Current genetic engineering strategies for the production of antihypertensive ACEI peptides.

Hypertension is a major and highly prevalent risk factor for various diseases. Among the most frequently prescribed antihypertensive first-line drugs are synthetic angiotensin I-converting enzyme inhibitors (ACEI). However, since  their use in hypertension therapy has been linked to various side effects, interest in the application of food-derived ACEI peptides (ACEIp) as antihypertensive agents is rapidly growing. Although promising, the industrial production of ACEIp through conventional methods such as chemical synthesis or enzymatic hydrolysis of food proteins has been proven troublesome. We here provide an overview of current antihypertensive therapeutics, focusing on ACEI, and illustrate how biotechnology and bioengineering can overcome the limitations of ACEIp large-scale production. Latest advances in ACEIp research and current genetic engineering-based strategies for heterologous production of ACEIp (and precursors) are also presented. Cloning approaches include tandem repeats of single ACEIp, ACEIp fusion to proteins/polypeptides, joining multivariate ACEIp into bioactive polypeptides, and producing ACEIp-containing modified plant storage proteins. Although bacteria have been privileged ACEIp heterologous hosts, particularly when testing for new genetic engineering strategies, plants and microalgae-based platforms are now emerging. Besides being generally safer, cost-effective and scalable, these "pharming" platforms can perform therelevant posttranslational modifications and produce (and eventually deliver) biologically active protein/peptide-based antihypertensive medicines.

Understanding the Binding Specificity of G-Protein Coupled Receptors toward G-Proteins and Arrestins: Application to the Dopamine Receptor Family.

G-Protein coupled receptors (GPCRs) are involved in a myriad of pathways key for human physiology through the formation of complexes with intracellular partners such as G-proteins and arrestins (Arrs). However, the structural and dynamical determinants of these complexes are still largely unknown. Herein, we developed a computational big-data pipeline that enables the structural characterization of GPCR complexes with no available structure. This pipeline was used to study a well-known group of catecholamine receptors, the human dopamine receptor (DXR) family and its complexes, producing novel insights into the physiological properties of these important drug targets. A detailed description of the protein interfaces of all members of the DXR family (D1R, D2R, D3R, D4R, and D5R) and the corresponding protein interfaces of their binding partners (Arrs: Arr2 and Arr3; G-proteins: Gi1, Gi2, Gi3, Go, Gob, Gq, Gslo, Gssh, Gt2, and Gz) was generated. To produce reliable structures of the DXR family in complex with either G-proteins or Arrs, we performed homology modeling using as templates the structures of the ß2-adrenergic receptor (ß2AR) bound to Gs, the rhodopsin bound to Gi, and the recently acquired neurotensin receptor-1 (NTSR1) and muscarinic 2 receptor (M2R) bound to arrestin (Arr). Among others, the work demonstrated that the three partner groups, Arrs and Gs- and Gi-proteins, are all structurally and dynamically distinct. Additionally, it was revealed the involvement of different structural motifs in G-protein selective coupling between D1- and D2-like receptors. Having constructed and analyzed 50 models involving DXR, this work represents an unprecedented large-scale analysis of GPCR-intracellular partner interface determinants. All data is available at www.moreiralab.com/resources/dxr.

A Complete Assessment of Dopamine Receptor- Ligand Interactions through Computational Methods.

Background: Selectively targeting dopamine receptors (DRs) has been a persistent challenge in the last years for the development of new treatments to combat the large variety of diseases involving these receptors. Although, several drugs have been successfully brought to market, the subtype-specific binding mode on a molecular basis has not been fully elucidated. Methods: Homology modeling and molecular dynamics were applied to construct robust conformational models of all dopamine receptor subtypes (D1-like and D2-like). Fifteen structurally diverse ligands were docked. Contacts at the binding pocket were fully described in order to reveal new structural findings responsible for selective binding to DR subtypes. Results: Residues of the aromatic microdomain were shown to be responsible for the majority of ligand interactions established to all DRs. Hydrophobic contacts involved a huge network of conserved and non-conserved residues between three transmembrane domains (TMs), TM2-TM3-TM7. Hydrogen bonds were mostly mediated by the serine microdomain. TM1 and TM2 residues were main contributors for the coupling of large ligands. Some amino acid groups form electrostatic interactions of particular importance for D1R-like selective ligands binding. Conclusions: This in silico approach was successful in showing known receptor-ligand interactions as well as in determining unique combinations of interactions, which will support mutagenesis studies to improve the design of subtype-specific ligands.

