Lactones are important biological molecules that offer a new molecular scaffold to develop new and more selective inhibitors targeting glycosidases [1]. The chemical routes that can speed up their synthesis in a stereo- and regio-selective way have become a major demand. A new derivative of 2,4-O-alkylidene-D-erythrose, enclosing a C=C moiety into a 1,5-lactone ring, was found to induce a complete facial selectivity in 1,3-dipolar cycloadditions [2]. This new D-erythrosyl 1,5-lactone was studied as a Michael acceptor with sulfur and nitrogen nucleophiles and from which a complete facial selectivity was demonstrated in all reactions [3]. Sulfides attack exclusively at C-4, but primary amines and hydrazine attack both at C-2 and C-4. The sulfur adducts formed are 1 (D-erythrose derivative):1 (nucleophile), and the nitrogen adducts are 1:2. The theoretical and computational results clearly explain the origin of the stereo-selectivity, and the energetic course of the reactions, starting with nitrogen and sulfide nucleophiles. Considering that the 1,4-lactones obtained in this work offer a new molecular scaffold for organic synthesis, these new results provide a solid theoretical platform that can be used to speed up synthesis of other derivatives in a stereo- and regio-selective way.

References

[1]Rocha, J. F.; Cerqueira, N. M. F. S. A. Glucosidases and galactosidases as drug targets. Curr. Top. Biochem. Res. 2017, 18, 117− 126.

[2]Sousa, C. E.; Ribeiro, A. M.; Gil Fortes, A.; Cerqueira, N. M.; Alves, M. J. Total Facial Discrimination of 1,3-Dipolar Cycloadditions in a d-Erythrose 1,3-Dioxane Template: Computational Studies of a Concerted Mechanism. J. Org. Chem. 2017, 82 (2), 982−991.

[3]Rocha, J. F.; Freitas, D. S.; Noro, J.; Teixeira, C. S. S.; Sousa, C. E.; Alves, M. J.; Cerqueira, N. M. F. S. A. Total Stereoselective Michael Addition of N- and S- Nucleophiles to a d-Erythrosyl 1,5-Lactone Derivative. Experimental and Theoretical Studies Devoted to the Synthesis of 2,6-Dideoxy-4-functionalized-d-ribono-hexono-1,4-lactone. J. Org. Chem. 2018, 83 (15), 8011-8019 DOI: 10.1021/acs.joc.8b00769.

In the 40’s, Erwin Chargaff was the first to observe the parity between Adenines (A) and Timinies (T) and Citosines (C) and Guanines (G), in the molecule of DNA. In the 60’s, Chargaff found a second parity rule. This time in a single strand of DNA. The amounts of A’s and T’s, and the amounts of C’s and G’s is similar. The explanation of the first rule is the complementary nature of the double stranded helix of the DNA molecule. However, for the 2nd rule, a biological explanation has remained a mystery. In the last 40 years, a generalization of the second rule was proposed, to explain the 2nd rule as a particular case. This generalization states that for any given k-mer and its reverse complement (RC), the number of times both are found is similar in a single strand of DNA. Two measures have been proposed to test the generalized Chargaff’s 2nd rule (gC2r), both include an artifact regarding the length of the genomes. This has led the authors to think there is a minimum length of a genome and a maximum k-mer for compliance. We propose a new way to measure the compliance of any given genome to the gC2r. The measure is the proportion of the genome which complies with gC2r. The compliance is measured per pair of kmer/k-merRC, using the natural logarithm of the number of times the k-mer is found, divided by the number of times its reverse complement is found in the genome or ln(#k-mer/k-merRC). This measure is independent of the size of the analyzed k-mer and the size of the genome. This measure has been implemented in a software, ChargaffCracker, which can rapidly analyze sequences and deliver a statistical report. We have generated random genomes based on the proportions and lengths of biological prokaryote genome sequences and compared them. We conclude hypothesizing that: 1. The compliance of the gC2r is a consequence, not cause of the 2nd rule and; 2. Although Chargaff’s 2nd rule might be a consequence of transpositions and inversions, the limits of compliance of the gC2r is a property of the sequence model of genomes, not of the biology of organisms. However, this property might have been selected to fulfill biological needs in genome evolution.

Sulfur oxides emission is one of the major causes for the formation of acid rain and other noxious atmospheric pollutants. Fossil fuels have sulfur containing molecules that, upon combustion, are degraded producing sulfur oxides. Conventional methods become unprofitable in order to achieve the level of desulfurization required by legislation. One alternative is the use of sulfur metabolizing bacteria that possess enzymatic machinery capable of removing sulfur, without degrading the calorific content of the molecule. Rhodococcus erythropolis is capable of performing such task using four different enzymes: DszA, DszB, DszC and DszD. DszD is an HBPS desulfinase which ultimately cleaves the carbon-sulfur bond.

