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Valeria Márquez-Miranda,1,2,* Johanna Abrigo,3,4,* Juan Carlos Rivera,3,4 Ingrid Araya-Durán,1 Javier Aravena,3,4 Felipe Simon,3,4 Nicolás Pacheco,1 Fernando Danilo González-Nilo,1,2,5 Claudio Cabello-Verrugio3,4 1Center for Bioinformatics and Integrative Biology (CBIB), Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago, 2Fundación Fraunhofer Chile Research, Las Condes, 3Departamento de Ciencias Biologicas, Facultad de Ciencias Biologicas & Facultad de Medicina, Universidad Andres Bello, 4Millennium Institute on Immunology and Immunotherapy, Santiago, 5Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile *These authors contributed equally to this work Abstract: Angiotensin (1–7) (Ang-(1–7)) is a bioactive heptapeptide with a short half-life and has beneficial effects in several tissues – among them, skeletal muscle – by preventing muscle atrophy. Dendrimers are promising vehicles for the protection and transport of numerous bioactive molecules. This work explored the use of a neutral, non-cytotoxic hydroxyl-terminated poly(amidoamine) (PAMAM-OH) dendrimer as an Ang-(1–7) carrier. Bioinformatics analysis showed that the Ang-(1–7)-binding capacity of the dendrimer presented a 2:1 molar ratio. Molecular dynamics simulation analysis revealed the capacity of neutral PAMAM-OH to protect Ang-(1–7) and form stable complexes. The peptide coverage ability of the dendrimer was between ~50% and 65%. Furthermore, an electrophoretic mobility shift assay demonstrated that neutral PAMAM-OH effectively bonded peptides. Experimental results showed that the Ang-(1–7)/PAMAM-OH complex, but not Ang-(1–7) alone, had an anti-atrophic effect when administered intraperitoneally, as evaluated by muscle strength, fiber diameter, myofibrillar protein levels, and atrogin-1 and MuRF-1 expressions. The results of the Ang-(1–7)/PAMAM-OH complex being intraperitoneally injected were similar to the results obtained when Ang-(1–7) was systemically administered through mini-osmotic pumps. Together, the results suggest that Ang-(1–7) can be protected for PAMAM-OH when this complex is intraperitoneally injected. Therefore, the Ang-(1–7)/PAMAM-OH complex is an efficient delivery method for Ang-(1–7), since it improves the anti-atrophic activity of this peptide in skeletal muscle. Keywords: muscle wasting, peptide delivery, carrier, anti-atrophic peptide

The role of ventral hippocampus (vHipp) astroglial gliotransmission in depression was studied using chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS) rodent models. CRS increased Cx43 hemichannel activity and extracellular glutamate levels in the vHipp and blocking astroglial Cx43 hemichannel-dependent gliotransmission during CRS prevented the development of depression and glutamate buildup. Moreover, the acute blockade of Cx43 hemichannels induced antidepressant effects in rats previously subjected to CRS or CUMS. This antidepressant effect was prevented by coinjection of glutamate and D-serine. Furthermore, Cx43 hemichannel blockade decreased postsynaptic NMDAR currents in vHipp slices in a glutamate and D-serine-dependent manner. Notably, chronic microinfusion of glutamate and D-serine, L-serine, or the NMDAR agonist NMDA, into the vHipp induced depressive-like symptoms in nonstressed rats. We also identified a small molecule, cacotheline, which blocks Cx43 hemichannels and its systemic administration induced rapid antidepressant effects, preventing stress-induced increases in astroglial Cx43 hemichannel activity and extracellular glutamate in the vHipp, without sedative or locomotor side effects. In conclusion, chronic stress increases Cx43 hemichannel-dependent release of glutamate and D-/L-serine from astrocytes in the vHipp, overactivating postsynaptic NMDARs and triggering depressive-like symptoms. This study highlights the critical role of astroglial gliotransmitter release in chronic stress-induced depression and suggests it can be used as a target for the prevention and treatment of depression., Competing Interests: Competing interests statement:M.A.R., D.G.-N., and J.S. authored a Patent application filed in 2013-05-31 (WO 2013/179264), which was granted in the US (US-9879058-B2) on the date 2018/01/30, entitled “Use of compounds that selectively modulate astrocytic release of substances through hemichannels of connexins and pannexins, without influencing gap junctions, for the treatment of psychiatric disorders,” which included cacotheline as an example. All other authors report no conflicts of interest.

