 202.93 s -1 , respectively. Lk CPR displayed a specific activity versus cytochrome C of 402.07 µmol min -1 mg 1 , the highest activity value described for a CPR up to date (3.2-4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, Lk CPR offers great potential to be used as a suitable biocatalyst.

Enzyme-mediated processes have proven to be a valuable and sustainable alternative to traditional chemical methods. In this regard, the use of multi-enzymatic systems enables the realization of complex synthetic schemes, while also introducing a number of additional advantages, including the conversion of reversible reactions into irreversible processes, the partial or complete elimination of product inhibition problems, and the minimization of undesirable by-products. In addition, the immobilization of biocatalysts on magnetic supports allows for easy reusability and streamlines the downstream process. Herein we have developed a cascade system for cladribine synthesis based on the sequential action of two magnetic biocatalysts. For that purpose, purine 2'-deoxyribosyltransferase from Leishmania mexicana ( Lm PDT) and Escherichia coli hypoxanthine phosphoribosyltransferase ( Ec HPRT) were immobilized onto Ni 2+ -prechelated magnetic microspheres (MagReSyn ® NTA). Among the resulting derivatives, M Lm PDT3 (activity: 11,935 IU/g support , 63% retained activity, operational conditions: 40 °C and pH 5-7) and M Ec HPRT3 (12,840 IU/g support , 45% retained activity, operational conditions: pH 5-8 and 40-60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the M Lm PDT3/M Ec HPRT3 system was biochemically characterized and successfully applied to the one-pot synthesis of cladribine under various conditions. This methodology not only displayed a 1.67-fold improvement in cladribine synthesis (compared to M Lm PDT3), but it also implied a practically complete transformation of the undesired by-product into a high-added-value product (90% conversion of Hyp into IMP). Finally, M Lm PDT3/M Ec HPRT3 was reused for 16 cycles, which displayed a 75% retained activity.

An anticipatory stress response develops before an internal or external stimulus, which initiates a homeostasis process through a chain of responses that enable human organisms to face different threats, thus allowing them to adapt to a continuous and eliciting environment. In the current research, we analyzed the psychophysiological anticipatory anxiety response of professional soldiers prior to a real mission in an actual theater of operation. Autonomic modulation through the heart rate variability values, muscular strength manifestation, and psychological stress of 53 military personnel of Army Airmobile Forces (age: M = 35.4 years, SD = 5.88 years; height: M = 1.75 m, SD = 6.87 cm; body mass: M = 77.33 kg, SD = 11.95 kg; military duty = 14.44 years, SD = 6.43; military operation experience = 4 months, SD = 4.25 months) and a control group of 33 civil participants were analyzed. The military personnel presented significant differences in some HRV values related to the activation of sympathetic systems. We found that the military personnel presented an anticipatory anxiety response only at an autonomic level, showing an increased sympathetic modulation, but not at a psychological level, since their anxiety levels were not significantly different than those of the control civilians. In addition, this anticipatory anxiety response did not affect muscular strength manifestation, as it presented no significant differences between the military personnel and the control group., Competing Interests: The authors declare no conflicts of interest.

One of the major drawbacks of the industrial implementation of enzymatic processes is the low operational stability of the enzymes under tough industrial conditions. In this respect, the use of thermostable enzymes in the industry is gaining ground during the last decades. Herein, we report a structure-guided approach for the development of novel and thermostable 2′-deoxyribosyltransferases (NDTs) based on the computational design of disulfide bonds on hot spot positions. To this end, a small library of NDT variants from Lactobacillus delbrueckii (LdNDT) with introduced cysteine pairs was created. Among them, LdNDTS104C (100% retained activity) was chosen as the most thermostable variant, displaying a six- and two-fold enhanced long-term stability when stored at 55 °C (t1/255 °C ≈ 24 h) and 60 °C (t1/260 °C ≈ 4 h), respectively. Moreover, the biochemical characterization revealed that LdNDTS104C showed >60% relative activity across a broad range of temperature (30−90 °C) and pH (5−7). Finally, to study the potential application of LdNDTS104C as an industrial catalyst, the enzymatic synthesis of nelarabine was successfully carried out under different substrate conditions (1:1 and 3:1) at different reaction times. Under these experimental conditions, the production of nelarabine was increased up to 2.8-fold (72% conversion) compared with wild-type LdNDT.