Sidestream superoxygenation for wastewater treatment: Oxygen transfer in clean water and mixed liquor.

The performance of a pilot-scale superoxygenation system was evaluated in clean water and mixed liquor. A mass balance was applied over the pilot-scale system to determine the overall oxygen mass transfer rate coefficient (KLa, h-1), the standard oxygen transfer rate (SOTR, kg O2 d-1), and the standard oxygen transfer efficiency (SOTE, %). Additionally, the alpha factor (α) was determined at a mixed liquor suspend solids (MLSS) concentration of approximately 5 g L-1. SOTEs of nearly 100% were obtained in clean water and mixed liquor. The results showed that at higher oxygen flowrates, higher transfer rates could be achieved; this however, at expenses of the transfer efficiency. As expected, lower transfer efficiencies were observed in mixed liquor compared to clean water. Alpha factors varied between 0.6 and 1.0. However, values of approximately 1.0 can be obtained in all cases by fine tuning the oxygen flowrate delivered to the system.

Persistent Left Superior Vena Cava: Rare Location of Hemodialysis Catheter.

When the left cardinal vein fails to involute during fetal life, a persistent left superior vena cava (PLSVC) develops. PLSVC is a rare vascular anomaly, and the reported incidence is 0.3-0.5% in healthy individuals. It is usually asymptomatic and does not cause hemodynamic disturbances unless associated with cardiac malformations. If the PLSVC drains adequately into the right atrium and there are no cardiac abnormalities, catheterization of this vessel, including temporary and cuffed HD catheter insertion, is deemed safe. We present the case of a 70-year-old woman with acute kidney injury (AKI), in which the necessity to place an HD central venous catheter (CVC) through the left internal jugular vein led to the discovery of a PLSVC. Once it was shown that the vessel was adequately draining into the right atrium, this catheter was changed to a cuffed tunneled HD catheter, which was successfully utilized for HD sessions for three months and removed after the recuperation of renal function without complications.

MUG: A mutation overview of GPCR subfamily A17 receptors.

G protein-coupled receptors (GPCRs) mediate several signaling pathways through a general mechanism that involves their activation, upholding a chain of events that lead to the release of molecules responsible for cytoplasmic action and further regulation. These physiological functions can be severely altered by mutations in GPCR genes. GPCRs subfamily A17 (dopamine, serotonin, adrenergic and trace amine receptors) are directly related with neurodegenerative diseases, and as such it is crucial to explore known mutations on these systems and their impact in structure and function. A comprehensive and detailed computational framework - MUG (Mutations Understanding GPCRs) - was constructed, illustrating key reported mutations and their effect on receptors of the subfamily A17 of GPCRs. We explored the type of mutations occurring overall and in the different families of subfamily A17, as well their localization within the receptor and potential effects on receptor functionality. The mutated residues were further analyzed considering their pathogenicity. The results reveal a high diversity of mutations in the GPCR subfamily A17 structures, drawing attention to the considerable number of mutations in conserved residues and domains. Mutated residues were typically hydrophobic residues enriched at the ligand binding pocket and known activating microdomains, which may lead to disruption of receptor function. MUG as an interactive web application is available for the management and visualization of this dataset. We expect that this interactive database helps the exploration of GPCR mutations, their influence, and their familywise and receptor-specific effects, constituting the first step in elucidating their structures and molecules at the atomic level.

The World of GPCR dimers - Mapping dopamine receptor D2 homodimers in different activation states and configuration arrangements.

