Loop dynamics and the evolution of enzyme activity.

In the early 2000s, Tawfik presented his 'New View' on enzyme evolution, highlighting the role of conformational plasticity in expanding the functional diversity of limited repertoires of sequences. This view is gaining increasing traction with increasing evidence of the importance of conformational dynamics in both natural and laboratory evolution of enzymes. The past years have seen several elegant examples of harnessing conformational (particularly loop) dynamics to successfully manipulate protein function. This Review revisits flexible loops as critical participants in regulating enzyme activity. We showcase several systems of particular interest: triosephosphate isomerase barrel proteins, protein tyrosine phosphatases and ß-lactamases, while briefly discussing other systems in which loop dynamics are important for selectivity and turnover. We then discuss the implications for engineering, presenting examples of successful loop manipulation in either improving catalytic efficiency, or changing selectivity completely. Overall, it is becoming clearer that mimicking nature by manipulating the conformational dynamics of key protein loops is a powerful method of tailoring enzyme activity, without needing to target active-site residues.

Effect of 4 weeks of plyometric training in the pre-competitive period on volleyball athletes' performance.

We aimed to evaluate the effect of 4 weeks of plyometric training (PT), performed in the pre-competitive period, on the vertical jump performance of professional volleyball athletes. We recruited 17 professional female volleyball players (age: 19 ± 3 years; weight: 67.2 ± 5.50 kg; height: 1.81 ± 0.22 m; body fat: 14.4 ± 2.12%; squat 1RM test: 75.5 ± 7.82 kg; training time experience: 6.2 ± 3.4 years) to participate in four weeks of training and assessments. They were divided into an experimental group (EG = 9) and a control group (CG = 8). Both groups were submitted to friendly matches, technical, tactical and resistance training (4 weeks/˜9 sessions per week), and internal load monitoring was carried out. The EG performed PT twice a week. At the beginning and end of the four weeks, jump tests were performed. The main findings are: 1) PT when incorporated into the pre-competitive period can induce greater improvements in jumping performance (EG = 28.93 ± 3.24 cm to 31.67 ± 3.39 cm; CG = 27.91 ± 4.64 cm to 28.97 ± 4.58 cm; when comparing the percentage delta, we found a difference between groups with ES of 1.04 and P = 0.02); 2) this result is observed when the training load is similar between groups and increases over the weeks, respecting the linear progression principle. Therefore, including plyometric training in the preparatory period for volleyball, with low monotony and training strain increment, is an effective strategy for further CMJ performance improvement.

Q-RepEx: A Python pipeline to increase the sampling of empirical valence bond simulations.

The exploration of chemical systems occurs on complex energy landscapes. Comprehensively sampling rugged energy landscapes with many local minima is a common problem for molecular dynamics simulations. These multiple local minima trap the dynamic system, preventing efficient sampling. This is a particular challenge for large biochemical systems with many degrees of freedom. Replica exchange molecular dynamics (REMD) is an approach that accelerates the exploration of the conformational space of a system, and thus can be used to enhance the sampling of complex biomolecular processes. In parallel, the empirical valence bond (EVB) approach is a powerful approach for modeling chemical reactivity in biomolecular systems. Here, we present an open-source Python-based tool that interfaces with the Q simulation package, and increases the sampling efficiency of the EVB free energy perturbation/umbrella sampling approach by means of REMD. This approach, Q-RepEx, both decreases the computational cost of the associated REMD-EVB simulations, and opens the door to more efficient studies of biochemical reactivity in systems with significant conformational fluctuations along the chemical reaction coordinate.

LoopGrafter: a web tool for transplanting dynamical loops for protein engineering.

The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.

Exploiting enzyme evolution for computational protein design.

Recent years have seen an explosion of interest in understanding the physicochemical parameters that shape enzyme evolution, as well as substantial advances in computational enzyme design. This review discusses three areas where evolutionary information can be used as part of the design process: (i) using ancestral sequence reconstruction (ASR) to generate new starting points for enzyme design efforts; (ii) learning from how nature uses conformational dynamics in enzyme evolution to mimic this process in silico; and (iii) modular design of enzymes from smaller fragments, again mimicking the process by which nature appears to create new protein folds. Using showcase examples, we highlight the importance of incorporating evolutionary information to continue to push forward the boundaries of enzyme design studies.