Droplet microfluidics enables the ultrahigh-throughput screening of the natural or man-made genetic diversity for industrial enzymes, with reduced reagent consumption and lower costs than conventional robotic alternatives. Here we describe an example of metagenomic screening for nucleoside 2'-deoxyribosyl transferases using FACS as a more widespread and accessible alternative than microfluidic on-chip sorters. This protocol can be easily adapted to directed evolution libraries by replacing the library construction steps and to other enzyme activities, e.g., oxidases, by replacing the proposed coupled assay., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Nucleoside 2'-deoxyribosyltransferases (NDTs) catalyze the cleavage of glycosidic bonds of 2'-deoxynucleosides and the following transfer of the 2'-deoxyribose moiety to acceptor nucleobases. Here, we report the crystal structures and biochemical properties of the first tetrameric NDTs: the type I NDT from the mesophilic bacterium Enterococcus faecalis V583 (EfPDT) and the type II NDT from the bacterium Desulfotalea psychrophila (DpNDT), the first psychrophilic NDT. This novel structural and biochemical data permitted an exhaustive comparative analysis aimed to shed light into the basis of the high global stability of the psychrophilic DpNDT, which has a higher melting temperature than EfPDT (58.5 °C versus 54.4 °C) or other mesophilic NDTs. DpNDT possesses a combination of unusual structural motifs not present neither in EfPDT nor any other NDT that most probably contribute to its global stability, in particular, a large aliphatic isoleucine-leucine-valine (ILV) bundle accompanied by a vicinal disulfide bridge and also an intersubunit disulfide bridge, the first described for an NDT. The functional and structural features of DpNDT do not fit the standard features of psychrophilic enzymes, which lead us to consider the implication of (sub)cellular levels together with the protein level in the adaptation of enzymatic activity to low temperatures., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nowadays, enzymatic synthesis of nucleotides is an efficient and sustainable alternative to chemical methodologies. In this regard, after the biochemical characterization of wild-type and mutant uracil phosphoribosyltransferases from Toxoplasma gondii (TgUPRT, TgUPRT 2, and TgUPRT 3 ), TgUPRT 2 was selected as the optimal candidate (69.5 IU mg -1 , UMP synthesis) for structure-guided immobilization onto Ni 2+ chelate (M Ni UPRT 2 ) and onto glutaraldehyde-activated microparticles (M Gl UPRT 2 ). Among resulting derivatives, M Ni UPRT 2 3 (6127 IU g -1 biocat ; 92% retained activity; 3-5 fold enhanced stability at 50-60 °C) and M Gl UPRT 2 N (3711 IU g -1 biocat ; 27% retained activity; 8-20 fold enhanced stability at 50-60 °C) displayed the best operability. Moreover, the enzymatic synthesis of different pyrimidine NMPs was performed. Finally, the reusability of both derivatives in 5-FUMP synthesis (M Ni UPRT 2 3, 80% retained activity after 7 cycles, 5 min; M Gl UPRT 2 N, 70% retained activity after 10 cycles, 20 min) was carried out at short times., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Nowadays, pharmaceutical industry demands competitive and eco-friendly processes for active pharmaceutical ingredients (APIs) manufacturing. In this context, enzyme and whole-cell mediated processes offer an efficient, sustainable and cost-effective alternative to the traditional multi-step and environmentally-harmful chemical processes. Particularly, 2'-deoxyribosyltransferases (NDTs) have emerged as a novel synthetic alternative, not only to chemical but also to other enzyme-mediated synthetic processes. This review describes recent findings in the development and scaling up of NDTs as industrial biocatalysts, including the most relevant and recent examples of single enzymatic steps, multienzyme cascades, chemo-enzymatic approaches, and engineered biocatalysts. Finally, to reflect the inventive and innovative steps of NDT-mediated bioprocesses, a detailed analysis of recently granted patents, with specific focus on industrial synthesis of nucleoside-based APIs, is hereunder presented., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fenneropenaeus merguiensis (commonly named banana shrimp) is one of the most important farmed crustacean worldwide species for the fisheries and aquaculture industry. Besides its nutritional value, it is a good source of chitinase, an enzyme with excellent biological and catalytic properties for many industrial applications. In the present study, a putative chitinase-encoding cDNA was synthesized from mRNA from F. merguiensis hepatopancreas tissue. Subsequently, the corresponding cDNA was cloned, sequenced and functionally expressed in Escherichia coli, and the recombinant F. merguiensis chitinase (rFmCHI) was purified by His-tag affinity chromatography. The bioinformatics analysis of aminoacid sequence of rFmCHI displayed a cannonical multidomain architecture in chitinases which belongs to glycoside hydrolase family 18 (GH18 chitinase). Biochemical characterization revealed rFmCHI as a monomeric enzyme of molecular weight 52 kDa with maximum activity at 40 °C and pH 6.0 Moreover, the recombinant enzyme is also stable up to 60 °C, and in the pH range 5.0-8.0. Steady-state kinetic studies for colloidal chitin revealed K M , V max and k cat values of 78.18 μM, 0.07261 μM. min -1 and 43.37 s -1 , respectively. Overall, our results aim to demonstrate the potential of rFmCHI as suitable catalyst for bioconversion of chitin waste., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2'-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT ( Ld NDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiG PEI 25000 - Ld NDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5-9) and temperature (30-60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiG PEI 25000 - Ld NDT-Alg was successfully employed in the green production of cladribine at mg scale.