Living organisms require detecting the environmental thermal clues for survival, allowing them to avoid noxious stimuli or find prey moving in the dark. In mammals, the Transient Receptor Potential ion channels superfamily is constituted by 27 polymodal receptors whose activity is controlled by small ligands, peptide toxins, protons and voltage. The thermoTRP channels subgroup exhibits unparalleled temperature dependence -behaving as heat and cold sensors. Functional studies have dissected their biophysical features in detail, and the advances of single-particle Cryogenic Electron microscopy provided the structural framework required to propose detailed channel gating mechanisms. However, merging structural and functional evidence for temperature-driven gating of thermoTRP channels has been a hard nut to crack, remaining an open question nowadays. Here we revisit the highlights on the study of heat and cold sensing in thermoTRP channels in the light of the structural data that has emerged during recent years., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Cancer cells have high demands for energy to maintain their exceedingly proliferative growth. However, the mechanism of energy expenditure in cancer is not well understood. We hypothesize that cancer cells might utilize energy-rich inorganic polyphosphate (polyP), as energetic reserve. PolyP is comprised of orthophosphates linked by phosphoanhydride bonds, as in ATP. Here, we show that polyP is highly abundant in several types of cancer cells, including brain tumor-initiating cells (BTICs), i.e., stem-like cells derived from a mouse brain tumor model that we have previously described. The polymer is avidly consumed during starvation of the BTICs. Depletion of ATP by inhibiting glycolysis and mitochondrial ATP-synthase (OXPHOS) further decreases the levels of polyP and alters morphology of the cells. Moreover, enzymatic hydrolysis of the polymer impairs the viability of cancer cells and significantly deprives ATP stores. These results suggest that polyP might be utilized as a source of phosphate energy in cancer. While the role of polyP as an energy source is established for bacteria, this finding is the first demonstration that polyP may play a similar role in the metabolism of cancer cells., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2020 Boyineni et al.)

The incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision in vitro and in vivo . To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, Xenopus oocytes, and HEK-293T cells. Here, we describe the rational generation of a novel orthogonal aminoacyl-tRNA synthetase based on the E. coli tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells., (© 2020 Published by Elsevier Ltd.)

Motivation: Molecular docking is aimed at predicting the conformation of small-molecule (ligands) within an identified binding site (BS) in a target protein (receptor). Protein-ligand docking plays an important role in modern drug discovery and biochemistry for protein engineering. However, efficient docking analysis of proteins requires prior knowledge of the BS, which is not always known. The process which covers BS identification and protein-ligand docking usually requires the combination of different programs, which require several input parameters. This is furtherly aggravated when factoring in computational demands, such as CPU-time. Therefore, these types of simulation experiments can become a complex process for researchers without a background in computer sciences., Results: To overcome these problems, we have designed an automatic computational workflow (WF) to process protein-ligand complexes, which runs from the identification of the possible BSs positions to the prediction of the experimental binding modes and affinities of the ligand. This open-access WF runs under the Galaxy platform that integrates public domain software. The results of the proposed method are in close agreement with state-of-the-art docking software., Availability and Implementation: Software is available at: https://pistacho.ac.uma.es/galaxy-bitlab., Contact: euv@uma.es., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The use of herbicides plays a vital role in controlling weeds and conserving crops; however, its usage generates both environmental and economic problems. For example, herbicides pose a financial issue as farmers must apply large quantities to protect crops due to absorption rates of less than 0.1%. Therefore, there is a great need for the development of new methods to mitigate these issues. Here, we report for the first time the synthesis of poly(lactic- co-glycolic-acid) (PLGA) nanoherbicides loaded with atrazine as an active ingredient. We used potato plants as a biological model to assess the herbicidal activity of the engineered PLGA nanoherbicides. Our method produced nanoherbicides with an average size of 110 ± 10 nm prior to lyophilization. Fifty percent of the loaded atrazine in the PLGA matrix is released in 72 h. Furthermore, we performed Monte Carlo simulations to determine the chemical interaction among atrazine, PLGA, and the solvent system. One of the most significant outcomes of these simulations was to find the formation of a hydrogen bond of 1.9 Å between PLGA and atrazine, which makes this interaction very stable. Our in vitro findings showed that as atrazine concentration is increased in PLGA nanoparticles, potato plants undergo a significant decrease in stem length, root length, fresh weight, dry weight, and the number of leaves, with root length being the most affected. These experimental results suggest the herbicidal effectiveness of atrazine-loaded PLGA nanoherbicides in inhibiting the growth of the potato plant. Hence, we present the proof-of-concept for using PLGA nanoherbicides as an alternative method for inhibiting weed growth. Future studies will involve a deep understanding of the mechanism of plant-nanoherbicide interaction as well as the role of PLGA as a growth potentiator.