G protein-coupled receptors (GPCRs) are known to dimerize, but the molecular and structural basis of GPCR dimers is not well understood. In this study, we developed a computational framework to generate models of symmetric and asymmetric GPCR dimers using different monomer activation states and identified their most likely interfaces with molecular details. We chose the dopamine receptor D2 (D2R) homodimer as a case study because of its biological relevance and the availability of structural information. Our results showed that transmembrane domains 4 and 5 (TM4 and TM5) are mostly found at the dimer interface of the D2R dimer and that these interfaces have a subset of key residues that are mostly nonpolar from TM4 and TM5, which was in line with experimental studies. In addition, TM2 and TM3 appear to be relevant for D2R dimers. In some cases, the inactive configuration is unaffected by the partnered protomer, whereas in others, the active protomer adopts the properties of an inactive receptor. Additionally, the ß-arrestin configuration displayed the properties of an active receptor in the absence of an agonist, suggesting that a switch to another meta-state during dimerization occurred. Our findings are consistent with the experimental data, and this method can be adapted to study heterodimers and potentially extended to include additional proteins such as G proteins or ß-arrestins. In summary, this approach provides insight into the impact of the conformational status of partnered protomers on the overall quaternary GPCR macromolecular structure and dynamics.

Proteomic Tools for the Analysis of Cytoskeleton Proteins.

Proteomic analyses have become an essential part of the toolkit of the molecular biologist, given the widespread availability of genomic data and open source or freely accessible bioinformatics software. Tools are available for detecting homologous sequences, recognizing functional domains, and modeling the three-dimensional structure for any given protein sequence, as well as for predicting interactions with other proteins or macromolecules. Although a wealth of structural and functional information is available for many cytoskeletal proteins, with representatives spanning all of the major subfamilies, the majority of cytoskeletal proteins remain partially or totally uncharacterized. Moreover, bioinformatics tools provide a means for studying the effects of synthetic mutations or naturally occurring variants of these cytoskeletal proteins. This chapter discusses various freely available proteomic analysis tools, with a focus on in silico prediction of protein structure and function. The selected tools are notable for providing an easily accessible interface for the novice while retaining advanced functionality for more experienced computational biologists.

Correcting optic capture with 2 flanged 6-0 sutures after intrascleral haptic fixation with ViscoNeedling.

A 6-0 polypropylene suture was introduced in the eye through an ophthalmic viscosurgical device syringe in a completely closed system. The procedure was used to correct optic capture of the intraocular lens by the pupil after 2 cases of Yamane technique.

Decoding Partner Specificity of Opioid Receptor Family.

This paper describes an exciting big data analysis compiled in a freely available database, which can be applied to characterize the coupling of different G-Protein coupled receptors (GPCRs) families with their intracellular partners. Opioid receptor (OR) family was used as case study in order to gain further insights into the physiological properties of these important drug targets, known to be associated with the opioid crisis, a huge socio-economic issue directly related to drug abuse. An extensive characterization of all members of the ORs family (µ (MOR), δ (DOR), κ (KOR), nociceptin (NOP)) and their corresponding binding partners (ARRs: Arr2, Arr3; G-protein: Gi1, Gi2, Gi3, Go, Gob, Gz, Gq, G11, G14, G15, G12, Gssh, Gslo) was carried out. A multi-step approach including models' construction (multiple sequence alignment, homology modeling), complex assembling (protein complex refinement with HADDOCK and complex equilibration), and protein-protein interface characterization (including both structural and dynamics analysis) were performed. Our database can be easily applied to several GPCR sub-families, to determine the key structural and dynamical determinants involved in GPCR coupling selectivity.

In Silico End-to-End Protein-Ligand Interaction Characterization Pipeline: The Case of SARS-CoV-2.