The actual COVID-19 pandemic scenario has generated a context of uncertainty, helplessness, and inequality. Yet, the perception of COVID-19 risk has influenced nutritional, psychological, and physical activity patterns depending on gender. We conducted the present research with the aim of studying gender differences of university students in the perceived risk of the COVID-19 pandemic, and in psychological, nutritional, oral health, and physical activity habits. To reach the study's aim, 300 volunteer university students completed an online questionnaire which analyzed variables of perceived risk of the COVID-19 pandemic, psychological profiles, and nutritional, oral health, and physical activity habits. Results showed that females presented a higher perception of danger to the COVID-19 virus than males but showed no differences in how the pandemic has affected personal lives. Females showed higher values of anxiety, conscientiousness, neuroticism, and openness to experience, while males presented higher values of extraversion. Nutritionally, males presented greater consumption of soft drinks, meat, and pasta or rice, and lower buccal hygiene. Yet, no differences were found regarding physical activity patterns. Results from the present study could be used by various educational institutions to implement multidisciplinary interventions to reduce the stress and risk perception.

The use of magnetic biocatalysts is highly beneficial in bioprocesses technology, as it allows their easy recovering and enhances biocatalyst lifetime. Thus, it simplifies operational processing and increases efficiency, leading to more cost-effective processes. The use of small-size matrices as carriers for enzyme immobilization enables to maximize surface area and catalysts loading, also reducing diffusion limitations. As highly expensive nanoparticles (nm size) usually aggregate, their application at large scale is not recommended. In contrast, the use of magnetic micro-macro (µm-mm size) matrices leads to more homogeneous biocatalysts with null or very low aggregation, which facilitates an easy handling and recovery. The present review aims to highlight recent trends in the application of medium-to-high size magnetic biocatalysts in different areas (biodiesel production, food and pharma industries, protein purification or removal of environmental contaminants). The advantages and disadvantages of these above-mentioned magnetic biocatalysts in bioprocess technology will be also discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Herein we report the first proof for the application of type II 2'-deoxyribosyltransferase from Lactobacillus delbrueckii ( Ld NDT) in suicide gene therapy for cancer treatment. To this end, we first confirm the hydrolytic ability of Ld NDT over the nucleoside-based prodrugs 2'-deoxy-5-fluorouridine (dFUrd), 2'-deoxy-2-fluoroadenosine (dFAdo), and 2'-deoxy-6-methylpurine riboside (d6MetPRib). Such activity was significantly increased (up to 30-fold) in the presence of an acceptor nucleobase. To shed light on the strong nucleobase dependence for enzymatic activity, different molecular dynamics simulations were carried out. Finally, as a proof of concept, we tested the Ld NDT/dFAdo system in human cervical cancer (HeLa) cells. Interestingly, Ld NDT/dFAdo showed a pronounced reduction in cellular viability with inhibitory concentrations in the low micromolar range. These results open up future opportunities for the clinical implementation of nucleoside 2'-deoxyribosyltransferases (NDTs) in cancer treatment.

Profiling RNA expression in a cell-specific manner continues to be a grand challenge in biochemical research. Bioorthogonal nucleosides can be utilized to track RNA expression; however, these methods currently have limitations due to background and incorporation of analogs into undesired cells. Herein, we design and demonstrate that uracil phosphoribosyltransferase can be engineered to match 5-vinyluracil for cell-specific metabolic labeling of RNA with exceptional specificity and stringency.