The way DszB performs this reaction is still an object of discussion. Different mutagenesis studies have revealed key aminoacid residues for the reaction to occur of which can be named Cys27, His60 and Arg70.

Through the use of MD we obtain different conformations of the active site which are then used to elaborate QM/MM models in order to study of the desulfination reaction of DszB. Our results assure the importance of Cys27, His60 and Arg70 for the reactivity of the enzyme, as well as re vealing other important residues such as Gly73, which functions as a stabilizer of Cys27.

Protein structure is not static; residues undergo conformational rearrangements and, in doing so, create, stabilize or break non-covalent interactions. Molecular Dynamics (MD) is a technique used to simulate these movements with atomic resolution. However, given the data-intensive nature of the technique, athering relevant information from MD simulations is a complex and time consuming process requiring several computational tools to perform these analyses. Among different approaches, the study of Residue Interaction Networks (RINs) has proven to facilitate the study of protein structures. In a RIN, nodes represent amino-acid residues and the connections between them depict non-covalent interactions.

Here, we describe RIP-MD, a Visual Molecular Dynamics (VMD) plugin to facilitate the study of RINs using trajectories obtained from MD simulations of proteins. Our software generates RINs from MD trajectory files. The non-covalent interactions defined by RIP-MD include H-bonds, Salt bridges, VdWs, cation-π, π-π, Arginine-Arginine and Coulomb interactions. In addition, RIP-MD also computes interactions based on distances between Cαs and disulfide bridges. The results of the analysis are shown in an user friendly interface. Moreover, the user can take advantage of the VMD visualization capacities, whereby through some effortless steps, it is possible to select and visualize interactions described for a single, several or all residues in a MD trajectory. Network and descriptive table files are also generated, allowing their further study in other specialized platforms. Our method was written in python in a parallelized fashion. This characteristic allows the analysis of large systems impossible to handle otherwise. RIP-MD is available at http://www.dlab.cl/ripmd.

The cellular membrane is an essential component of all cells. Distributed on the cellular membrane are many different proteins, either integrated or in interaction, with a variety of functions, namely assisting in membrane transport. GALA is a helical peptide that is prone to protonation, at acidic pH, followed by insertion in the membrane [1]. In its folded form, GALA has an amphiphilic structure, and shows a concentration-dependent tendency to aggregate in multimeric structures inside the membrane, such that the polar glutamate residues face toward each other and the apolar residues face toward the membrane’s lipid tails [1].

In this preliminary study, we used Constant-pH molecular dynamics (CpHMD) simulations [2] to study the conformation and protonation behaviour of GALA as a monomer and as a dimer (parallel or anti-parallel configurations) in the membrane. From these results, we obtained the effects of the initial step of GALA aggregation, particularly, in the secondary structure stabilization and the shifts in the pKa values of the different glutamate residues [2]. In the future, these results will serve as the basis for expanding this study to larger aggregated complexes with 3, 5 or 10 monomer subunits.

The present study was designed to investigate the bioactive compounds in Rumex tunetanus extracts (polygonaceae), a plant growing in Garâa Sejnane region (NW-Tunisia). Hydro-methanol extracts of flowers and stems of Rumex tunetanus were analyzed by RP-UHPLC-ESI-QTOF-MS in the negative mode to identify the maximum of bioactive compounds. Applied the aforementioned method, a total of 60 bioactive compounds were characterized for the first time in Rumex tunetanus between them , 18 photochemical were firstly identified in the Polygonaceae family in negative ionization mode. Quantification of the identified compounds revealed that quercetin-3-O-glucuronide and (-)-epicatechin gallate were the most abundant phenolic compounds in flowers and stems, respectively. Moreover, positive correlations were found between the antioxidant activity measured by DPPH and FRAP assays with the total phenolic compounds (r = 0.98; r = 0.99, respectively) and the abundance of some phenolic subfamilies such as hydroxycinnamic acids, hydroxybenzoic acids, flavonols and flavones with r > 0.86. The compounds displaying significant (P < 0.01) and good correlations with the antioxidant activity (r > 0.93) were hydroxybenzoic acid, rutin, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, quercetin and luteolin-7-O-rutinoside.
In addition , the flowers and stems of Rumex tunetanus showed different bioactive compound profiles and significant antioxidant properties of extracts were evaluated in terms of single electron transfer as DPPH and FRAP assay and Rumex tunetanus flowers exhibited significantly higher antioxidant activities than the stems and could be further used in food and nutraceutical industries.
These results highlight the potential of the RP-UHPLC-ESI-QTOF-MS and MS/MS system to identify untargeted metabolic profiling of Rumex tunetanus. Overall, these results contribute to the clear explanation of the past and current usage of genus Rumex in folk medicine. Future investigations are necessary to develop purified antioxidant extracts, with the application of more selective extraction techniques as well as the use of other spectroscopy tools to confirm the preliminary predicted structures.