Virtual screening of potential anticancer drugs based on microbial products.

The development of microbial products for cancer treatment has been in the spotlight in recent years. In order to accelerate the lengthy and expensive drug development process, in silico screening tools are systematically employed, especially during the initial discovery phase. Moreover, considering the steadily increasing number of molecules approved by authorities for commercial use, there is a demand for faster methods to repurpose such drugs. Here we present a review on virtual screening web tools, such as publicly available databases of molecular targets and libraries of ligands, with the aim to facilitate the discovery of potential anticancer drugs based on microbial products. We provide an entry-level step-by-step description of the workflow for virtual screening of microbial metabolites with known protein targets, as well as two practical examples using freely available web tools. The first case presents a virtual screening study of drugs developed from microbial products using Caver Web, a web tool that performs docking along a tunnel. The second case comprises a comparative analysis between a wild type isocitrate dehydrogenase 1 and a mutant that results in cancer, using the recently developed web tool PredictSNPOnco. In summary, this review provides the basic and essential background information necessary for virtual screening experiments, which may accelerate the discovery of novel anticancer drugs.

LoopGrafter: Visual Support for the Grafting Workflow of Protein Loops.

In the process of understanding and redesigning the function of proteins in modern biochemistry, protein engineers are increasingly focusing on the exploration of regions in proteins called loops. Analyzing various characteristics of these regions helps the experts to design the transfer of the desired function from one protein to another. This process is denoted as loop grafting. As this process requires extensive manual treatment and currently there is no proper visual support for it, we designed LoopGrafter: a web-based tool that provides experts with visual support through all the loop grafting pipeline steps. The tool is logically divided into several phases, starting with the definition of two input proteins and ending with a set of grafted proteins. Each phase is supported by a specific set of abstracted 2D visual representations of loaded proteins and their loops that are interactively linked with the 3D view onto proteins. By sequentially passing through the individual phases, the user is shaping the list of loops that are potential candidates for loop grafting. In the end, the actual in-silico insertion of the loop candidates from one protein to the other is performed and the results are visually presented to the user. In this way, the fully computational rational design of proteins and their loops results in newly designed protein structures that can be further assembled and tested through in-vitro experiments. LoopGrafter was designed in tight collaboration with protein engineers, and its final appearance reflects many testing iterations. We showcase the contribution of LoopGrafter on a real case scenario and provide the readers with the experts' feedback, confirming the usefulness of our tool.

Performance and carcass quality traits of Iberian × Duroc crossbred pig subject to gender and age at the beginning of the free-range finishing phase.

The traditional production of the Iberian breed pig involves a long production cycle. It might be shortened by using Iberian pigs crossed with Duroc and by reducing the growing phase, but the age-related changes on productive performance and carcass quality should be addressed. Thus, productive performance, live measurements and carcass and primal cut traits were evaluated on Iberian × Duroc 50:50 crossed pigs according to animal age at the beginning of the free-range finishing phase (Montanera): 10, 12 and 14 months old (IBxD10 (n = 15), IBxD12 (n = 17) and IBxD14 (n = 18) animal batches, respectively) and gender (immunologically castrated female -consisted of the Improvac® vaccination- and surgically castrated males). During the growing period, animals were fed with restrictions; 1.49, 1.29 and 1.20 ± 0.023 (mean ± SEM) kg/day of commercial feeds to start Montanera with similar BW; 103.9, 102.9 and 102.1 ± 0.22 kg, for IBxD10, IBxD12 and IBxD14, respectively. IBxD14 animals yielded the highest average daily gain (ADG) and BW after Montanera, as well as larger rump height and croup width. In contrast, these animals had the lowest carcass yield. Although animals from IBxD10 yielded hams of inferior size, this could be of interest to the sector, as there is a certain segment of the market that demands hams of smaller size and, generally, this is difficult to obtain with the traditional Montanera production system. The gender had no major effects on performance and carcass and primal cut traits, so both immunologically castrated female and surgically castrated males are suitable for finishing in Montanera.

Engineering the protein dynamics of an ancestral luciferase.