With the aim to explore cultural differences in stress-related psychological, nutrition, physical activity, and oral health factors between Spanish and Latin American professors, we analysed stress-related factors in 598 professors (39.9% male, 60.1% female, 41.3 ± 9.8 years) by a collection of questionnaires, which involved psychological, nutritional, physical activity and oral health items. Results showed how Spanish professors presented significantly ( p ≤ 0.05) higher scores than Latin American professors in perceived stress (Spanish: 21.40 ± 4.32 vs. Latin American: 20.36 ± 4.31), teaching stress (Spanish: 6.59 ± 2.28 vs. Latin American: 6.00 ± 2.99) and neuroticism (Spanish: 5.40 ± 2.10 vs. Latin American: 4.58 ± 1.72). Spanish professors also showed healthier nutritional and physical activity habits than their Latin American counterparts, presenting higher consumption of milk products and a higher numbers of meals per day, greater weekly meat and fish consumption and higher weekly resistance training, as well as less eating between hours and snacking consumption. Nevertheless, Spanish professors brushed their teeth less and showed a higher smoking habit than Latin American professors. We concluded that there were cultural differences between Spanish and Latin American professors. In the present research, Spanish professors showed significantly higher burnout levels, teaching stress, perceived stress, and neuroticism than Latin American professors, and several differences were also found around health behaviours. These differences in perceived stress, teaching stress and burnout syndrome may be due to the habituation process of Latin American professors, and probably are associated with a higher stressful and demanding socio-cultural context.

In this work, a mono- and a bi-enzymatic analytical immobilized enzyme reactors (IMERs) were developed as prototypes for biosynthetic purposes and their performances in the in-flow synthesis of nucleoside analogues of pharmaceutical interest were evaluated. Two biocatalytic routes based on nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) and uridine phosphorylase from Clostridium perfrigens (CpUP)/purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were investigated in the synthesis of 2'-deoxy, 2',3'-dideoxy and arabinonucleoside derivatives. LrNDT-IMER catalyzed the synthesis of 5-fluoro-2'-deoxyuridine and 5-iodo-2'-deoxyuridine in 65-59% conversion yield, while CpUP/AhPNP-IMER provided the best results for the preparation of arabinosyladenine (60% conversion yield). Both IMERs proved to be promising alternatives to chemical routes for the synthesis of nucleoside analogues. The developed in-flow system represents a powerful tool for the fast production on analytical scale of nucleosides for preliminary biological tests., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from Zobellia galactanivorans ( Zg HGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C. Thermal denaturation experiments revealed that Zg HGPRT/AMPK exhibits an apparent unfolding temperature ( Tm ) of 45°C and a retained activity of around 80% when incubated at 40°C for 240 min. This bifunctional enzyme shows a dependence on divalent cations, with a remarkable preference for Mg 2+ and Co 2+ as cofactors. More interestingly, substrate specificity studies revealed Zg HGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of the substrate. Finally, to assess the potential of Zg HGPRT/AMPK as biocatalyst for the synthesis of nucleoside-5'-mono, di- and triphosphates, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of water-miscible solvents on enzyme activity were studied., (Copyright © 2020 Acosta, Del Arco, Del Pozo, Herrera-Tapias, Clemente-Suárez, Berenguer, Hidalgo and Fernández-Lucas.)

Nucleoside-2'-deoxyribosyl-transferases (NDTs) catalyze a transglycosylation reaction consisting of the exchange of the 2'-deoxyribose moiety between a purine and/or pyrimidine nucleoside and a purine and/or pyrimidine base. Because NDTs are highly specific for 2'-deoxyribonucleosides they generally display poor activity on modified C2' and C3' nucleosides and this limitation hampers their applicability as biocatalysts for the synthesis of modified nucleosides. We now report the production and purification of a novel NDT from Archaeoglobus veneficus that is endowed with native ribosyltransferase activity and hence it is more properly classified as an N -ribosyltransferase ( Av NRT). Biophysical and biochemical characterization revealed that Av NRT is a homotetramer that displays maximum activity at 80°C and pH 6 and shows remarkably high stability at high temperatures (60-80°C). In addition, the activity of Av NRT was found to increase up to 2-fold in 4 M NaCl aqueous solution and to be retained in the presence of several water-miscible organic solvents. For completeness, and as a proof of concept for possible industrial applications, this thermophilic and halotolerant biocatalyst was successfully employed in the synthesis of different purine ribonucleoside analogs., (Copyright © 2020 Acosta, Del Arco, Pisabarro, Gago and Fernández-Lucas.)