SARS-CoV-2 triggered a worldwide pandemic disease, COVID-19, for which an effective treatment has not yet been settled. Among the most promising targets to fight this disease is SARS-CoV-2 main protease (Mpro), which has been extensively studied in the last few months. There is an urgency for developing effective computational protocols that can help us tackle these key viral proteins. Hence, we have put together a robust and thorough pipeline of in silico protein-ligand characterization methods to address one of the biggest biological problems currently plaguing our world. These methodologies were used to characterize the interaction of SARS-CoV-2 Mpro with an α-ketoamide inhibitor and include details on how to upload, visualize, and manage the three-dimensional structure of the complex and acquire high-quality figures for scientific publications using PyMOL (Protocol 1); perform homology modeling with MODELLER (Protocol 2); perform protein-ligand docking calculations using HADDOCK (Protocol 3); run a virtual screening protocol of a small compound database of SARS-CoV-2 candidate inhibitors with AutoDock 4 and AutoDock Vina (Protocol 4); and, finally, sample the conformational space at the atomic level between SARS-CoV-2 Mpro and the α-ketoamide inhibitor with Molecular Dynamics simulations using GROMACS (Protocol 5). Guidelines for careful data analysis and interpretation are also provided for each Protocol.

Potential Distribution of Nysius simulans (Hemiptera: Lygaeidae) in Soybean Crops in South America Under Current and Future Climate.

Nysius simulans (Stål) is a suctorial, fluid feeding herbivore that can transmit toxins and spread pathogens via saliva and is an economically important pest for soybean in South America. Currently, N. simulans in soybean is predominantly found in Argentina, but future changes in the distribution from both dispersal and range shifts due to climate change may affect soybean cultivation in southern South America. We developed a species distribution model to examine the distribution range of N. simulans. We compared the potential distribution of N. simulans under current and future projected climatic conditions in order to identify future areas of natural occurrence with ecological niche models using Maxent. Current records of N. simulans show that while the species is present in Argentina, and some areas of Brazil, Paraguay, Peru, and Uruguay, our models suggest that many new suitable areas will be available for N. simulans under climate change including other regions of Argentina, and southern Chile. Our results also predict potential future range shifts and distributions into Bolivia, but not Peru nor Brazil. In our model, seasonal trends in temperature were shown to have the greatest contribution to the potential distribution, whereas isothermality (i.e., temperature variability) was correlated to potential future distribution ranges. We conclude that current populations of N. simulans may be expanding its distribution range by diffusion (i.e., range expansion over generations at the margins of populations), and regions with potential future N. simulans distribution should be closely monitored.

Hemodialysis Catheter-Related Thrombi: A Challenging Patient.

Patients requiring renal replacement therapy remain a significant burden on the healthcare system. A substantial amount of hospitalization costs for these patients are related to vascular complications, especially catheter-related thrombosis, which is associated with a significant increase in morbidity and mortality. We report the case of a male patient with multiple myeloma (MM) and dialysis-dependent renal failure due to light-chain cast nephropathy, who presented recurrent early catheter dysfunction. A large thrombus was detected, extending from the superior vena cava (SVC) to the right atrium (RA) and later at the inferior vena cava (IVC), ultimately leading to exhaustion of vascular capital. To this date, there is limited evidence regarding the best approach to catheter-related thrombosis, which frequently leads to treatment dilemmas in clinical practice and demands careful evaluation and individualized decisions. In patients with a highly thrombotic profile, peritoneal dialysis may be considered earlier to prevent further vascular capital damage.

Prediction and targeting of GPCR oligomer interfaces.

GPCR oligomerization has emerged as a hot topic in the GPCR field in the last years. Receptors that are part of these oligomers can influence each other's function, although it is not yet entirely understood how these interactions work. The existence of such a highly complex network of interactions between GPCRs generates the possibility of alternative targets for new therapeutic approaches. However, challenges still exist in the characterization of these complexes, especially at the interface level. Different experimental approaches, such as FRET or BRET, are usually combined to study GPCR oligomer interactions. Computational methods have been applied as a useful tool for retrieving information from GPCR sequences and the few X-ray-resolved oligomeric structures that are accessible, as well as for predicting new and trustworthy GPCR oligomeric interfaces. Machine-learning (ML) approaches have recently helped with some hindrances of other methods. By joining and evaluating multiple structure-, sequence- and co-evolution-based features on the same algorithm, it is possible to dilute the issues of particular structures and residues that arise from the experimental methodology into all-encompassing algorithms capable of accurately predict GPCR-GPCR interfaces. All these methods used as a single or a combined approach provide useful information about GPCR oligomerization and its role in GPCR function and dynamics. Altogether, we present experimental, computational and machine-learning methods used to study oligomers interfaces, as well as strategies that have been used to target these dynamic complexes.