Temperature sensing is one of the oldest capabilities of living organisms, and is essential for sustaining life, because failure to avoid extreme noxious temperatures can result in tissue damage or death. A subset of members of the transient receptor potential (TRP) ion channel family is finely tuned to detect temperatures ranging from extreme cold to noxious heat, giving rise to thermoTRP channels. Structural and functional experiments have shown that thermoTRP channels are allosteric proteins, containing different domains that sense changes in temperature, among other stimuli, triggering pore opening. Although temperature-dependence is well characterized in thermoTRP channels, the molecular nature of temperature-sensing elements remains unknown. Importantly, thermoTRP channels are involved in pain sensation, related to pathological conditions. Here, we provide an overview of thermoTRP channel activation. We also discuss the structural and functional evidence supporting the existence of an intrinsic temperature sensor in this class of channels, and we explore the basic thermodynamic principles for channel activation. Finally, we give a view of their role in painful pathophysiological conditions.

Large-conductance Ca 2+ - and voltage-activated K + (BK) channels play many physiological roles ranging from the maintenance of smooth muscle tone to hearing and neurosecretion. BK channels are tetramers in which the pore-forming α subunit is coded by a single gene (Slowpoke, KCNMA1). In this review, we first highlight the physiological importance of this ubiquitous channel, emphasizing the role that BK channels play in different channelopathies. We next discuss the modular nature of BK channel-forming protein, in which the different modules (the voltage sensor and the Ca 2+ binding sites) communicate with the pore gates allosterically. In this regard, we review in detail the allosteric models proposed to explain channel activation and how the models are related to channel structure. Considering their extremely large conductance and unique selectivity to K + , we also offer an account of how these two apparently paradoxical characteristics can be understood consistently in unison, and what we have learned about the conduction system and the activation gates using ions, blockers, and toxins. Attention is paid here to the molecular nature of the voltage sensor and the Ca 2+ binding sites that are located in a gating ring of known crystal structure and constituted by four COOH termini. Despite the fact that BK channels are coded by a single gene, diversity is obtained by means of alternative splicing and modulatory β and γ subunits. We finish this review by describing how the association of the α subunit with β or with γ subunits can change the BK channel phenotype and pharmacology., (Copyright © 2017 the American Physiological Society.)

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Adjuvants used in vaccine aquaculture are frequently harmful for the fish, causing melanosis, granulomas and kidney damage. Along with that, vaccines are mostly administered by injection, causing pain and stress to the fish. We used the DNA coding for the replicase of alphavirus as adjuvant (Ad) of a vaccine against ISAV. The Ad and an inactivated ISAV (V) were loaded in chitosan nanoparticles (NPs) to be administered orally to Atlantic salmon. NP-Ad was able to deliver the DNA ex vivo and in vivo. Oral administration of the NPs stimulated the expression of immune molecules, but did not stimulate the humoral response. Although the vaccination with NP-V results in a modest protection of fish against ISAV, NP-V administered together with NP-Ad caused a protection of 77%. Therefore, the DNA coding for the replicase of alphavirus could be administered orally and can potentiate the immuneprotection of a virine against infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A series of chalcones and aurones were synthesized and evaluated in vitro as monoamine oxidase inhibitors (MAOi). Our results show that aurones, which had not been previously reported as MAOi, are MAO-B inhibitors. Thus, both families inhibited selectively the B isoform of MAO in the micromolar range, offering novel scaffolds for the design of new and potent MAO inhibitors. The main structural requirements for their activity were characterized with the aid of 3D-QSAR and docking studies., (© 2014 John Wiley & Sons A/S.)