Nowadays enzymatic synthesis of nucleic acid derivatives is gaining momentum over traditional chemical synthetic processes. Biotransformations catalyzed by whole cells or enzymes offer an ecofriendly and efficient alternative to the traditional multistep chemical methods, avoiding the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Herein we report for the first time the covalent immobilization a uracil phosphoribosyltransferase (UPRT). In this sense, UPRT from Thermus thermophilus HB8 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtUPRT). According to the catalyst load experiments, MTtUPRT3 was selected as optimal biocatalyst for further studies. MTtUPRT3 was active and stable in a broad range of temperature (70-100 °C) and in the pH interval 6-8, displaying maximum activity at 100 °C and pH 7 (activity 968 IU/g support , retained activity 100%). In addition, MTtUPRT3 could be reused up to 8 times in the synthesis of uridine-5'-monophosphate (UMP). Finally, MTtUPRT3 was successfully applied in the sustainable synthesis of different 5-modified uridine-5'-monophosphates at short times. Taking into account these results, MTtUPRT3 would emerge as a valuable biocatalyst for the synthesis of nucleoside monophosphates through an efficient and environmentally friendly methodology., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The use of nucleoside 2'-deoxyribosyltransferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2'-deoxyribonucleosides. It is shown herein that a highly versatile purine NDT from Trypanosoma brucei (TbPDT) can also accept ribonucleosides as substrates; this is most likely because of the distinct role played by Asn53 at a position that is usually occupied by Asp in other NDTs. Moreover, this unusual activity was improved about threefold by introducing a single amino acid replacement at position 5, following a structure-guided approach. Biophysical and biochemical characterization revealed that the TbPDT Y5F variant is a homodimer that displays maximum activity at 50 °C and pH 6.5 and shows a remarkably high melting temperature of 69 °C. Substrate specificity studies demonstrate that 6-oxopurine ribonucleosides are the best donors (inosine>guanosine≫adenosine), whereas no significant preferences exist between 6-aminopurines and 6-oxopurines as base acceptors. In contrast, no transferase activity could be detected on xanthine and 7-deazapurines. TbPDT Y5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present work aims to develop a magnetic biocatalyst for customized production of nucleoside analogues using mutant His-tagged purine 2'-deoxyribosyltransferase from Trypanosoma brucei (TbPDT V11S ) immobilized onto Ni 2+ chelate magnetic iron oxide porous microparticles (MTbPDT V11S ). Biochemical characterization revealed MTbPDT V11S 5 as optimal candidate for further studies (10,552 IU g -1 ; retained activity 54% at 50 °C and pH 6.5). Interestingly, MTbPDT V11S 5 displayed the highest activity value described up to date for an immobilized NDT. Moreover, MTbPDT V11S 5 was successfully employed in the one-pot, one-step production of different therapeutic nucleoside analogues, such as cladribine or 2'-deoxy-2-fluoroadenosine, among others. Finally, MTbPDT V11S 5 proved to be stable when stored at 50 °C for 8 h and pH 6.0 and reusable up to 10 times without negligible loss of activity in the enzymatic production of the antitumor prodrug 2'-deoxy-2-fluoroadenosine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Insight into the catalytic mechanism of Lactobacillus leichmannii nucleoside 2'-deoxyribosyltransferase (LlNDT) has been gained by calculating a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape of the reaction within the enzyme active site. Our results support an oxocarbenium species as the reaction intermediate and thus an SN1 reaction mechanism in this family of bacterial enzymes. Our mechanistic proposal is validated by comparing experimental kinetic data on the impact of the single amino acid replacements Tyr7, Glu98 and Met125 with Ala, Asp and Ala/norLeu, respectively, and accounts for the specificity shown by this enzyme on a non-natural substrate. This work broadens our understanding of enzymatic C-N bond cleavage and C-N bond formation.

The study of facial emotional expressions not only gives information about communicative and adaptive processes, but also provides valuable knowledge about some aspects of human behaviour, such as emotions and intentions. Taking into account the proved evidence that deficits in processing emotional information shows difficulties in executive functions and social behavior, the present work aims to compare the perceptual and dimensional similarities between the emotions transmitted by the faces (facial emotions) and those typically associated with two particular emotional contexts (offenders and non-offenders). To evaluate the contextual influence on emotional face categorization, the emotional recognition in 69 male young (35 offenders (17.22 ± 1.5 years) and 34 non-offenders (16.90 ± 1.56 years) from Barranquilla (Colombia) was analyzed. Experimental results displayed significant differences among the two groups, mainly focused in the recognition of anger and disgust faces. In this sense, the analysis of abovementioned data may lead to the development of more specific and cost-effective therapeutic treatments for offender population., (Copyright © 2019. Published by Elsevier Inc.)