An Overview of Antiretroviral Agents for Treating HIV Infection in Paediatric Population.

Paediatric Acquired ImmunoDeficiency Syndrome (AIDS) is a life-threatening and infectious disease in which the Human Immunodeficiency Virus (HIV) is mainly transmitted through Mother-To- Child Transmission (MTCT) during pregnancy, labour and delivery, or breastfeeding. This review provides an overview of the distinct therapeutic alternatives to abolish the systemic viral replication in paediatric HIV-1 infection. Numerous classes of antiretroviral agents have emerged as therapeutic tools for downregulation of different steps in the HIV replication process. These classes encompass Non- Nucleoside Analogue Reverse Transcriptase Inhibitors (NNRTIs), Nucleoside/Nucleotide Analogue Reverse Transcriptase Inhibitors (NRTIs/NtRTIs), INtegrase Inhibitors (INIs), Protease Inhibitors (PIs), and Entry Inhibitors (EIs). Co-administration of certain antiretroviral drugs with Pharmacokinetic Enhancers (PEs) may boost the effectiveness of the primary therapeutic agent. The combination of multiple antiretroviral drug regimens (Highly Active AntiRetroviral Therapy - HAART) is currently the standard therapeutic approach for HIV infection. So far, the use of HAART offers the best opportunity for prolonged and maximal viral suppression, and preservation of the immune system upon HIV infection. Still, the frequent administration of high doses of multiple drugs, their inefficient ability to reach the viral reservoirs in adequate doses, the development of drug resistance, and the lack of patient compliance compromise the complete HIV elimination. The development of nanotechnology-based drug delivery systems may enable targeted delivery of antiretroviral agents to inaccessible viral reservoir sites at therapeutic concentrations. In addition, the application of Computer-Aided Drug Design (CADD) approaches has provided valuable tools for the development of anti-HIV drug candidates with favourable pharmacodynamics and pharmacokinetic properties.

Prediction and Targeting of Interaction Interfaces in G-protein Coupled Receptor Oligomers.

BACKGROUND: Communication within a protein complex is mediated by physical interactions made among the protomers. Evidence for both the allosteric regulation present among the protomers of the protein oligomer and of the direct effect of membrane composition on this regulation has made it essential to investigate the underlying molecular mechanism that drives oligomerization, the type of interactions present within the complex, and to determine the identity of the interaction interface. This knowledge allows a holistic understanding of dynamics and also modulation of the function of the resulting oligomers/signalling complexes. G-Protein-Coupled Receptors (GPCRs), which are targeted by 40% of currently prescribed drugs in the market, are widely involved in the formation of such physiological oligomers/signalling complexes. SCOPE: This review highlights the importance of studying Protein-Protein Interactions (PPI) by using a combination of data obtained from cutting-edge experimental and computational methods that were developed for this purpose. In particular, we focused on interaction interfaces found at GPCR oligomers as well as signalling complexes, since any problem associated with these interactions causes the onset of various crucial diseases. CONCLUSION: In order to have a holistic mechanistic understanding of allosteric PPIs that drive the formation of GPCR oligomers and also to determine the composition of interaction interfaces with respect to different membrane compositions, it is essential to combine both relevant experimental and computational data. In this way, efficient and specific targeting of these interaction interfaces in oligomers/ complexes can be achieved. Thus, effective therapeutic molecules with fewer side effects can be designed to modulate the function of these physiologically important receptor family.