The rapid development of aquaculture, its intensification, and the occurrence of fish health problems on farms push to develop alternative methods to antibiotics and chemotherapy for controlling fish diseases. Probiotics may provide a potential alternative method to protect fish from opportunistic and pathologic bacteria and promote a balanced environment.

In this work, we have assessed the in vitro probiotic properties of twenty one bacteria from aquatic and fish origin, for their application in aquaculture. Selection was based on their antimicrobial activity (Bacteriocin) against fish pathogens and their in vitro safety assessment. This includes the evaluation of their haemolytic, proteolytic and mucinolytic activities, bile salt deconjugation ability and antibiotic susceptibility.

Twelve of the twenty one bacteria isolated from several showed strong antibacterial activity against several pathogenic species such as Lactococcus garvieae, Vibrio anguillarum, Vibrio harveyi, Aeromonas hydrophila and Aeromonas salmonicida, and were taxonomically identified by partial 16S rDNA gene sequencing. The cell-free culture supernatants from cultures of these twelve strains were treated with proteinase K (10 mg/ml; 37ºC, 1h) and submitted to heat treatment (100ºC, 10 min), which showed that eleven strains exert extracellular antimicrobial activity against fish pathogens due to the production and secretion of thermo-stable antimicrobial peptides (i.e., Bacteriocins). The tested strains showed a great heterogeneity respect to their safety and antibiotic susceptibility.

Given their antimicrobial (Bacteriocin) activity against fish pathogens and their safety, some of the tested strains may be considered as potential fish probiotics, and their effectiveness will be further tested in vivo.

TRPV5 and TRPV6 are highly calcium-selective channels from the Transient Receptor Potential (TRP) family¹. These channels are considered gatekeepers of epithelial calcium transport, essential for calcium homeostasis¹. At negative potentials, the channels exhibit a two-phase calcium-dependent inactivation where the slow component is shared and determined by the binding of Ca²⁺-Calmodulin complex to the C-terminal region of the channel ^(2,3). In contrast, the rapid phase of inactivation depends on the binding of calcium ions and allows differentiating both channels from a functional point of view; while TRPV6 shows a very robust inactivation, at the same calcium concentrations, the inactivation of TRPV5 conductance is modest⁴. The intracellular loop S2-S3⁵ and residues downstream the transmembrane segment S6⁶ has been associated to the differences observed in the kinetics of the rapid phase of inactivation. However, the exact location of the putative calcium-binding site and the molecular mechanism governing this process are not known. A thorough phylogenetic reconstruction in vertebrates suggest that the genes encoding for these channels duplicates more than once during evolution, naturally introducing the same set of mutations within a HLH domain located at the N terminal region. Further sequence analysis unveiled that the HLH domain acts as a fingerprinting in both channels. Molecular dynamics simulations, allowed us to identify a putative calcium-binding site that put together three different portions of the folded channel. By means of site-directed mutagenesis and patch clamp electrophysiology we reversed the phenotype of inactivation in these channels, confirming that the HLH sequence serves as modulator for the calcium-induced inactivation process. We conclude that subtle evolutionary-related variations within the binding region affect the phenotype of the fast inactivation phase.

The Ubiquitin Proteasome Pathway (UPP) plays a pivotal role in intracellular protein degradation and turnover in eukaryotic cells. [1] Therefore, modulation of the UPP emerged as a rational therapeutic approach in cancer, neurodegenerative diseases, among others. [2] During the last two decades academia and pharmaceutical industry made huge efforts to develop natural and synthetic proteasome inhibitors (PI). However, despite the enormous potential of PI, their use is still limited to certain types of blood cancer and shows severe side effects, limited activity in solid tumor and innate or acquired drug resistance. [3] This work encompasses a computational drug discover campaign to find new small molecules that inhibit proteasomal activity, with the goal of obtaining new anti-cancer drugs. A set of several compounds were identified in our lab as PIs obtained from virtual screening procedure. Since the proteasome can be found both on the cell cytoplasm and nucleus, inhibitors developed to target it, must be able to cross the membrane barrier. To acquire more information on how they interact with the lipid bilayer restrained (PMF) and unrestrained MD simulations at the water/membrane interface. The results showed that our compounds have similar permeability rates and behavior in the lipid bilayer when compared with known proteasome inhibitors. Furthermore, one of the major challenges with the approved PIs is acquired resistance, possibly from point mutations in the catalytic subunits of the proteasome. We have used MD simulations to focused our analysis on three different point mutations in the β5 catalytic subunit, with recognized importance in PI’s resistance: Ala49Thr, Ala50Val and Cys52Phe.Hopefully, our studies will be able to shed the light on the structural key determinants that regulate the observed PI’s resistance in the different mutations, and ultimately use the acquired knowledge in the development of new alternative and efficient proteasome inhibitors.

Acknowledgements:We thank the Fundação para a Ciência e a Tecnologia for financial support PTDC/QEQ-MED/7042/2014, UID/DTP/04138/2013 and SAICTPAC/0019/2015.

References:

1. Sommer, T.; Wolf, D. H. Biochim. Biophys. Acta - Mol. Cell Res. 2014, 1843 (1), 1.

2. Nalepa, G.; Rolfe, M.; Harper, J. W. Nat. Rev. Drug Discov. 2006, 5 (7), 596–613.

3. Crawford, L. J.; Walker, B.; Irvine, A. E. J. Cell Commun. Signal. 2011, 5 (2), 101–110.

Glutathione (GSH; γ-L-glutamyl-L-cysteinyl-glycine) is the most abundant endogenous antioxidant present in mammalian cells (0.1 to 15 mM) and plays a protective role for exogenous toxins and endogenous, especially in the central nervous system. Biochemistly knowns that biosynthesis pathway have two consecutive reactions that consume ATP, including two enzymes; glutamate cysteine ligase (GCL), [E-6.3.2.2], formerly known as ganma-glutamylcysteine synthetase (GCS) and glutathione synthetase (GSS), [E-6.3 .2.3] to generate GSH. The defense against the toxic effects of reactive oxygen species (ROS) is an essential task within the brain during a long human life, which indicates the presence of an effective antioxidant system. However, the balance between ROS generation and antioxidant processes can be altered, causing neurological disorders such as Alzheimer's and Parkinson's. The same way, markers of oxidative stress are strongly associated with severe mitochondrial dysfunction in autism pathology. Previous studies indicate that ASD is associated with deficits in the antioxidant defense of glutathione in selective regions of the brain, however, the molecular mechanisms of oxidative stress continue being unclear. In our previous studies we described the kinetic imbalance in tri-cellular metabolism of N-acetyl-aspartyl glutamate (NAAG), in anterior (ACC) and posterior (PCC) cingulated cortices relate to the executive control networks and the attention alert functions respectively, linked to ASD pathogenesis. In the present study, we use resonance magnetic spectroscopy (1H-MRS) to study the specie reduced of glutathione (GSH) biosynthesis in the cingulated cortices, as target of oxidative stress in individuals with ASD. The single voxel of 1H-MRS in bilateral anterior (ACC) and posterior (PCC) cingulated cortices, in adults with ASD and controls with typical development (n = 21 and n = 46 respectively) were assessed. Glutathione (GSH) concentration were significantly decreased in ACC (P = 0.05). The affinity between enzyme and substrate associated with the biosynthesis of reduced species at glutathione was calculate by Michaelis Menten constant (Km) showing that glutathione biosynthesis decreased significant (1.1e^-12 mM; R² = 0.001) in anterior cingulate cortex in autism and, the dissociation constant (ki) was reduced by 67.22% in consequence. On the other hand, maximum rate (Vmax) of the appearance of the product, which depends on the slowest pathway of the enzymatic reaction was significantly decreased (15.12 µM / min; R² = 0.51) in PCC. Our findings indicate that, with a small amount of substrate, the rate increases rapidly and linearly in ACC, suggesting that the active sites of the enzyme are saturated with the substrate, whereas the enzyme-substrate complex is very tight and rarely dissociates without the substrate reacting to give the product. Imbalance enzymatic kinetic in glutathione biosynthesis in the autism cingulated cortices is a novel finding indicative of a chronic neuroinflamatory state in these regions. We further conclude that a better understanding of the enzymatic activity in the synthesis of glutathione in the cingulated cortices can lead us to a new therapeutic pathway in the treatment of individuals with ASD.