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

Screening of world approved drugs against highly dynamical spike glycoprotein of SARS-CoV-2 using CaverDock and machine learning.

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes pathological pulmonary symptoms. Most efforts to develop vaccines and drugs against this virus target the spike glycoprotein, particularly its S1 subunit, which is recognised by angiotensin-converting enzyme 2. Here we use the in-house developed tool CaverDock to perform virtual screening against spike glycoprotein using a cryogenic electron microscopy structure (PDB-ID: 6VXX) and the representative structures of five most populated clusters from a previously published molecular dynamics simulation. The dataset of ligands was obtained from the ZINC database and consists of drugs approved for clinical use worldwide. Trajectories for the passage of individual drugs through the tunnel of the spike glycoprotein homotrimer, their binding energies within the tunnel, and the duration of their contacts with the trimer's three subunits were computed for the full dataset. Multivariate statistical methods were then used to establish structure-activity relationships and select top candidate for movement inhibition. This new protocol for the rapid screening of globally approved drugs (4359 ligands) in a multi-state protein structure (6 states) showed high robustness in the rate of finished calculations. The protocol is universal and can be applied to any target protein with an experimental tertiary structure containing protein tunnels or channels. The protocol will be implemented in the next version of CaverWeb (https://loschmidt.chemi.muni.cz/caverweb/) to make it accessible to the wider scientific community.

Computational design of enzymes for biotechnological applications.

Enzymes are the natural catalysts that execute biochemical reactions upholding life. Their natural effectiveness has been fine-tuned as a result of millions of years of natural evolution. Such catalytic effectiveness has prompted the use of biocatalysts from multiple sources on different applications, including the industrial production of goods (food and beverages, detergents, textile, and pharmaceutics), environmental protection, and biomedical applications. Natural enzymes often need to be improved by protein engineering to optimize their function in non-native environments. Recent technological advances have greatly facilitated this process by providing the experimental approaches of directed evolution or by enabling computer-assisted applications. Directed evolution mimics the natural selection process in a highly accelerated fashion at the expense of arduous laboratory work and economic resources. Theoretical methods provide predictions and represent an attractive complement to such experiments by waiving their inherent costs. Computational techniques can be used to engineer enzymatic reactivity, substrate specificity and ligand binding, access pathways and ligand transport, and global properties like protein stability, solubility, and flexibility. Theoretical approaches can also identify hotspots on the protein sequence for mutagenesis and predict suitable alternatives for selected positions with expected outcomes. This review covers the latest advances in computational methods for enzyme engineering and presents many successful case studies.

A scenario-based analysis of the effect of carbon pricing on organic livestock farm performance: A case study of Spanish dehesas and rangelands.

The current livestock farm production model is being questioned due to its excessive use of resources and impacts on the environment, and it has played a major role in climate change due to the excessive level of greenhouse gas (GHG) emissions. A valid tool in the reduction of such emissions is the imposition of a tax on CO2 emissions that can act as an economic and financial instrument. Additionally, livestock production based on grazing animals is proposed as a more sustainable model that involves improved environmental practices and provides society with various ecosystem services, including carbon sequestration. The main purpose of this paper is to estimate the maximum price per tonne of CO2 equivalent (eq) that could be borne by the various models of organic livestock farms in the dehesas and rangelands of southwestern Spain. With this purpose in mind, we have made a scenario-based estimation of the environmental-economic balance in three different scenarios considering farm emissions and CO2 sequestration levels. The results show that the maximum price that farms can bear is within a range of € 0.20 to € 792/tn of CO2 eq depending on the scenario analysed and the production model. In the cases in which carbon sequestration balances GHG emissions, the implementation of carbon pricing implies additional economic income for farm accounts.

Transcription and Translation Inhibitors in Cancer Treatment.

Transcription and translation are fundamental cellular processes that govern the protein production of cells. These processes are generally up regulated in cancer cells, to maintain the enhanced metabolism and proliferative state of these cells. As such cancerous cells can be susceptible to transcription and translation inhibitors. There are numerous druggable proteins involved in transcription and translation which make lucrative targets for cancer drug development. In addition to proteins, recent years have shown that the "undruggable" transcription factors and RNA molecules can also be targeted to hamper the transcription or translation in cancer. In this review, we summarize the properties and function of the transcription and translation inhibitors that have been tested and developed, focusing on the advances of the last 5 years. To complement this, we also discuss some of the recent advances in targeting oncogenes tightly controlling transcription including transcription factors and KRAS. In addition to natural and synthetic compounds, we review DNA and RNA based approaches to develop cancer drugs. Finally, we conclude with the outlook to the future of the development of transcription and translation inhibitors.

The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step.

Transport of ligands between bulk solvent and the buried active sites is a critical event in the catalytic cycle of many enzymes. The rational design of transport pathways is far from trivial due to the lack of knowledge about the effect of mutations on ligand transport. The main and an auxiliary tunnel of haloalkane dehalogenase LinB have been previously engineered for improved dehalogenation of 1,2-dibromoethane (DBE). The first chemical step of DBE conversion was enhanced by L177W mutation in the main tunnel, but the rate-limiting product release was slowed down because the mutation blocked the main access tunnel and hindered protein dynamics. Three additional mutations W140A + F143L + I211L opened-up the auxiliary tunnel and enhanced the product release, making this four-point variant the most efficient catalyst with DBE. Here we study the impact of these mutations on the catalysis of bulky aromatic substrates, 4-(bromomethyl)-6,7-dimethoxycoumarin (COU) and 8-chloromethyl-4,4'-difluoro-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (BDP). The rate-limiting step of DBE conversion is the product release, whereas the catalysis of COU and BDP is limited by the chemical step. The catalysis of COU is mainly impaired by the mutation L177W, whereas the conversion of BDP is affected primarily by the mutations W140A + F143L + I211L. The combined computational and kinetic analyses explain the differences in activities between the enzyme-substrate pairs. The effect of tunnel mutations on catalysis depends on the rate-limiting step, the complementarity of the tunnels with the substrates and is clearly specific for each enzyme-substrate pair.

Fast Screening of Inhibitor Binding/Unbinding Using Novel Software Tool CaverDock.

Protein tunnels and channels are attractive targets for drug design. Drug molecules that block the access of substrates or release of products can be efficient modulators of biological activity. Here, we demonstrate the applicability of a newly developed software tool CaverDock for screening databases of drugs against pharmacologically relevant targets. First, we evaluated the effect of rigid and flexible side chains on sets of substrates and inhibitors of seven different proteins. In order to assess the accuracy of our software, we compared the results obtained from CaverDock calculation with experimental data previously collected with heat shock protein 90α. Finally, we tested the virtual screening capabilities of CaverDock with a set of oncological and anti-inflammatory FDA-approved drugs with two molecular targets-cytochrome P450 17A1 and leukotriene A4 hydrolase/aminopeptidase. Calculation of rigid trajectories using four processors took on average 53 min per molecule with 90% successfully calculated cases. The screening identified functional tunnels based on the profile of potential energies of binding and unbinding trajectories. We concluded that CaverDock is a sufficiently fast, robust, and accurate tool for screening binding/unbinding processes of pharmacologically important targets with buried functional sites. The standalone version of CaverDock is available freely at https://loschmidt.chemi.muni.cz/caverdock/ and the web version at https://loschmidt.chemi.muni.cz/caverweb/.

Correction: SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior.

This article was originally published under standard licence, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the paper have been modified accordingly.

SSRIs target prefrontal to raphe circuits during development modulating synaptic connectivity and emotional behavior.