Natural radionuclides in electronic waste: an initial approach.

Some radionuclides are present in trace amount in the raw material of electronic devices that suffer obsolescence and are improperly disposed of. Obsolete personal computers become electronic waste and may contain potential environmental contaminants. Furthermore, there may be an additional concern: the ionizing radiation emission. This study aimed to assess qualitatively the possibility of alpha and beta particles and gamma rays reaching somehow workers involved in the handling of such wastes. Using gamma spectrometry method, we analyzed a set of 10 microprocessors randomly taken from motherboards incorrectly discarded. The analysis resulted in the occurrence of primordial radioisotopes in the microchips.

Standardization of DNA Residual Quantification Method of Vero Cell Rabies Vaccine for Human Use.

OBJECTIVES: Normalize the quantification of residual DNA from Vero cells in the rabies vaccine for use in human VAHV I, by quantitative PCR in real time and the design of primers that amplified, highly repetitive sequences of Cercopithecus aethiops and a constitutive gene according to sequences reported in the GenBank and quantifying the residual DNA in the vaccine VAHV I in three consecutive batches according to the standard set by the World Health Organization. METHODS: A real time quantitative method based on SYBR Green chemistry has been applied for the quantification of residual DNA (resDNA) using highly repetitive DNA (Alu) and a housekeeping gene (B-actin) as target sequences. RESULTS: The sensitivity achieved with this white sequence is within the reported limits and who are between 5 and 50 pg. For real time PCR optimization with Alu-p53, different concentrations of MgCl2 (0.5, 0.75, 1.0, 1.25 and 1.5 mm) in combination with three different concentrations of primers (75, 100 and 150nM) were used. pDNA in concentration of 1x107 copies / ul was used as template. Optimal concentrations were 1.25 mM MgCl2 and 100nM primers. To level of detection of 1.53 ng/ul was found for p53-Alu and Alu-Glob and 0.39 ng/ul for B-actin with gDNA curves. CONCLUSION: Quantification of resDNA of vaccine VAHV I with close-ups of B-actin was normalized. Reached a sensitivity of 30 pg of resDNA/dose VAHV I, with close-ups of B-actin. Found, in three consecutive batches, an amount less than 10 ng/dose, these results suggest that the production process ensures vaccine resDNA removal, meeting international requirements for biological products for use in humans that use continuous cell lines for production.

Does pre- and post-angioplasty Doppler ultrasound evaluation help in predicting vascular access outcome?

BACKGROUND: Kidney Disease - Improving Global Outcomes (KDIGO) recommends post-percutaneous transluminal angioplasty (PTA) <30% residual stenosis (RS) and hemodynamic parameters improvement. Primary end point: how post-PTA access blood flow (ABF) improvement predicts vascular access (VA) outcome. Secondary: compare Doppler ultrasound (DU) and angiography diagnostic accuracy; determine how other factors predict outcome. METHODS: Eighty patients. DU evaluation performed pre- and post-PTA. Several parameters recorded. Secondary patency verified after 6 months. RESULTS: Initial ABF 537 ± 248 mL/min; final ABF 1013 ± 354 mL/min. Number and location of stenosis was highly correlated between DU and angiography (p<0.001); central vessels mismatching. First semester overall survival was 63%; significantly better for fistulas (76%) than grafts (51.7%), p 0.044. Final RS>30% associated to better survival, p 0.038. Initial ABF<500 mL/min and multiple stenosis did not affect outcome (p>0.05). A >2-fold ABF increase had no significant impact on fistulas (p>0.05) but was significantly associated with worst outcomes in grafts (23.1% vs. 73.5%, p 0.009). Grafts had lower survival (HR 3.3, p 0.034). CONCLUSIONS: Although less accurate for central lesions, DU has a key role on VA surveillance, allowing a morphologic and hemodynamic assessment. Angioplasty is effective in preserving VA; however, it may increase restenosis due to accelerated neointimal hyperplasia. Current parameters are not useful. Trials addressing this issue are needed.