The psychophysiological response due to stress of soldiers in actual combat operations have been determined by recent researchers, but there is a lack of knowledge about the most effective training methodologies to prepare these population for these new psychophysiological and tactical requirements. The aim of this study was to analyze the effect of an operative high intensity interval training (HIIT) in the psychophysiological response and shooting performance of professional soldiers. We analyzed 20 soldiers of the Spanish Army which performed an operative HIIT composed by 3 series of 7 repetitions of exercises based on operative military procedures with 30 s of workload and 30 s of resting between repetitions and 5 min resting between series. Blood lactate, rate of perceived exertion and stress, upper and lower limbs, and respiratory muscle strength, skin temperature, blood oxygen saturation, heart rate, cortical arousal, short-term memory and anxiety response were measured before and after the training. After the training rated of perceived exertion and stress, leg strength, heart rate and lactate presented a significant increase and blood oxygen saturation and cortical arousal significantly decreased. An operative HIIT achieves similar psychophysical response than the evaluated in combat simulations in professional soldiers, producing a decrease in cortical arousal and lactate values over the anaerobic threshold., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The present work describes the functional and structural characterization of adenine phosphoribosyltransferase 2 from Thermus thermophilus HB8 (TtAPRT2). The combination of structural and substrate specificity data provided valuable information for immobilization studies. Dimeric TtAPRT2 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles by two different strategies: a) an enzyme immobilization at pH 8.5 to encourage the immobilization process by N-termini (MTtAPRT2A, MTtAPRT2B, MTtAPRT2C) or b) an enzyme immobilization at pH 10.0 to encourage the immobilization process through surface exposed lysine residues (MTtAPRT2D, MTtAPRT2E, MTtAPRT2F). According to catalyst load experiments, MTtAPRT2B (activity: 480 IU g -1 biocatalyst , activity recovery: 52%) and MTtAPRT2F (activity: 507 IU g -1 biocatalyst , activity recovery: 44%) were chosen as optimal derivatives. The biochemical characterization studies demonstrated that immobilization process improved the thermostability of TtAPRT2. Moreover, the potential reusability of MTtAPRT2B and MTtAPRT2F was also tested. Finally, MTtAPRT2F was employed in the synthesis of nucleoside-5'-monophosphate analogues., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Chitinolytic enzymes are capable to catalyze the chitin hydrolysis. Due to their biomedical and biotechnological applications, nowadays chitinolytic enzymes have attracted worldwide attention. Chitinolytic enzymes have provided numerous useful materials in many different industries, such as food, pharmaceutical, cosmetic, or biomedical industry. Marine enzymes are commonly employed in industry because they display better operational properties than animal, plant, or bacterial homologs. In this mini-review, we want to describe marine chitinolytic enzymes as versatile enzymes in different biotechnological fields. In this regard, interesting comments about their biological role, reaction mechanism, production, functional characterization, immobilization, and biotechnological application are shown in this work.

Due to their similarity to natural counterparts, nucleic acid derivatives (nucleobases, nucleosides, and nucleotides, among others) are interesting molecules for pharmaceutical, biomedical, or food industries. For this reason, there is increasing worldwide demand for the development of efficient synthetic processes for these compounds. Chemical synthetic methodologies require numerous protection-deprotection steps and often lead to the presence of undesirable by-products or enantiomeric mixtures. These methods also require harsh operating conditions, such as the use of organic solvents and hazard reagents. Conversely, enzymatic production by whole cells or enzymes improves regio-, stereo-, and enantioselectivity and provides an eco-friendly alternative. Because of their essential role in purine and pyrimidine scavenging, enzymes from purine and pyrimidine salvage pathways are valuable candidates for the synthesis of many different nucleic acid components. In recent years, many different enzymes from these routes, such as nucleoside phosphorylases, nucleoside kinases, 2'-deoxyribosyltransferases, phosphoribosyl transferases, or deaminases, have been successfully employed as biocatalysts in the production of nucleobase, nucleoside, or nucleotide analogs. Due to their great activity and stability at extremely high temperatures, the use of enzymes from thermophiles in industrial biocatalysis is gaining momentum. Thermophilic enzymes not only display unique characteristics such as temperature, chemical, and pH stability but also provide many different advantages from an industrial perspective. This mini-review aims to cover the most representative enzymatic approaches for the synthesis of nucleic acid derivatives. In this regard, we provide detailed comments about enzymes involved in crucial steps of purine and pyrimidine salvage pathways in thermophiles, as well as their biological role, biochemical characterization, active site mechanism, and substrate specificity. In addition, the most interesting synthetic examples reported in the literature are also included.

In our search for thermophilic and acid-tolerant nucleoside 2'-deoxyribosyltransferases (NDTs), we found a good candidate in an enzyme encoded by Chroococcidiopsis thermalis PCC 7203 (CtNDT). Biophysical and biochemical characterization revealed CtNDT as a homotetramer endowed with good activity and stability at both high temperatures (50-100 °C) and a wide range of pH values (from 3 to 7). CtNDT recognizes purine bases and their corresponding 2'-deoxynucleosides but is also proficient using cytosine and 2'-deoxycytidine as substrates. These unusual features preclude the strict classification of CtNDT as either a type I or a type II NDT and further suggest that this simple subdivision may need to be updated in the future. Our findings also hint at a possible link between oligomeric state and NDT's substrate specificity. Interestingly from a practical perspective, CtNDT displays high activity (80-100%) in the presence of several water-miscible co-solvents in a proportion of up to 20% and was successfully employed in the enzymatic production of several therapeutic nucleosides such as didanosine, vidarabine, and cytarabine.

Caffeine is one of the ergogenic substances most used by warfighters in current operation areas, but the effect on the organic response and operational performed is already poor knowledge. This research aimed to study the acute effect of 400 mg of caffeine monohydrate on the psycho-physiological response and rifle marksmanship of warfighters during a close quarter combat simulation. We analysed parameter of heart rate, blood lactate, cortical arousal, state anxiety and marksmanship of 20 Spanish Army veteran warfighters before and after a close quarter combat simulation in a double-blind procedure, also a correlation analysis was conducted between all the study variables. Marksmanship of warfighters did not improve with the caffeine ingestion, but it produced an increase in blood lactate concentration (caffeine: 1.9 ± 0.5 vs. 9.2 ± 1.1 mmol.l -1 ; placebo: 1.8 ± 0.7 vs. 6.9 ± 2.2 mmol.l -1 ), cortical arousal (% of change: caffeine: 2.51; placebo: -1.96) and heart rate (caffeine: 80.0 ± 7.2 vs. 172.9 ± 28.2 bpm; placebo: 79.3 ± 6.4 vs. 154.0 ± 26.8 bpm). In addition, higher heart rate values correlated negatively with marksmanship in close quarter combat. We concluded that caffeine intake did not improve the warfighters rifle marksmanship in close quarter combat possibly due to the increase in the physiological response.

Clemente-Suárez, Vicente Javier, José Juan Robles-Pérez, Ketty Herrera-Mendoza, Beliña Herrera-Tapias, and Jesús Fernández-Lucas. Psychophysiological response and fine motor skills in high-altitude parachute jumps. High Alt Med Biol 18:392-399, 2017.-We analyzed the psychophysiological response and specific fine motor skill of an experienced jumper in HALO (high altitude low opening) and HAHO (high altitude high opening) parachute jumps. Eight HALO and eight HAHO jumpers were analyzed. They jumped at 5500 m, HALO jumpers opened the parachute at 500 m and HAHO jumpers at 4300 m of altitude. Before and after the jumps, parameters of muscle strength, cortical arousal, blood creatine kinase (CK) and glucose, blood oxygen saturation, rate of perceived exertion (RPE), and specific fine motor skills of an experienced jumper were assessed; during the jump, heart rate (HR), HR variability, and speed were evaluated. HALO and HAHO jumps produced a significant increase in CK, lactate, and RPE, and a decrease in glucose. HAHO decreased cortical arousal, presented a higher sympathetic modulation, and a higher HR during the jump than HALO. HALO and HAHO produced an increase in the physiological, sympathetic modulation and muscle destruction, and a decrease in cortical arousal and a higher blood lactate concentration only in the HAHO jump. Also, somatic and cognitive anxiety correlated with higher strength manifestation and muscle destruction. This novel research could be used to improve actual training systems in both civil and military parachute jumpers.

Background: In recent years, enzymatic methods have shown to be an efficient and sustainable alternative for the synthesis of nucleosides and nucleoside-5'-monophosphates (NMPs) to the traditional multistep chemical methods, since chemical glycosylation reactions include several protection-deprotection steps and the use of chemical reagents and organic solvents that are expensive and environmentally harmful., Results: In this mini-review, we want to illustrate the application of phosphoribosyltransferases (PRTs) in enzymatic synthesis of NMPs. In this sense, many different examples about the use of PRTs as biocatalysts, as whole cells or enzymes, are described. In addition, it also includes detailed comments about structure and catalytic mechanism of described PRTs, as well as their possible biological role and therapeutic use, substrate specificity and advances in detection of new enzyme specificities towards different substrates. In addition, several examples about the use of PRTs in mono or multi-enzymatic synthesis of NMP analogues are shown. Finally, a brief discussion about advantages and drawbacks of the use of PRTs as industrial biocatalyst of NMPs has been commented., Conclusion: Despite the great potential of PRTs as biocatalysts for industrial synthesis of NMPs, several drawbacks must be overcome before reaching a suitable industrial application. In this sense, multi-enzyme systems provide an appropriate framework for this purpose. Moreover, future advances in different disciplines as protein engineering, bioinformatics and -omics will help to reach this goal., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly water-soluble purine bases, such as guanine, xanthine, or hypoxanthine, requires alkaline pH and/or high temperatures in order to solubilize the substrate. In this work, we demonstrate that the 2'-deoxyribosyltransferase from Leishmania mexicana (LmPDT) exhibits an unusually high activity and stability under alkaline conditions (pH 8-10) across a broad range of temperatures (30-70 °C) and ionic strengths (0-500 mM NaCl). Conversely, analysis of the crystal structure of LmPDT together with comparisons with hexameric, bacterial homologues revealed the importance of the relationships between the oligomeric state and the active site architecture within this family of enzymes. Moreover, molecular dynamics and docking approaches provided structural insights into the substrate-binding mode. Biochemical characterization of LmPDT identifies the enzyme as a type I NDT (PDT), exhibiting excellent activity, with specific activity values 100- and 4000-fold higher than the ones reported for other PDTs. Interestingly, LmPDT remained stable during 36 h at different pH values at 40 °C. In order to explore the potential of LmPDT as an industrial biocatalyst, enzymatic production of several natural and non-natural therapeutic nucleosides, such as vidarabine (ara A), didanosine (ddI), ddG, or 2'-fluoro-2'-deoxyguanosine, was carried out using poorly water-soluble purines. Noteworthy, this is the first time that the enzymatic synthesis of 2'-fluoro-2'-deoxyguanosine, ara G, and ara H by a 2'-deoxyribosyltransferase is reported.

We aimed to analyse the effect of experience and type of parachute jump on the psychophysiological responses of jumpers. We analysed blood oxygen saturation, heart rate, blood glucose, lactate and creatinkinase, leg strength, isometric hand strength, cortical arousal, specific fine motor skills, self-confidence and cognition, and somatic and state anxiety, before and after four different parachute jumps: a sport parachute jump, a manual tactical parachute jump, tandem pilots, and tandem passengers. Independently of the parachute jump, the psychophysiological responses of experienced paratroopers were not affected by the jumps, except for an increase in anaerobic metabolism. Novice parachute jumpers presented a higher psychophysiological stress response than the experienced jumpers, together with a large anticipatory anxiety response before the jump; however, this decreased after the jump, although the high physiological activation was maintained. This information could be used by civil and military paratroopers' instructors to improve their training programmes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Parachute jump is an extreme activity that elicits an intense stress response that affects jumpers' body systems being able to put them at risk. The present research analysed modifications in blood oxygen saturation (BOS), heart rate (HR), cortisol, glucose, lactate, creatine kinase (CK), muscles strength, cortical arousal, autonomic modulation, pistol magazine reload time (PMRT) and state anxiety before and after an automatic open parachute jump in 38 male Spanish soldiers (25.6 ± 5.9 years). A MANOVA with samples as a fixed factor and Effect Size (ES) were conducted. MANOVA showed (Wilks lambda = .225; F = 5.980; P = .000) a significantly increase in cortisol (6.2 ± 3.2 vs. 8.2 ± 4.3 nmol/l; P = .025; ES = .47), HR (75.0 ± 14.6 vs. 87.4 ± 17.3 bpm; P = .004; ES = .72), lactate (1.8 ± 1.2 vs. 4.4 ± 2.2 mmol · l -1 ; P = .002; ES = 1.18), sympathetic nervous system and leg strength manifestation after the parachute jump. By contrary BOS, PMRT (55.6 ± 27.6 vs. 48.0 ± 16.7 s; P = .021; ES = .46) and somatic anxiety (SA), evaluated by CSAI2R questionnaire, decreased. An automatic parachute jump increased physiological and cortical response and decreased SA of participants. This stress response can affect the jumpers' abilities and allow us to have a better understanding of the organism stress response and to improve training for both military and sport parachute jumps.