Antidepressants that block the serotonin transporter, (Slc6a4/SERT), selective serotonin reuptake inhibitors (SSRIs) improve mood in adults but have paradoxical long-term effects when administered during perinatal periods, increasing the risk to develop anxiety and depression. The basis for this developmental effect is not known. Here, we show that during an early postnatal period in mice (P0-P10), Slc6a4/SERT is transiently expressed in a subset of layer 5-6 pyramidal neurons of the prefrontal cortex (PFC). PFC-SERT+ neurons establish glutamatergic synapses with subcortical targets, including the serotonin (5-HT) and GABA neurons of the dorsal raphe nucleus (DRN). PFC-to-DRN circuits develop postnatally, coinciding with the period of PFC Slc6a4/SERT expression. Complete or cortex-specific ablation of SERT increases the number of functional PFC glutamate synapses on both 5-HT and GABA neurons in the DRN. This PFC-to-DRN hyperinnervation is replicated by early-life exposure to the SSRI, fluoxetine (from P2 to P14), that also causes anxiety/depressive-like symptoms. We show that pharmacogenetic manipulation of PFC-SERT+ neuron activity bidirectionally modulates these symptoms, suggesting that PFC hypofunctionality has a causal role in these altered responses to stress. Overall, our data identify specific PFC descending circuits that are targets of antidepressant drugs during development. We demonstrate that developmental expression of SERT in this subset of PFC neurons controls synaptic maturation of PFC-to-DRN circuits, and that remodeling of these circuits in early life modulates behavioral responses to stress in adulthood.

Evaluation of leucocytes from sputum samples of pulmonary tuberculosis patients using flow cytometry.

SETTING: Information about the sputum cells of pulmonary tuberculosis (PTB) patients is scarce. The analysis of sputum cells using optical microscopy (OM) is a well-established method, but it has some serious limitations. OBJECTIVE: To establish a new flow cytometry (FC) protocol for the leucocyte evaluation of sputum samples from PTB patients. DESIGN: A new FC protocol using 0.1% dithiothreitol and 0.5% paraformaldehyde was developed to fluidise sputum samples and kill Mycobacterium tuberculosis, respectively, to allow the analysis of sputum samples collected from TB patients. The protocol was validated by comparing it with OM, and the cellularity of 30 sputum samples from patients with PTB was evaluated. RESULTS: The comparison between leucocyte subsets analysed using OM and FC showed agreement. Immunophenotyping of leucocytes from sputum samples showed that neutrophils (95.7%) comprised the largest proportion of sputum cells, followed by monocytes/macrophages (2.6%) and lymphocytes (1.6%). Among the total T-lymphocytes (100%), 12.3% were T-helper cells, 24.1% were cytotoxic T-cells and 62.9% were gamma/delta T; none of the T lymphocytes had the CD4+/CD8+ phenotype. CONCLUSION: FC is a useful method for evaluating the different subtypes of leucocytes present in the sputum samples of PTB patients.

First nationwide antimicrobial susceptibility surveillance for Neisseria gonorrhoeae in Brazil, 2015-16.

Objectives: Gonorrhoea and antimicrobial resistance (AMR) in Neisseria gonorrhoeae are major public health concerns globally. Enhanced AMR surveillance for gonococci is essential worldwide; however, recent quality-assured gonococcal AMR surveillance in Latin America, including Brazil, has been limited. Our aims were to (i) establish the first nationwide gonococcal AMR surveillance, quality assured according to WHO standards, in Brazil, and (ii) describe the antimicrobial susceptibility of clinical gonococcal isolates collected from 2015 to 2016 in all five main regions (seven sentinel sites) of Brazil. Methods: Gonococcal isolates from 550 men with urethral discharge were examined for susceptibility to ceftriaxone, cefixime, azithromycin, ciprofloxacin, benzylpenicillin and tetracycline using the agar dilution method, according to CLSI recommendations and quality assured according to WHO standards. Results: The levels of resistance (intermediate susceptibility) to tetracycline, ciprofloxacin, benzylpenicillin and azithromycin were 61.6% (34.2%), 55.6% (0.5%), 37.1% (60.4%) and 6.9% (8.9%), respectively. All isolates were susceptible to ceftriaxone and cefixime using the US CLSI breakpoints. However, according to the European EUCAST cefixime breakpoints, 0.2% (n = 1) of isolates were cefixime resistant and 6.9% (n = 38) of isolates had a cefixime MIC bordering on resistance. Conclusions: This study describes the first national surveillance of gonococcal AMR in Brazil, which was quality assured according to WHO standards. The high resistance to ciprofloxacin (which promptly informed a revision of the Brazilian sexually transmitted infection treatment guideline), emerging resistance to azithromycin and decreasing susceptibility to extended-spectrum cephalosporins necessitate continuous surveillance of gonococcal AMR and ideally treatment failures, and increased awareness when prescribing treatment in Brazil.