Ferritins are ubiquitous iron-storage proteins found in all kingdoms of life. They share a common architecture made of 24 subunits of five α-helices. The recombinant Chlorobium tepidum ferritin (rCtFtn) is a structurally interesting protein since sequence alignments with other ferritins show that this protein has a significantly extended C-terminus, which possesses 12 histidine residues as well as several aspartate and glutamic acid residues that are potential metal ion binding residues. We show that the macromolecular assembly of rCtFtn exhibits a cage-like hollow shell consisting of 24 monomers that are related by 4-3-2 symmetry; similar to the assembly of other ferritins. In all ferritins of known structure the short fifth α-helix adopts an acute angle with respect to the four-helix bundle. However, the crystal structure of the rCtFtn presented here shows that this helix adopts a new conformation defining a new assembly of the 4-fold channel of rCtFtn. This conformation allows the arrangement of the C-terminal region into the inner cavity of the protein shell. Furthermore, two Fe(III) ions were found in each ferroxidase center of rCtFtn, with an average FeA-FeB distance of 3 Å; corresponding to a diferric μ-oxo/hydroxo species. This is the first ferritin crystal structure with an isolated di-iron center in an iron-storage ferritin. The crystal structure of rCtFtn and the biochemical results presented here, suggests that rCtFtn presents similar biochemical properties reported for other members of this protein family albeit with distinct structural plasticity., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na(+) channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H(+)) or sodium (Na(+)) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na(+) and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na(+) recognition domain was conserved in both SxtF/M, indicating that Na(+) can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na(+) incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na(+) in toxin export, as well as the motifs L(398)XGLQD(403) (SxtM) and L(390)VGLRD(395) (SxtF) in toxin recognition.

Nanoinformatics has recently emerged to address the need of computing applications at the nano level. In this regard, the authors have participated in various initiatives to identify its concepts, foundations and challenges. While nanomaterials open up the possibility for developing new devices in many industrial and scientific areas, they also offer breakthrough perspectives for the prevention, diagnosis and treatment of diseases. In this paper, we analyze the different aspects of nanoinformatics and suggest five research topics to help catalyze new research and development in the area, particularly focused on nanomedicine. We also encompass the use of informatics to further the biological and clinical applications of basic research in nanoscience and nanotechnology, and the related concept of an extended "nanotype" to coalesce information related to nanoparticles. We suggest how nanoinformatics could accelerate developments in nanomedicine, similarly to what happened with the Human Genome and other -omics projects, on issues like exchanging modeling and simulation methods and tools, linking toxicity information to clinical and personal databases or developing new approaches for scientific ontologies, among many others.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a classical glycolytic protein that can promote the fusion of phospholipid vesicles and can also play a vital role on in vivo fusogenic events. However, it is not clear how this redox enzyme, which lack conserved structural or sequence motifs related to membrane fusion, catalyze this process. In order to detect if this ability is present in other NAD(P)H dehydrogenases with available structure, spectroscopic studies were performed to evaluate the capability of alcohol dehydrogenase (ADH), glutamic dehydrogenase (GDH) and sorbitol dehydrogenase (SDH) to bind, aggregate, destabilize and fuse vesicles. Based on finite difference Poisson-Boltzmann calculations (FDPB) the protein-membrane interactions were analyzed. A model for the protein-membrane complex in its minimum free energy of interaction was obtained for each protein and the amino acids involved in the binding processes were suggested. A previously undescribed relationship between membrane destabilization and crevices with high electropositive potential on the protein surface was proposed. The putative implication of the non-specific electrostatics on NAD(P)H dehydrogenases induced membrane fusion is discussed.

The p53 tumor suppressor gene (TP53; OMIM: 191170) plays an important role in tumorigenesis in lung epithelial cells. TP53 encodes a sequence-specific DNA-binding protein that regulates transcription of several genes in response to DNA damage promoting cell cycle arrest, DNA repair or apoptosis. A mutation does not necessarily alter the protein function and since not all altered tumor protein p53 (TP53) conformations lead to the same biological properties, we studied Cys135Arg TP53 gene mutation in squamous cell type of non-small cell lung cancers (NSCLCs), by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. Cys135Arg TP53 mutation, rare in databases (11/23544 in R11, IARC TP53 database), was detected. We chose p.C135R in order to examine DNA-TP53 interaction. A comparison with the wild-type after 1 nano-second molecular dynamic simulation analysis revealed a significant structural change (over 4A displacement) in the contact loop Lys-Ser-Val which lies upstream and next to the mutated site in the TP53, that sterically prevents its DNA-binding activity. Additionally, the mutation produced a change in the electrostatic potential surface of the protein in the same loop where the structural modification took place. To demonstrate the degree of loss of function, functional assays in yeast and bacteria with oligonucleotides for competitive electrophoretic mobility shift assays (EMSAs) were done proving that this mutation decreases TP53 ability to bind DNA of the TP53 response element from the human p21 gene. These results demonstrate that the amino acid change C135R in the human TP53 generates the loss of TP53 DNA-binding activity directly affecting its role as a transcription factor and suggests that this observation can explain part of the phenotype described in patients affected by this type of tumor.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between cold-sensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel-PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis.