Leishmania genus’ survival against oxidative stress. Thus, it poses as an attractive drug target, yet due to the size and features of its catalytic pocket, modeling techniques such as molecular docking focusing on that region is not convenient. Herein, we present a computational study using several structure-based approaches to assess the druggability of TryR from L. mexicana, the predominant Leishmania species in Mexico, beyond its catalytic site. Using this consensus methodology, three relevant pockets were found, of which the one we call σ-site promises to be the most favorable one. These findings may help the design of new drugs of trypanothione-related diseases.Human leishmaniasis is a neglected tropical disease which affects nearly 1.5 million people every year, with Mexico being an important endemic region. One of the major defense mechanisms of these parasites is based in the polyamine metabolic pathway, as it provides the necessary compounds for its survival. Among the enzymes in this route, trypanothione reductase (TryR), an oxidoreductase enzyme, is crucial for the  Leishmania infections are reported yearly [Leishmaniasis is a neglected tropical disease caused by protozoan parasites, often transmitted by hematophagous sandflies ,2. This d yearly . This fid yearly ; in factd yearly ,6, with d yearly .® and meglumine antimoniate/Glucantime® , amphotcapacity ,19), millcholine  and acyllcholine ), paromolcholine ,23,24 anlcholine ), and pelcholine , interfelcholine , inhibitlcholine , and mitlcholine ). Accordlcholine .L. mexicana causes up to 90% of the cases reported in the country [Among the different variants of this disease, the cutaneous one is of particular interest to our research group. Almost 95% of cutaneous leishmaniasis cases occur in the Americas, Mediterranean basin, the Middle East, and Central Asia . Althoug country , making 2) and the polyamine pathway [L. mexicana and other parasites start polyamine synthesis from L-arginine, which is converted to ornithine by arginine deiminase (ARG); subsequently, the resulting substrate is transformed to putrescine by ornithine decarboxylase (ODC). Spermidine follows from the conversion of putrescine by spermidine synthase (SPSyn) in presence of S-adenosylmethionine decarboxylase (ADOMETDC). Finally, the latter is converted to T(SH)2 by the action of trypanothione synthetase (TryS) [Leishmania parasites depend on trypanothione regeneration [Leishmania is remarkably sensitive to reactive oxygen species (ROS), in the absence of the T(SH)2/TryR system [Leishmania and Trypanosoma parasites [Several metabolic pathways have been examined as means of parasite inactivation, including, but not limited to: sterol biosynthesis, glycolytic pathway, purine salvage, glycosylphosphatidylinositol (GPI) glycolipid biosynthesis, folate biosynthesis, topoisomerases, hypusine, trypanothione (T(SH) pathway . The lat pathway  and have pathway ,35,36. T pathway , and sta pathway ,39. L. me (TryS) . This mee (TryS) . Therefoneration . It has R system . Here isarasites . Hence, arasites ,46. Stilarasites ,47. It fL. mexicana (TryRLmex) at a concentration of 100 µM. Additionally, it showed lower toxicity when compared to amphotericin B and higher activity than Glucantime® [Our research group recently published a pioneering work on this topic, which resulted in the discovery of compound ZINC12151998 , achievicantime® . Thus, ccantime® ,50.Structurally, TryR is a homodimer containing two FAD molecules and two NADPH molecules as cofactors . Each moTo date, TryRLmex has not been crystallized, making it a virtually unexplored target. Therefore, most reports in the literature include in silico characterizations . HoweverWe built two homology models, for the first one, we used the Swiss Model server and the second one was built with AlphaFold, the latter being one of the best methods to predict the structure of a protein available as of the writing of this work . The SwiThe 2JK6 and 2W0H showed superior results to TRLMS, but similar ones with respect to TRLMA in all the quality standards used. QMEANDisCO, due to its superiority to QMEAN4  and RMSDTo test whether the differences between TryRLmex (TRLMA and TRLMS) and TryRLinf (2W0H and 2JK6) models have a deeper impact, we decided to strengthen the analysis of the models using molecular dynamics and thereby assess the relative stability and overall quality of the models using the classic representative metrics such as RMSD, RMSF and radius of gyration. In addition, the Ramachandran number (𝓡) as implemented in the Backmap library was used . The latBased on RMSD, RMSF, and radius of gyration of alpha carbons see , no signRamachandran number plots see , on the Based on the results from molecular dynamics simulations, we chose the TRLMS model and its experimental analogue 2W0H to continue the characterization of this target. Then, we explored the potential of the surface to capture ligands by mapping the presence of putative hotspots. These small regions are crucial to track and identify binding sites , since thttps://ftmap.bu.edu/login.php (accessed on 24 April 2023)) was used for hotspot mapping [The FTMap server  and residues 396, 398, 399, 462, 463, and 464 (chain B) or vice versa. According to FTMap, it presented van der Waals and hydrogen bond interactions with the probes. Henceforth, we will refer to this area as the sigma site (σ-site) to facilitate its description. This is important because few co-crystallized inhibitors seem to bind to this pocket in close proximity to the His461-Glu466 pair , which is a vital part of the catalytic mechanism . More imhttps://fragment-hotspot-maps.ccdc.cam.ac.uk/ (accessed on 6 August 2023)) [Therefore, we sought to gather more data in order to reinforce these preliminary results; thus, we opted to use the Fragment Hotspot Maps server (FHM) (t 2023)) , which iT. brucei (TryRTbru) [FHM highlighted in both templates a similar distribution of hotspots compared to FTMap; in contrast, however, the σ-site showed a poor ranking. Nevertheless, FHM identified two rather attractive zones near the NADPH site. In 2W0H, the important residues were 198, 230, 364, and 374 (first zone) and 377, 378, 423, and 425 (second zone); in TRLMS, only 198, 378, and 425 were relevant see . A fragmryRTbru) , confirmryRTbru) . Residueholo structure, but not necessarily in an apo structure [Neglecting protein’s flexibility is an overarching problem in finding putative binding sites, as these depend on the conformations of the target. An example of this came from hotspot analysis of 2W0H, where FTMap showed that, apparently, there were no sufficiently favored areas for hot spots in this target. Even though this is not true, as the configuration of the PDB used was decisive for this result. In such circumstances, hotspots are not enough to define appropriate areas to design new molecules and, therefore, it is necessary to rely on an extension of these: cryptic sites; metastable pockets present in a tructure . These ttructure  usually tructure . The latFor this purpose, the CryptoSite server was used, which is based on the selection of 93 pairs of proteins from the Protein Data Bank in which each unbound structure had a site considered cryptic and each bound structure had a functionally relevant ligand bound to the site, all performed within a machine learning context . AlthougThe distribution of probable cryptic sites found by CryptoSite in 2W0H and TRLMS was very similar see . To our In the light of our findings, these areas are particularly attractive for targeted drug design. As such, our interest shifted to the NADPH site since, as of now, only one crystallized inhibitor contacts the border zone of this pocket (PDBID code 6ER5). In addition, we are dealing with an area located near the cofactor, but significantly different from previous reports .So far, we have discussed relatively unknown sites found in TRLMS that could be use for drug design. Hotspots and putative cryptic sites have proven to be very useful tools. However, even if a protein has either, it is not a guarantee that it can be effectively exploited .Another requirement to consider is geometry, since size and shape of the binding sites are key factors to assess binding affinity . TherefoDogSite3 revealed that the σ-site and the NADPH site boundaries are favorable zones. Once more, the similarity trend in both templates was preserved see . LikewisFor the NADPH site, residues 198, 229, and 230 were not marked as a favorable pocket. In contrast, 360, 361, 362, and 446, among others, were the residues reported by the server in both cases; therefore, this made us question any further consideration of this site.Previous methods allowed us to extensively examine the surface of 2W0H and TRLMS, finding broad similarities in both structures; particularly, the zone already described the catalytic cavity and the NADPH site were pointed out. However, these approaches do not account for target’s flexibility ,84,87,88To cope with these limitations, we decided to carry out cosolvent molecular dynamics, as a final test for molecular probes and thereby determine further differences, if any, between 2W0H and TRLMS. As the name suggests, cosolvent molecular dynamics uses a discrete concentration of organic solvents with different chemical properties and with high solubility in water, to avoid aggregation phenomena; this allows the study of putative pockets in the protein . FurtherN-methylacetamide , residues 458, 459, 461, 466, 470, and 472 (chain B), and vice versa. This cavity was partially suggested by FTMap and CryptoSite servers. Henceforth, we shall refer to this as the lambda site (λ-site), which is depicted in Despite the similarity of results between production times, we set ourselves the task of examining which of these proved optimal to achieve robust analysis and mapped pockets in the shortest simulation time and with the minimum number of replicates. When comparing the surfaces of the pockets found and production times, we observed that there was a great similarity of results between 20 and 40 ns , both inOn the other hand, 5 ns had significant differences compared to the latter A, despitThe results showed that the affinity of both templates for selected probes is very similar, yet the distribution of these on the targets’ surface suggests that TRLMS has a greater facility to place probes in the vicinity of the NADPH site and the σ-site than 2W0H. In addition, the latter exhibits more distant locations from critical areas of the target as relevant than TRLMS, which suggests that 2W0H may retain some ligands in these areas before they reach the essential sites related to the inhibition of the target. The residues of each cavity, as well as their location, can be consulted in the The relevance of the previous results could be observed by comparing the target residues that interacted with the ZINC12151998 in a classical molecular dynamics simulation with a 50 ns trajectory carried out in our previous work . Where rCosolvents molecular dynamics simulations allowed us to determine that the σ-site, the λ-site, and the contours of the NADPH site were the best candidates for rational design of drugs, since they have most of the required features by a cavity to capture and retain a ligand in its vicinity. Due to its proximity to the His461-Glu466 pair, we chose the σ-site among them to carry out a final test: metadynamics. Briefly, metadynamics allows the recovery of free energy values and characterization of folding events using an enhanced potential along a given choice of reaction coordinates, also known as collective variables (CVs) .For this study, we chose the well-tempered variation of the method to avoid the system transients on high-energy regions. For our analysis, six replicas of 50 ns were obtained.Naturally, this production time is insufficient to yield a reliable estimate on either free energy or the overall shape of the potential energy surface. However, we did not aim want to estimate them precisely, but rather gain intuition into the general flexibility of the region and exploit it to reinforce the feasibility of using this region as a suitable ligand design. The first system to which metadynamics were applied was TRLMS, which showed that the σ-site presents some metastability .As observed, there is a clear trend for a local minimum, with average values of ~2.5 Å and ~6.5 Å for RMSD and radius of gyration, respectively. Only one replica showed the presence of additional minima, yet such behavior was not observed in 2W0H see . This suFor comparison, Considering these results, we made a comparison to assess whether metadynamics indeed revealed something that conventional molecular dynamics simulations would not have shown. Based on radius of gyration values, the site shows metastability with small but consistent fluctuations, a behavior also observed in TRLMA . In the L. infantum (PDBID code 2JK6), T. cruzi (PDBID code 1BZL), and T. brucei (PDBID code 2WBA). Hence, we compared the residues from the σ-site, λ-site, NADPH site, and doorstop pocket, including their contours, to examine the degree of conservation of these pockets; already known areas were also included.With the aid of computational tools, highly relevant pockets were found in TryRLmex. However, this by itself is not enough, as it is our intention to explore their significance for drug design, even if preliminary. To do this, we considered TryR from other parasites such as T. cruzi and T. brucei.We found that, in general, residues from already known pockets are largely conserved see . NotablyIn sharp contrast, the σ-site is also completely conserved among the trypanosomatids mentioned above; thus, it remains a strong candidate to initiate a rational drug design against TryR. In addition, the experimental evidence in the RCSB database shows several crystallized inhibitors that bind to this pocket. Of note, their effectiveness seems not to be conditioned only to binding to the σ-site, but to an interplay with additional sub-pockets in the catalytic cavity . This suggests that the effectiveness of the σ-site as a binding site will also depend on the haptoforic regions of TryR. In this case, almost all of the previously described pockets in the catalytic cavity  are conserved in the trypanosomatids already mentioned. Thus, the premise of broad-spectrum drug gains higher relevance.Finally, it is necessary to consider another critical point: the potential risk of collateral inhibition of glutathione reductase (GR), the enzyme analogous to TryR in humans. Although it has been reported that the catalytic cavity of GR is highly conserved with respect to that of TryR , there aRegarding the σ-site in TRLMS and GR, it is conserved in six out of seven residues, with the only difference being the substitution of Leu399 in TRLMS for Met406 in GR. This by itself does not provide us with much information about the potential behavior of the σ-site in both targets, so we conducted final comparison between proteins using metadynamics and molecular docking.71 were selected  and Leu399 (hydrogen bond) through the phenyl and amide residues of the ligand; it also highlights that the orientation with respect to the native pose of 71 is inverted, but the type of interactions is similar ) [L. mexicana (strain MHOM/GT/2001/U1103). This sequence was submitted to the Swiss Model server (https://swissmodel.expasy.org/ (accessed on 12 August 2019)) [Leishmania infantum (L. infantum), were also downloaded for comparative purposes [https://saves.mbi.ucla.edu/ (accessed on 14 august 2019)) [The TryR sequence was obtained from the UniProt server (t 2019))  entry E9t 2019))  and, frot 2019)) ,123 inclt 2019)) ,125,126,purposes . The Propurposes  was usedpurposes , and thepurposes . Qualityt 2019)) ,133,134 t 2019)) .Molecular dynamics simulations and its visualization were performed with Desmond (v.2021-4) ,127, begThese simulations were then analyzed using Backmap (v.0.2.6)  to asseshttps://ftmap.bu.edu/login.php (accessed on 24 April 2023)) [Mapping and searching for hotspots was performed using the FTMap server (l 2023)) . For thel 2023)) .https://fragment-hotspot-maps.ccdc.cam.ac.uk/ (accessed on 6 August 2023)) [Additionally, the Fragment Hotspot Maps server (FHM) (t 2023))  was usedhttps://modbase.compbio.ucsf.edu/cryptosite/ (accessed on 30 April 2023)) [The search for cryptic sites was performed with the CryptoSite server (l 2023)) . PDB fill 2023)) .https://proteins.plus/#dogsite (accessed on 3 May 2023)) [The prediction of pockets and their relevance was carried out with the DoGSite3 server (y 2023)) . PDB fily 2023)) .N-methylacetamide; these were selected by fragment analysis of ZINC12151998 [xyzZ is the occupancy of functional groups of the probes, converted to Z-scores using the following equation:xyzX comes from the raw bin count for each grid and 〈X〉 is the mean value for all the bins and σ is their standard deviation [The search for pockets and hot spots using molecular dynamics was carried out with Desmond . For thi12151998 . Hotspoteviation .Additionally, the same protocol was applied to 2W0H and TRLMS, with a production time of 20 ns (Experiment B and C). A total of 3 simulations per template were performed, accumulating 60 ns per system, for comparison purposes. Visualizations of the results were made with UCSF ChimeraX .σ and w, respectively);To test the behavior of the σ-site, we conducted enhanced sampling using the well-tempered metadynamics algorithm . BrieflyT is the heart of the well-tempered algorithm, as it establishes the rate at which the Gaussian height decreases. It follows that for To date, there is no systematic nor explicit way to determine what may constitute a pertinent choice for CVs; reasonable choices should still be robust enough to distinguish reactants, transition state, and products ,146. Forrelevant .Simulations accounted for 50 nanoseconds; additional settings were kept from the initial simulations. Six replicas were obtained under these conditions for the homology model obtained from the Swiss Model and 2W0H for comparison. Free energy surfaces were analyzed using Maestro.71 was carried out with the PLANTS and LeDock programs, recognized for their precision and accuracy [Molecular docking of ZINC12151998 and compound accuracy ,148.PLANTS uses the well-known ChemPLP scoring function, which comes from:plpf) is used to model steric complementarity of the protein and the ligand. To avoid internal ligand clashes, an empirical heavy-atom potential (clashf) is evaluated for each ligand pose. The torsional potential (torsf) is calculated for all rotatable bonds available in the ligand molecule except for terminal hydrogen bond donor groups. Finally, as it is outside the binding site definition, which is defined by a sphere in PLANTS, no interaction potentials are calculated, and a mechanism is needed to guide the search algorithm toward the interesting regions located at the predefined binding site. A quadratic potential is calculated for the reference point of the ligand (origin of the ligand’s coordinate system) if this point lies outside the sphere, that is, the distance r of the reference point to the center of the sphere is greater than the binding site radius (siteC) [The scoring function of the piecewise linear potential function ( (Csite) . PLANTS On the other hand, LeDock uses the following equation as a scoring function:vdw interaction vdwE and hydrogen bonding energy hbE, where Θ is the Heaviside step function and COE is the cutoff energy to allow for soft docking. The second term is the electrostatic interaction energy with a distance function β(r) accounting for both electrostatic screening and desolvation effects, where q is the partial atomic charge and r is the distance between a pair of atoms. The last term is the ligand conformational strain upon binding, and is contributed by the intra-molecular clash and/or torsion strain. Coefficients α, β, and γ were empirically determined by referring to the previous scoring function [The first term is the sum of function .x, y and z of 25 Å centered on the catalytic cavity of both targets, in such a way that all of it was part of search space.Proteins were prepared using LePro  and, forJust like the initial metadynamics simulations, Desmond was used with similar parameters and settings. Selected binding modes from both docking programs were used as starting point for these simulations. In this case, collective variables were defined by the distance between the center of mass from the σ-site and the ligands; the second reaction coordinate was ligand’s RMSD; for both CVs, the width was set to 0.1 Å and gaussians were deposited every picosecond. The bias factor was set to 8.0. Two replicas of 20 ns were carried out for each ligand and program yielding four simulations for comparison; additional FES diagrams can be found in the L. mexicana was carried out. The study included the search for hotspots, cryptic sites, and overall druggable pockets in static models, later reinforced with molecular dynamics simulations and enhanced sampling. To the best of our knowledge, this is the first study of its kind on this target. Our study sought to find pockets that satisfied the following criteria: binding free energy contribution, flexibility, and a favorable geometry. Despite not finding significant differences between the homologue and 2W0H, we found three putative pockets, two of which were found in the vicinity of the catalytic cavity and one in the vicinity of the NADPH site. Certainly, we consider that, due to its proximity to the His461-Glu466 pair, the pocket denominated as the σ-site was the most appropriate to perform additional analyses. Preliminary metadynamics studies showed us that this zone presents acceptable metastability and flexibility, desirable characteristics in a pocket that is intended to place a ligand, for which reason we consider this zone critical in the targeted design of drugs. Furthermore, the fact that it remains highly conserved in the trypanothione reductase of other parasites and the broad differences between TryR and GR make this site an extremely attractive area. Finally, enhanced sampling around this pocket suggested a notable cooperativity of previously identified sub-pockets to allow binding around the interface region. Moreover, the role of Lys61 has been highlighted as a putative gatekeeper and haptoforic interaction that allows ligand orientation and overall affinity for the aforementioned subpockets and σ-site.In the present work, the analysis and comparison of a homologue of the TryR from In summary, this information can serve as a starting point for drug design efforts, since now it would not be necessary to consider the entire catalytic cavity to design an inhibitor, which would facilitate its design.


Varroa destructor salivary secretion plays a vital role in mite–bee interactions. In this study, we found that the salivary cystatin gene was highly expressed in mites during the reproductive phase when they fed on pupal bees. Injection of prokaryotic-expressed cystatin into white-eyed pupal bees downregulated the metabolism of carbohydrates, fatty acids, and amino acids, and ATP production, which caused the pupal bees to fail to emerge and decreased the weight of newly emerged bees. Downregulation could save nutrients and energy for V. destructor to maximize its reproduction potential, implying that Varroa destructor could manipulate the metabolism of host bees through the injected salivary secretion. These results provide new insights into mite–bee interactions, providing a basis for Varroa destructor control in apiculture.Varroa destructor injects a salivary secretion into honeybees during their feeding process. The salivary secretion plays a vital role in mite–bee interactions and is the main cause of honeybee illness. To determine the biological function of cystatin-L2-like, one of the components of V. destructor salivary secretion, its gene expression in mites during the reproductive phase and dispersal phase was quantified using RT-qPCR, respectively. Moreover, the E. coli-expressed and -purified cystatin was injected into the white-eyed honeybee pupae, and its effects on the survival, the weight of the newly emerged bee, and the transcriptome were determined. The results showed that cystatin was significantly upregulated in mites during the reproductive phase. Cystatin significantly shortened the lifespan of pupae and decreased the weight of the newly emerged bees. Transcriptome sequencing showed that cystatin upregulated 1496 genes and downregulated 1483 genes in pupae. These genes were mainly enriched in ATP synthesis, the mitochondrial respiratory chain, and cuticle structure and function. Cystatin comprehensively downregulated the metabolism of carbohydrates, fatty acids, and amino acids, and energy production in the pupae. The downregulation of metabolic activity could save more nutrients and energy for V. destructor, helping it to maximize its reproduction potential, implying that the mite could manipulate the metabolism of host bees through the injected salivary secretion. The results provide new insights into mite–bee interactions, providing a basis for related studies and applications. Honeybees can produce many nutritious, healthy, and valuable bee products for human beings . MoreoveVarroa destructor is always the greatest threat to European honeybees (Apis mellifera L.) worldwide [V. destructor, as an ectoparasitic mite native to the Asian honeybee (Apis cerana), spread to the European honeybee probably around the 1950s in Asia, and then rapidly spread in the 1970s in Europe and the 1980s in America [A. mellifera, V. destructor has been linked to a worldwide decline in honeybee health and has had a devastating impact on apiculture [V. destructor currently remains a major beekeeping issue throughout the world.Among the multiples biotic factors threatening honeybee survival, orldwide ,10. Withiculture ,12,13. DV. destructor can be divided into the reproductive phase, which takes place inside honeybee brood cells, where a foundress mite rears her young, and the dispersal phase (or phoretic phase), in which mature female mites travel and feed on adult bees [V. destructor is a vector for various honeybee viruses [V. destructor can decrease the body weight and water content of young emerging bees [The life cycle of ult bees . The mitult bees , and canult bees . The repult bees . In addi viruses ,18,19. T viruses ,21. A suing bees , alter ting bees , downreging bees ,25,26, aing bees ,28.V. destructor feeds on the bodily fluids of brood or adult bees through a wound in the cuticle, which allows food uptake by the mother mite and its progeny [V. destructor feeds primarily on honeybee fat body tissues [V. destructor salivary secretion. So far, over 300 proteins have been determined from the salivary secretome via nano-liquid chromatography coupled with tandem mass spectrometry [V. destructor showed that there were six cystatin-related mRNAs among the nine salivary-related mRNAs, implying the vital roles of cystatin in mites. To determine the biological function of cystatin, which was not previously reported, V. destructor salivary cystatin-L2-like  was expressed and purified from the E. coli. Then, the purified protein was injected into white-eyed pupae, and its effects on honeybees were determined in detail. progeny ,30. Late tissues . The fee tissues . A toxic tissues . Salivar tissues . The est tissues . Moreove tissues ,36. All trometry . Except A. mellifera used in the experiment were collected from the colonies kept in the experimental apiary of the College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University. Ten colonies were intentionally kept without mite control, from which the mites in the study were collected.The honeybees of V. destructor infection were selected for mite sampling. Specifically, for collecting the Varroa mites of the reproductive phase, one frame with elder brood and capped brood was removed from each colony, the cell’s cap was opened with forceps and the white-eyed pupae were carefully pulled out of the cell; the mite on the white-eyed pupae was collected, and around 30 mites per colony were pooled into a 1.5 mL Eppendorf tube  as one sample. The Varroa mites of the dispersal phase were collected according to a previous report [To determine the difference in gene expression of cystatin in Varroa mites in the reproductive phase and the dispersal phase, three colonies with s report ,39, and ® Reagent  was used for extracting RNA from these stored mites as described by the protocol. After inspection by the NanoDrop One , the RNA was stored at −80 °C. cDNA was synthesized from these RNA samples using a reagent kit according to manual instructions , and then stored at −20 °C for use.TRIzolV. destructor has four isoforms. By comparing and analyzing the sequence of the four isoforms, we designed a cystatin coding DNA sequence containing the conserved sequence of the four isoforms by removing the variant part, and adjusting a part of the nucleic acid base based on the code usage bias of E. coli so that the protein could be efficiently expressed in E. coli. Restriction sites of NdeI (CATATG) and XhoI (CTCGAG) were added to the 5′- end and 3′- end of the sequence, respectively. The designed sequence was artificially de novo synthesized by Sangon Biotech Co., Ltd., Shanghai. For the optimized amino acid sequence and DNA sequence of cystatin, refer to Supplementary File S1. After digestion by NdeI and XhoI, the DNA fragments and the plasmid DNA of pET-22b (+)  were connected using ligase and then transformed into the competent cell of E. coli, DH 5α. The recombinant plasmid was screened and transformed into an E. coli of BL21 (DE3) to induce the expression of the objective protein. After being verified by Western blot using the anti-His body as the primary antibody, the expressed protein was purified by Ni-NTA (His-tag Affinity). The concentration of the purified protein was determined by the Lowry Protein Assay Kit  and then stored at −80 °C.To comprehensively determine the protein’s function on honeybees, a prokaryotic expression system was set up for obtaining the cystatin in the study. In detail, the salivary cystatin-L2-like gene of A. mellifera and A. cerana [We tested the injection of 400 ng of cystatin into pupal bees, referring to a previous report of injecting Varroa mite saliva into both . cerana . However®  according to the instructions. The libraries were subjected to an Illumina Hiseq™ platform for generating 150 bp paired-end raw reads, from which clean data (clean reads) were obtained by removing the adapter containing reads, ploy-N reads, and low-quality reads using Trimmomatic v0.36 [A. mellifera using Hisat2 v2.1.0 [A. mellifera.To determine the effect of cystatin on the gene expression of honeybee pupae, the pupae 3 days post injection from groups Cys200 and PBS were selected for transcriptome sequencing. In detail, 10 pupae were pooled as one sample from each 24-well plate. Three samples (repeats) were prepared for groups Cys200 and PBS, respectively. RNA was extracted from these samples as described above. RNA quality and quantitation were determined by the Qubit™ RNA HS Assay Kit according to the manuals . Sequencing libraries were generated using Hieff NGS™ MaxUp Dual-mode mRNA Library Prep Kit for Illuminaic v0.36 . The cle2 v2.1.0 . The map2 v2.1.0  referrinp-values using Benjamini and Hochberg’s approach was selected for controlling the false discovery rate. In addition, genes with a Q-value < 0.05 were assigned as differentially expressed.The number of reads mapped to each gene was counted using FeatureCounts v1.5.0 . The expGO enrichment was performed using topGO R package v2.24.0, and KEGG pathway enrichment was implemented using the cluster Profiler R package v3.0.5 , in whicV. destructor was used as a template. A pair of cystatin-specific primers was used in the PCR  were quantified via qRT-PCR to validate the reliability of the transcriptome sequencing using the specific primers . The survival curve was constructed based on the data of daily dead pupae, and the difference between the test groups was analyzed using the log-rank (Mantel–Cox) test. All the analyses were performed in GraphPad Prism , and p < 0.05 was set as the significance.The gene expression was calculated using the 2V. destructor using qRT-PCR, we found that the gene of cystatin was significantly more highly expressed in the mites in the reproductive phase than in the mites in the dispersal phase  (By quantifying the gene expression of cystatin in  0.0049) . This imE. coli of BL21 (DE3) to induce the expression of the cystatin protein. A protein band with a molecular weight similar to the predicted cystatin was successfully induced to express in the precipitation of cell lysis (The de novo synthesized DNA coding cystatin was restricted with NdeI and XhoI and then ligated with the expression vector of pET-22b (+). The recombinant plasmid was screened by agarose gel electrophoresis of plasmid DNA digested with NdeI and XhoI, as shown in ll lysis B. Westerll lysis C. The cyll lysis D. The orχ2 = 221.5, p < 0.001). Most of the pupal bees injected with cystatin died before emergence, with their cuticle turning dark . Tukey’s multiple comparisons tests showed that the weights of newly emerged honeybees from group CK (111.3 ± 0.8947 mg) and PBS (111.4 ± 1.338 mg) were not significantly different (p > 0.9999). However, the weights of the bees from all three cystatin-injected groups  were significantly lower than the bees from the CK and PBS groups . Moreover, the weights of honeybees among the three cystatin-injected groups were also different   . TherebyA. mellifera genome by Hisat2. The original sequencing data of the six libraries of pupae from the group PBS and Cys200 were deposited in the Sequence Read Archives (SRAs) under the BioProject of PRJNA1029743 at NCBI.For determining the comprehensive effect of cystatin on pupal bees, the pupae from the PBS and Cys200 groups were subjected to transcriptome sequencing. We finally obtained clean reads ranging from 64,357,262 to 95,132,698 from the six libraries, with an average Q30 value of 94.16. A total of 13,173 unigenes were assembled using StringTie (v1.3.3b) from the clean reads mapped to the Differentially expressed genes in pupal bees between the PBS and Cys200 were analyzed using the DESeq2 R package. Among the 13,173 unigenes assembled, cystatin caused 1496 genes to be upregulated, and 1483 genes to be downregulated A. The diGO enrichment analysis showed that the differentially expressed genes were enriched in 133 items of biological process, 63 items of cellular components, and 48 items of molecular function, respectively . The mosKEGG enrichment showed that a total of 23 items were significantly enriched . Among tTo further reveal the relation between the KEGG-enriched functions and differentially expressed genes, an interaction network of the top 10 enriched functions and involved genes was constructed. The result showed that nine functions involving the metabolism of carbon, fatty acids, amino acids, starch, sucrose, and energy production-related processes such as oxidative phosphorylation and the citrate cycle (TCA cycle) were well interconnected. Only ECM–receptor interaction was not included in the network. Moreover, most of the circle node was green in color, which means the genes involved in the enriched functions were downregulated . Such a t-test, for all the sixteen genes, p > 0.05), which validated the accuracy of the RNA-seq results.Considering that the downregulated genes were well enriched in KEGG analysis, 16 downregulated genes from the KEGG-enriched pathways were selected for the validation of their expression by qRT-PCR using gene specific primers . The resV. destructor completely relies on feeding on the hemolymph and fat body of honeybees for its survival and reproduction [V. destructor plays a vital role in this process. Thereby, research on the component and function of the specific protein in salivary secretion is necessary for understanding the interaction between V. destructor and the honeybee host [V. destructor, with undetermined functions. To explore it, we expressed the cystatin from E. coli and injected it into white-eyed pupal bees. Its effects on pupal bee survival, the weight of newly emerged bees, and the transcriptome response were investigated in detail. The results showed that cystatin was lethal to pupae in a dose-dependent manner, could decrease the weight of newly emerged bees, and could fundamentally alter the host bee’s gene expression profile. Moreover, a previous report showed that the injection of 432 ng of Varroa mite saliva caused deformed wings in newly emerged bees of A mellifera, which was equal to the symptoms caused by two Varroa mites in the lab [V. destructor with effects on host bees determined except for in the previous reports [V. destructor and the interaction between V. destructor and honeybees.As an obligate parasite of honeybees, oduction . The salbee host . Previoubee host ,35,36,37 reports ,32,33,34V. destructor on the honeybee host involves two distinct stages: the reproductive phase on pupae and the dispersal phase on adult bees [V. destructor was positively related to its feeding activity and nutrient ingestion in the reproductive phase.The life of ult bees . The mitult bees . HoweverE. coli-expressed and purified cystatin was injected into the white-eyed pupae, and the growth and development of the treated bees were determined. The result showed that the survival of cystatin-injected pupae was significantly decreased compared to pupae from the control group and that most of the pupae died before emergence  and 36 h (in drones), respectively, after cell capping, were able to stimulate the mite’s oogenesis [V. destructor with a longer time window for oogenesis and egg laying. From this point, V. destructor could manipulate (slow down) the development process of the host pupal bee to maximize its reproduction potential by injecting the salivary secretion.From the view of ogenesis . TherebyV. destructor could intervene in the normal process of metamorphosis through blocking the disruption of storage protein in the brood and the construction of pupae tissues. This leads to nutrient and energy conservation, meaning V. destructor can maximize its reproduction potential by injecting salivary cystatin into host bees in the feeding process.Cysteine is a common amino acid, and the disulfide bond formed between two cysteine residues is essential for forming the tertiary and quaternary structure of all kinds of functional proteins. As holometabolous insects, brood bees store large amounts of nutrients in the fat body, which are completely disrupted for the purposes of pupae tissue building in metamorphosis . The disV. destructor could act as a vector and disseminator of honeybee viruses between and within bee colonies, and as an activator of virus multiplication in infected individuals. The deformed wing virus (DWV) in V. destructor-infested colonies is now considered to be one of the key contributors to the final collapse [V. destructor, the amount of DWV is often sharply increased in individual honeybees [V. destructor has dramatically altered DWV epidemiology worldwide [V. destructor feeds on honeybees, DWV in the salivary secretion can be simultaneously injected into the host bee, which might, in part, account for the sharp increase in DWV [V. destructor salivary secretion could activate DWV replication in the host bee, causing the DWV to increase too [collapse . Followioneybees ,57. For orldwide . To detee in DWV ,59. In aease too . In our V. destructor and its host bees. The biological functions of specific salivary secretion proteins that are involved in this interaction have been continuously studied in the field over a long time period. In this study, cystatin was determined to be significantly highly expressed in the reproductive-stage of V. destructor and was verified to be virulent to pupal bees, shortening their life span and decreasing the weight of newly emerged bees. Cystatin changed the expression of nearly 3000 genes of pupal bees. These genes were mainly enriched in the downregulation of metabolic activity, affecting sugars, fats, and amino acids in the host pupal bees. Such downregulation implied that V. destructor could manipulate the metabolism of pupal bees through injected salivary secretion to save more nutrients and energy for self-reproduction. This result introduces new data on the interaction between V. destructor and honeybees, and provides an experimental basis for V. destructor control in apiculture.Salivary secretion mediated the interaction between


We previously reported the Rho-associated coiled-coil containing protein kinase (ROCK)-mediated di-phosphorylation of myosin light chain (MLC) and actin bundle formation at the cell periphery as early events of the endothelial barrier disruption. We herein examined the role of RhoA during early events of barrier disruption. Treatment of cultured porcine aortic endothelial cells with simvastatin prevented the decrease in trans-endothelial electrical resistance, MLC di-phosphorylation and peripheral actin bundle formation seen 3 min after thrombin stimulation. Co-treatment with geranylgeranyl pyrophosphate rescued the thrombin-induced events. Thrombin increased a GTP-bound form of RhoA and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at the ROCK site. The intracellular introduction of the inhibitory protein of RhoA inhibited the thrombin-induced di-phosphorylation of MLC. However, knockdown of either one of RhoA, RhoB or RhoC failed to inhibit thrombin-induced MLC di-phosphorylation. The findings suggest that Rho proteins play a critical role during early events of thrombin-induced barrier disruption.The online version contains supplementary material available at 10.1186/s12576-022-00857-x. Vascular endothelial cells form a monolayer sheet lining the luminal surface of the blood vessels, and play a critical role in the regulation of vascular permeability –5. UnderIn the previous study, we identified previously unappreciated early events during thrombin-induced barrier disruption . Namely,2+-calmodulin-dependent MLC kinase (MLCK), Rho-associated coiled-coil containing protein kinase (ROCK), zipper-interacting protein kinase (ZIPK) and integrin-linked protein kinase (ILK) [The protein kinases that are capable of inducing MLC di-phosphorylation include Case (ILK) –11. Our se (ILK) . Howeverse (ILK) , 13. ROCse (ILK) . The GTPse (ILK) . As a reThe present study addressed the critical involvement of RhoA in the early events of the thrombin-induced endothelial barrier disruption. In order to inhibit membrane localization of RhoA, cells were pretreated with simvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl CoA reductase that not only inhibits de novo synthesis of cholesterol, but also depletes farnesyl pyrophosphate (FPP), an intermediate metabolite of cholesterol synthesis, thereby depleting geranylgeranyl pyrophosphate (GGPP) . The inh2O, 2.2 mM GGPP in methanol, 2.3 mM FPP in methanol, 1 unit/µl thrombin in H2O, 10 mM Y27632 and 1 mM H1152 in H2O. When the concentration-dependent effects of simvastatin , simvastatin (S6196), GGPP ammonium salt G6025) and FPP (F6892) were purchased from Sigma . Y27632 and H1152 glycyl were purchased from Merck Millipore . The stock solutions of reagents were as follows: 10 mM simvastatin in H5 and FPPPorcine aortas were obtained at a local slaughterhouse, and porcine aortic endothelial cells (PAECs) were established and cultured in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum, as previously described , 17. The5 cells/60 mm-dish, the medium was replaced on the next day and the cells were exposure to 10 nM siRNA for 3 days. Cells were harvested and homogenized before and after thrombin stimulation as described below in the Preparation of cell lysates for immunoblot analysis section and MLC di-phosphorylation was analyzed with immunoblot.siRNAs targeting porcine RhoA (Accession No. NM_001244437.1), RhoB (NM_001123189.1) and RhoC (XM_003125846.4) were designed and synthesized by Sigma-Aldrich Japan . The sequences of sense siRNAs are AUA CUG AUG UUA UAC UGA U (RhoA), CCG ACG UGA UCC UUA UGU G (RhoB) and AUC CUC AUG UGC UUC UCC A (RhoC), all with overhang of two deoxythymidines. siRNA Universal Negative Control #1 (Sigma-Aldrich Japan) was used for negative control experiments. PAECs were plated at 3 × 104 cells in 0.32-cm2 culture inserts with polyethylene terephthalate membranes  in 24-well culture plates . TEER measurements were conducted in Hepes-buffered saline  at 37 °C with an EVOM2 voltohmmeter equipped with a STX2 electrode . The cells were equilibrated in HBS for 10 min prior to the experimental protocol. The change in the TEER (ΔTEER) from the value obtained in HBS just prior to agonist stimulation was expressed as the absolute value of the resistance normalized by the surface area of the culture insert membrane (Ω cm−2), unless otherwise specified.The endothelial barrier function was evaluated by assessing TEER, as previously described . In brie5 cells in 60-mm culture dishes. Cells were rinsed three times with pre-warmed phosphate-buffered saline , and then equilibrated for further 10 min and subjected to the experimental protocols in HBS at 37 °C. At each time point, the cells were rinsed once with ice-cold PBS containing 0.1 mM EDTA to remove residual extracellular Ca2+. The cells were then lysed in 70 µL ice-cold lysis buffer containing 50 mM Hepes, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µM 4-amidinophenylmethanesulfonyl fluoride (p-APMSF), 20 µM NaF, 50 µM calpain inhibitor I and 5 µM microcystin-LR. The lysates were immediately collected using a cell scraper, transferred to a microcentrifuge tube and snap-frozen in liquid N2. The lysates were then thawed on ice for 20 min and centrifuged at 12,000 rpm for 20 min on microcentrifuge  in a cold room. The supernatants were then made the samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentrations of supernatants were estimated with a Coomassie (Bradford) protein assay kit .The cells were plated at 3 × 102. After electrophoresis, the gels were soaked for 30 min at room temperature in transfer buffer  containing 2 mM EDTA to remove Mn2+ and then soaked in the transfer buffer for 15 min. Proteins were then transferred to polyvinylidene difluoride membranes (Merck Millipore). The membranes were post-fixed in PBS containing 0.5% formaldehyde for 45 min [The mono- and di-phosphorylation of MLC  were quantitatively analyzed using Phos-tag SDS-PAGE followed by immunoblot detection, as previously reported . In brier 45 min , rinsed The thrombin-induced di-phosphorylation of MLC in PAECs, which were subjected to knockdown of RhoA, RhoB and RhoC, was analyzed using anti-phospho-MLC at T18 and S19 antibody. The conventional SDS-PAGE with 3 μg protein loading and protein transfer to polyvinylidene difluoride membranes  were performed as previously described . Total lThe cell lysates (10 µg protein) were subjected to conventional SDS-PAGE on 10% polyacrylamide gels and then transferred to polyvinylidene difluoride membranes, as described above. After fixation with 0.5% formaldehyde in PBS for 45 min and rinsing in T-PBS, the membranes were blocked with Tris-buffered saline  containing 0.05% Tween 20 (T-TBS) supplemented with 5% bovine serum albumin for the detection of total MYPT1 or T-TBS supplemented with 5% non-fat dry milk for the detection of phosphorylated from of MYPT1. The membranes were then subjected to an immunoblot analysis using anti-MYPT1 antibody (x 5000 dilution) and anti-phospho-MYPT1 (Thr850) antibody (x 1000 dilution). The immune complexes were detected and analyzed, as described above. The level of MYTP1 phosphorylation was normalized by the level of total MYPT1, and then expressed as a relative value, while assigning the basal level obtained before thrombin stimulation a value of 1.The level of GTP-bound form of RhoA was quantified with a pull-down assay using a Rho Activation Assay Biochem Kit , according to the manufacturer’s instructions. The level of the GTP-bound form of RhoA was normalized by the level of total RhoA, and then the basal level of the GTP-bound form of RhoA was assigned a value of 1.5 cells on glass coverslips  in 35 mm culture dishes. Fluorescence staining of phosphorylated MLC, VE-cadherin and F-actin was performed, as previously reported [PAECs and HMVECs were plated at 1.5 × 10reported . 4′,6-di6 tag, cell-penetrating peptide of human immunodeficiency viral Tat protein and a hemagglutinin tag at their N-terminus for the inhibitory proteins of RhoA and Rac1/Cdc42, respectively, as previously described [E. coli strain BL21(DE3)LysS and affinity-purified with Ni2+-loaded Hi-Trap chelating column on Akta Prime . The column eluate  of the inhibitory protein of Rac1/Cdc42 was directly used in the experiment, while the column eluate of the inhibitory protein of RhoA was dialyzed against PBS and the dialysate was used in the experiment. The protein concentrations of the inhibitory proteins of RhoA and Rac1/Cdc42 were determined to be 186 μM and 475 μM, respectively, with the Bradford method using bovine serum albumin as the standard . PAECs were exposed to the protein preparations in HBS for 30 min prior to and during stimulation with 1 unit/mL thrombin.The RhoA-binding domain of ROCK (amino acid residues 941-1075 in humans) and the Rac1/Cdc42-binding domain of p21-activated protein kinase 1 (amino acid residues 67-150 in humans) were prepared as fusion proteins with a (His)escribed –21. The P < 0.05 was considered to be statistically significant.The data are presented as means ± standard error of the mean (SEM) of the indicated number of independent experiments. In bar graphs, the circles overlaid on columns indicate individual data points, while data points in the same color are derived from the same set of experiments. The StatView ver.5 software program  was used to evaluate the statistical significance by an analysis of variance (ANOVA), followed by the Dunnett's post hoc test for comparisons to control condition and the Fisher post hoc test for multiple comparisons, as indicated in the figure legends. A value of PAECs were treated with simvastatin for 16 h, and then stimulated with 1 unit/mL thrombin in the presence of simvastatin. The concentration of thrombin used in the present study is the optimal concentration to induce the maximal effects on TEER and MLC phosphorylation, as previously reported . SimvastTo obtain some mechanistic insight into the inhibitory effect of simvastatin on the thrombin-induced MLC phosphorylation and decrease in TEER, the antagonizing effects of GGPP and FPP were investigated. MLC di-phosphorylation induced by 1 unit/mL thrombin was significantly decreased in PAECs pretreated with 10 µM simvastatin for 16 h Fig. a, b. WheThe preventive effects of GGPP and FPP were also examined regarding the thrombin-induced decrease in TEER Fig. c. The inThrombin caused MLC di-phosphorylation and actin bundle formation at the cell periphery at 3 min after stimulation, both of which were absent before thrombin stimulation . Images of confocal microphotographs obtained before (Control) and 15 min after stimulation with 1 unit/mL thrombin in three independent experiments  show the staining of F-actin, VE-cadherin and nuclei (TO-PRO-3) in the indicated colors.


A network of autonomous, ice-tethered buoys was deployed around the Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC) experiment in late September 2019 for a year-long drift in the Arctic Transpolar Drift Stream. The buoys were deployed as part of the MOSAiC distributed network (DN) which included 12 multi-instrumented ice stations and an additional 116 GPS buoys distributed primarily within a 40 km radius of the MOSAiC Central Observatory. Buoy coverage within the DN was maintained with additional deployments throughout the year-long drift allowing for collection of data over a full sea ice growth and melt cycle. All GPS position data from buoys deployed within the DN have been assembled and processed into the collection of 216 quality-controlled buoy drift tracks presented in this dataset covering the period 26 September 2019 – 23 May 2021. The drift tracks in this collection are ideal for studies of dynamic sea ice motion around the MOSAiC experiment at cascading spatial scales ranging from 100s of meters to 100s of km. Organized networks of autonomous GPS buoys deployed on sea ice are used to compute strain rates by tracking the area change from arrays of buoys over time and buoys deployed within a hierarchical network allow for analyses of the horizontal deformation  of sea ice at a variety of nested spatial scales. In this data descriptor we present a data set of buoy drift tracks collected as part of the Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC) experiment2 that are suitable for analyses of sea ice drift and deformation.The dynamic drift and redistribution of sea ice is monitored using autonomous ice-tethered buoys by tracking time series of the buoy Global Positioning System (GPS) positionsPolarstern3 attached to a sea ice floe in the northern Laptev Sea  shortly after the onset of ice growth and began to drift across the central Arctic, eventually reaching the sea ice edge in Fram Strait  the following summer4  of autonomous buoys which were primarily deployed throughout a 40 km radius around the Polarstern and the CO2. Buoys were deployed at greater distances around the DN in coordination with the International Arctic Buoy Programme (IABP), increasing the DN sampling area to 100s of km around the MOSAiC site, covering cascading scales of differential motion2. The buoys that were initially deployed in October 2019 in a 40 km radius ring around the Polarstern were used in the Sea Ice Drift Forecast Experiment program .The MOSAiC field experiment was a year-long drift campaign from October 2019 – September 2020 during which the research ice breaker er4 Fig. . An ice Polarstern (ISPOL) 2004–2005 in the Weddell Sea7, Sea Ice Experiment: Dynamic Nature of the Arctic (SEDNA) 2007 in the Beaufort Sea8, and the Norwegian young sea Ice2015 (N-ICE2015) expedition in the Arctic9. The novelty of this collection of drift tracks from the MOSAiC experiment is that the DN was deployed and maintained for a full year, which can be used to produce a time series of strain rates for a full seasonal ice growth cycle. Further, the density of buoy deployments in the DN will allow for an analysis of sea ice drift and deformation over a range of spatial scales from 100s of m to 100s of km.This dataset contains 216 quality-controlled buoy drift tracks collected from all autonomous ice-tethered buoys deployed in the DN during the MOSAiC experiment. The drift track measurements from this data set are concentrated in the region of the Transpolar Drift Stream beginning in the high Arctic around 85 °N, north of the Laptev Sea and following the path of sea ice export through Fram Strait and along the eastern coast of Greenland Fig. . The dri2. A full description of the DN led by B. Rabe is in preparation; thus, only a brief description of buoy deployment sites is provided here. The DN was centered around the ice camp known as the CO which was established on the ice floe to which the Polarstern anchored, radiating outward from the CO at a distance of approximately 40 km  of autonomous ice-tethered buoys was deployed around the drifting MOSAiC experiment in the Arctic Ocean as it traversed the Transpolar Drift Stream Fig. 2. A full km Fig. . 11 remoAkademik Federov research ice breaker with helicopter support in late September and early October 20192. Leg 1 deployments established the locations of CO1, L sites, and M sites within the DN  to complete a personnel exchange in mid-May 2020 ending the first passive drift period2. The ship then returned to the original CO1 floe which had since broken into smaller floes to recommence passive drift on 19 June 2020 for leg 4  used to document instrument deployments at MOSAiC inaccurately describe the OptiCAL buoys as Light Ice Tethered Profilers (LITO) or Environmental Pope (Envipope) buoys , ocean conductivity, temperature, and depth buoy (CTD), drift-towing ocean profiler (DTOP), autonomous ocean flux buoy (Flux Buoy), modular sea ice buoy (IMBFlex), ice-tethered profiler (ITP), optical chain and logger (OptiCAL), radiation station, seasonal ice mass balance buoy , snow height beacon (Snow Buoy), surface velocity program (SVP) drifters, and thermistor string buoy (Thermistor). Table 12). The three L sites were instrumented with flux buoys, Seasonal IMB, radiation station, snow buoys, thermistor, ITP, and ASFS sleds (See L_site_buoy_summary.csv deposited at Arctic Data Center12). The eight M sites were equipped with CTD profilers, snow buoys, thermistor and DTOP ocean profiler buoys (See M_site_buoy_summary.csv deposited at Arctic Data Center12). The remainder of the DN included the P site buoys that were distributed throughout the study area. The 116 P site deployments in the DN included 110 SVP buoys, six thermistor, and 2 snow buoys (See P_site_buoy_summary.csv deposited at Arctic Data Center12).During MOSAiC, the CO1, CO2, CO3, and LM sites were equipped with a variety of buoy types including flux buoys, Seasonal IMB, OptiCAL, IMBFlex, radiation station, snow buoys, thermistor, CTD, an ASFS sled and SVP buoys  where preliminary buoy data from MOSAiC is hosted. The raw data source for each buoy type/model is provided in Table A significant portion of the development of this dataset involved obtaining the raw data for each buoy from one of three source types. The ASFS data and data from SVP buoys manufactured by Taiyuan University of Technology (TUT) were obtained directly from the data providers or instrument principal investigators (PIs) for use in this data set. The CTD, Snow Buoy, Thermistor, and SVP models manufactured by MetOcean Telematics, PacificGyre IT, and SAMS Enterprise Ltd were downloaded directly from the manufacturer’s online data portals Table  with pri13.The raw data included all measurements made by the instrument. We extract the GPS position observations including the latitude, longitude, and observation datetime from the files, reformat the datetimes if needed to a consistent format (YYYY-MM-DD HH:MM:SS) in UTC time, and compile the data into a single file per buoy Fig. . The res9 and Antarctic7 with further refinement as described in the next section. As a first pass to remove obviously erroneous data the raw drift track files from step 1 were sorted and filtered in the following order  are removed.Remove any observations with a latitude or longitude value outside of the valid range. Valid latitudes and longitudes range from 50 °N to 90 °N and −180 °W to 180 °E, respectively.The procedure used to clean the raw GPS data is the same methodology used by prior studies that derived horizontal sea ice deformation from buoy networks in the Arcticder Fig. .The obseIt should be noted that removing observations that fall outside of the fixed valid latitude range confines drift tracks to the Arctic domain but does not confine tracks to the sea ice region. The sea ice edge is well north of 50 °N latitude; thus, GPS observations were collected from buoys that were no longer tethered to sea ice and were instead drifting in the ocean as can be seen in Fig. https://sensor.awi.de/; Last accessed: 4 October 2022) during the field experiment or from deployment reports archived at www.meereisportal.de (Last accessed: 22 August 2022).Many of the buoys in the MOSAiC DN were turned on and collecting data before their deployment on the ice and several buoys were recovered and then redeployed at a new location. Thus, we extracted the data during valid buoy deployment periods from the cleaned time series. The timeseries for one buoy deployment begins with the first timestep following the deployment time and either [1] the last timestep when a good buoy position was reported before instrument failure or [2] the last timestep before the buoy was physically recovered. Buoy deployment and recovery times were obtained from entries in the buoy action log registered in the Alfred Wegner Institute (AWI) Sensor Information System , distance travelled (km), and mean drift speed (m/s) computed with a backward difference method. For many buoys, the regular sampling frequency varied over the deployment period; thus, the sampling frequencies reported in the summary tables, represent the highest frequency reporting interval of the drift track.Additional summaries of each buoy drift track in the dataset are found in four summary tables organized by deployment sites  deposited with the drift track data set at Arctic Data CenterProcessed drift tracks for individual buoys deployed in the DN and the raw GPS position files associated with each buoy are provided in comma-separated value (.csv) format. The processed data files contain sorted records of the date and time of the position observation in UTC, latitude, and longitude. Additional metadata providing details on the buoy type, manufacturer, model, sampling frequency, buoy owner or project PI, and authors of the source data for each drift track are provided in a separate .csv file that is archived with the processed drift tracks (DN_buoy_list_v2 .csv).12. Additionally, all archived data associated with this MOSAiC DN buoy drift track data set including the raw buoy data and processing code can be accessed at the Arctic Data Center Data Portal website .The full record of processed drift tracks from autonomous buoys in the MOSAiC DN from 26 September 2019 through 23 May 2021 is archived in the Arctic Data Center data repositoryThe .csv files for the processed data use descriptive filenames to identify the drift track data contained in the file. Filenames in the data set follow the structure “DNID_IMEI_SensorID.csv” where the DNID, International Mobile Equipment Identity (IMEI), and SensorID components correspond to the deployment site, instrument, and unique chronological deployment code, respectively, of the specific drift track contained in the file. The codes used in the file naming convention are described further in Table Specific buoy drift tracks or all tracks associated with a unique buoy can be identified in the data set using the identification codes in the filenames of the processed .csv files. The three identification codes used in the data set are described in detail below to help users identify specific tracks or buoys of interest and are summarized in Table Polarstern left the CO1 site and first ice floe to exchange personnel between legs 3 and 4. CO2 identifies buoys that were deployed after the Polarstern returned to the initial CO1 site during leg 4. CO3 identifies buoys deployed at a new ice floe in the CO that was established when the Polarstern repositioned for leg 5 following breakup of the initial MOSAiC floe at the sea ice edge in late July 2020.The first of the three identification codes, the DN ID, indicates the ice station or position of deployment within the DN , are known as “P buoys” for point or position buoys. The DN ID code for P buoys are numbered and preceded by a “P”. P buoys numbered P001 – P069 were deployed in leg 1, P070 – P087 were deployed in leg 3, and P090 – P120 were deployed in leg 5.The IMEI number is a 15-digit identification number that is unique to a specific buoy or instrument. During MOSAiC, many of the buoys were recovered and redeployed elsewhere in the DN at a later time; thus, the IMEI, like the DN id codes, cannot be used as a unique identifier for drift tracks. In this dataset the IMEI number identifies the instrument that collected the data for the drift track.The Sensor ID code is an identification code used to identify deployments of instruments on the AWI Sensor Information System (sensor.awi.de). All actions related to buoy deployment, servicing, recovery, and failure during the MOSAiC field campaign were registered in a tracking system and linked to the buoy Sensor ID code assigned on deployment.The Sensor ID code is composed of the deployment year 2019 or 2020), a single letter code to describe the general buoy type, and a number that corresponds to chronological deployment order collective of all instruments deployed during the MOSAiC expedition. Buoy types are codified in the Sensor ID with the letter listed in the buoy type column of Table 9 or 2020All buoy drift tracks in this dataset are associated with a Sensor ID code that was assigned on deployment with the exception of tracks from the ASFS. The ASFS are not buoys, but rather instrumented sleds that were deployed to collect a suite of atmospheric and radiation data at each of the L sites and in CO2 and CO3. The ASFS collected high frequency GPS positions which provide highly accurate drift tracks in this data set. However, the ASFS stations were not assigned Sensor ID codes like other buoys in the DN and do not have an associated IMEI number. Instead, we identify drift tracks from ASFS deployments using alternate codes unique to this data set. The three ASFS stations and one flux tower are identified as ASFS30UCB, ASFS40UCB, ASFS50UCB, and ASFSTUCB (“T” for tower) used in place of an IMEI number and appended with a number to differentiate redeployments of the systems in chronological order. For example, “ASFS50UCB3” is the code used to identify the third deployment of the ASFS50UCB station in lieu of a Sensor ID code.14 . As the autumn and winter seasons progressed, the ice pack in the region around the Polarstern thickened and consolidated through October and November 2019 and the observed dynamic ice motion decreased in December 2019 and January 202016. Dynamic ice motion increased to moderate levels in the early spring and increased suddenly as the MOSAiC site approached the marginal ice zone16 during leg 4  should have similar drift trajectories, especially during periods where the sea ice is fairly consolidated  and be less consistent when the sea ice is dynamically active and broken up such as during the summer melt season and as the DN neared the marginal ice zone during leg 4.In the early autumn when the DN was deployed in leg 1, the sea ice within a 40 km radius of CO1 was a collection of loosely assembled, first-year ice floes that had survived the summer melt seasong 4 Fig. . TherefoPolarstern during passive drift periods at CO1 (legs 1–3), CO2 (leg 4), and CO3  and with observation frequency of 4 hours or less were selected for comparison during each drift. Daily time series of drift speed and direction were obtained for each buoy track using a backward difference method. The drift speed was computed by dividing the geodesic distance based on the WGS-84 ellipsoid between two consecutive buoy position observations by the time step in s between the observations. The computed speed is then assigned to the later time step and is expressed in ms−1. Drift direction was computed as a forward azimuth toward the later time step and expressed in degrees with north at 0°. The circular mean and standard deviation of the drift speed and direction of the selected buoys for each CO was then computed. The daily mean Polarstern drift speed was computed as above using master tracks from the Polarstern MOSAiC cruises at 10 minute resolution21 and the direction was obtained from the Polarstern course field provided in the master track data. Figure Polarstern drift speed and direction, respectively, for the three passive drift periods of MOSAiC smoothed with a 3-day running mean. The mean of buoy drift in the vicinity of Polarstern during the passive drift periods at CO1, CO2, and CO3 are shown with the solid black curves. The cyan shading shows ±1 standard deviation to illustrate the variability of the buoy drift observed around the ship.To assess the variability in buoy drift tracks from the DN, we compare the mean drift speed and direction of buoys within the primary core of the DN to the drift of  CO2 leg , and CO3 CO2 leg , and CO3Polarstern (dotted red curves), the mean buoy drift speed and direction (black curves) are very similar indicating that the surrounding ice is drifting in a similar trajectory to that of Polarstern during the CO1 and CO3 drift periods . CO2 is also the period in which the largest standard deviations in buoy drift speed (0.1 ms−1) and direction (68.5°) occur. The CO2 drift occurred in leg 4 of the MOSAiC expedition  and larger differences between the mean buoy drift and Polarstern drift is consistent with what we expect for sea ice conditions during the summer melt season and as the DN passed through Fram Strait.When compared to the ods Fig. . When hiods Fig.  bottom, ion Fig.  when thePolarstern drift and the mean of buoys in the vicinity of the ship and COs, during the more consolidated sea ice periods (CO1 and CO3) and increased variability observed during CO2 corresponds well with the expected drift differences of more broken up floes that would be present with coincident sea ice conditions. This result gives us confidence that the buoy GPS drift tracks are accurately representative of the sea ice drift conditions in the DN. The drift comparison in Fig. Polarstern was during each leg of the drift experiment  such that buoy observations begin at the top of the hour to enable easier comparison of the positions of different buoys at the same time stamp. However, it is important to note that interpolating drift tracks from buoys that sampled less frequently  to hourly or finer time scales may produce unexpected results in the drift tracks.14 (Table 23. Users should be aware that position outliers in the drift tracks are not filtered out in the minor data cleaning procedures applied here.Although the expected GPS accuracies of buoy drift tracks are 3 m or less


Posterior pelvis ring injuries represent typical high-energy trauma injuries in young adults. Joint stabilization with two cannulated sacroiliac (SI) screws at the level of sacral vertebrae S1 and S2 is a well-established procedure. However, high failure- and implant removal (IR) rates have been reported. Especially, the washer recovery can pose the most difficult part of the IR surgery, which is often associated with complications.The aim of this biomechanical study was to evaluate the stability of S1-S2 fixation of the SI joint using three different screw designs.n = 6) for SI joint stabilization through S1 and S2 corridors using either two 7.5 mm cannulated compression headless screws (group CCH), two 7.3 mm partially threaded SI screws (group PT), or two 7.3 mm fully threaded SI screws (group FT). An SI joint dislocation injury type III APC according to the Young and Burgess classification was simulated before implantation. All specimens were biomechanically tested to failure in upright standing position under progressively increasing cyclic loading. Interfragmentary and bone-implant movements were captured via motion tracking and evaluated at four time points between 4000 and 7000 cycles.Eighteen artificial hemi-pelvises were assigned to three groups , followed by group CCH (9820 ± 597), and group FT (7202 ± 1087), being significantly lower in group FT compared to both groups CCH and PT, p ≤ 0.027.Combined interfragmentary angular displacement movements in coronal and transverse plane between ilium and sacrum, evaluated over the measured four time points, were significantly bigger in group FT versus both groups CCH and PT, From a biomechanical perspective, S1-S2 SI joint fixation using two cannulated compression headless screws or two partially threaded SI screws exhibited better interfragmentary stability compared to two fully threaded SI screws. The former can therefore be considered as a valid alternative to standard SI screw fixation in posterior pelvis ring injuries. In addition, partially threaded screw fixation was associated with less bone-implant movements versus fully threaded screw fixation. Further human cadaveric biomechanical studies with larger sample size should be initiated to understand better the potential of cannulated compression headless screw fixation for the therapy of the injured posterior pelvis ring in young trauma patients. High-energy pelvis trauma prevails in younger adults as opposed to low-energy trauma in elderly patients with poor bone quality associated with high morbidity and mortality rates . BiomechTherefore, the aim of this biomechanical study was to evaluate the stability of S1-S2 fixation of the SI joint with CCHS versus conventional fully and partially threaded SI screws in an artificial pelvic bone model. It was hypothesized that CCHS would provide equivalent stability as conventional SI screws.Nine anatomical composite pelvises  were used. A posterior SI joint dislocation injury type III APC according to the Young and Burgess classification was simulated on each side of all pelvises . For than = 6) for instrumentation as follows:Group CCH: Stabilization of the posterior pelvis ring with two 7.5 mmlong-threaded CCHS, 90 mm in length for S1 and 65 mm in length for S2;Group PT: Stabilization of the posterior pelvis ring with two 7.3 mm partially threaded cannulated SI screws and washers, 90 mm in length for S1 and 65 mm in length for S2;Group FT: Stabilization of the posterior pelvis ring with two 7.3 mm fully threaded cannulated SI screws and washers, 90 mm in length for S1 and 65 mm in length for S2.Each pelvis was considered for instrumentation and testing of its left and right site, resulting in eighteen available hemi-pelvis constructs, which were assigned to three groups of six specimens each (The screws in groups PT and FT were made of stainless steel (316LVM), whereas the screws in group CCH were made of titanium alloy (Ti-6Al-4V ELI). All implants were provided by the same manufacturer . A hemi-pelvis model was considered for biomechanical testing; therefore, both sides of the same pelvis were instrumented. The symphysis was discontinued by cutting the rami at midshaft on both sides. The ilium of the non-tested site was detached until the testing of the contralateral site was completed. Afterwards, the contralateral side was instrumented and the ilium of the tested side detached. The screw lengths were chosen such that their tips did not cross the midline of the sacrum, thus allowing for bilateral testing. Each group consisted of specimens with three left and three right SI joint fixations. The surgical treatment of the APC III injury was carried out according to the AO principles . An expeFollowing anatomical reduction of the SI joint, in all groups the screws were placed perpendicular to it within the bone specimen under avoidance of the nerve root tunnels using guidewires. All screws were placed with the help of a custom aiming device for standardized and repeatable wire- and screw placement. This approach assured avoidance of any perforations, via falsae, or cortical disruptions. The S1 screw was always placed first, followed by insertion of the S2 one. The screws just extended to the midline of the sacral body and  were tightened according to the best of the operator's knowledge. After instrumentation, true lateral, inlet and outlet projections were visualized via X-rays for documentation and verification of the positioning of the screws  equipped with a 5 kN/50 Nm load cell. The setup with a specimen mounted for testing is presented in Fig. The loading protocol commenced with an initial nondestructive quasi-static ramp from 20 N preload to 200 N at a rate of 18 N/s, followed by progressively increasing cyclic loading in axial compression with a physiological profile of each cycle at a rate of 2 Hz . KeepingMachine data in terms of axial displacement and axial load were acquired at 200 Hz from the machine transducer and load cell throughout the tests. Based on these, Initial Construct Stiffness was calculated from the ascending load–displacement curve of the initial quasi-static ramp.The positions of the optical markers attached to the sacrum, ilium, and the two SI screws were recorded at 20 Hz from a stereographic camera system  for motion tracking. Based on these, interfragmentary and bone-implant movements were analyzed in all six degrees of freedom. Total Displacement was defined as the magnitude of the deviating movement between the two fragments measured in the most superior fracture aspect. Gap Angle was defined as the combined angular displacement in sagittal and coronal plane of the sacrum relative to the ilium. Furthermore, Screw Tilt Ilium was defined as the angular displacement of the S1 screw relative to the ilium. Next, Screw Tip Cutout was defined as the displacement of the S1 screw tip relative to the sacrum perpendicular to the screw axis. The outcome measures were evaluated at four time points of cyclic testing after 4000, 5000, 6000, and 7000 cycles. The latter represented the highest rounded number of cycles when none of the specimens had failed. The outcome measures were considered with respect to their values at the beginning of the cyclic test and were calculated under peak loading condition. In addition, a clinically relevant criterion for specimen failure was arbitrarily set at 5° Gap Angle, and the number of cycles until its fulfillment – defined as Cycles To Failure – was evaluated together with the corresponding peak load – defined as Load At Failure.Statistical analysis was performed with SPSS software package . Normality of data distribution was screened and proved with Shapiro-Wilk test. Mean value and standard deviation (SD) were calculated for each parameter of interest and group separately. One-Way Analysis of Variance (ANOVA) and General Linear Model Repeated Measures tests with Bonferroni post-hoc test for multiple comparisons were conducted to detect significant differences among the treatment groups. Level of significance was set at 0.05 for all statistical tests.p = 0.137.Initial Construct Stiffness was 92.1 ± 29.1 N/mm for group CCH, 85.3 ± 20.2 N/mm for group PT, and 60.0 ± 14.9 N/mm for group FT, without significant differences among the groups, p ≤ 0.047. Similarly, instrumentation with fully threaded screws in group FT resulted in significantly bigger Screw Tilt Ilium compared to partially threaded screws in group PT, p = 0.038, without further significant differences among the groups, p ≥ 0.118. Finally, both Total Displacement and Screw Tip Cutout remained not significantly different among the groups, p ≥ 0.200.The outcome measures evaluated after 4000, 5000, 6000, and 7000 cycles are summarized in Table p ≤ 0.027 , followed by group CCH (9820 ± 597/691.0 ± 29.9 N), and group FT (7202 ± 1087/560.1 ± 54.4 N), with significantly lower values in group FT compared to both groups CCH and PT, The objective of this study was to assess the biomechanical potential of cannulated compression headless screws used for fixation of posterior pelvis ring injuries, and consecutively, to possibly decrease the rate of implant failures, consolidate minimally invasive procedures, and avoid secondary implant removal surgeries.With regard to the number of test cycles until failure and the corresponding failure load, both groups CCH and PT demonstrated considerably better stability versus group FT, and comparable stability versus each other. On the other hand, all three groups revealed similar initial construct stiffness.SI screw fixation of the posterior pelvis ring in unstable injuries is a well-established surgical procedure. SI screw fixation through S1 and S2 sacral body corridors was described to feature the best implant positioning . TherefoIt is known that the SI joint has a physiological movement from 2 mm to 4 mm –34. The For the partially threaded screws in groups PT and CCH, a movement of the joint around the threadless smooth part of the screw shafts is conceivable and could therefore explain their higher stability during testing. Likewise, as seen in fixations of ankle fractures, partially threaded screw designs have been proven to decrease both initial screw stiffness and yield load, compared to fully threaded screws . GeneralThe hypothetical target group for SI screw fixation with cannulated compression headless screws considers young patients with good bone quality. This study was able to show a partly superior and partly comparable stability of fixation with these screws versus conventional SI screws.There is very scarce literature on IR rates following SI fixation, with most of the studies discussing indications for IR regarding the anterior pelvis ring , 37.A complete IR may be necessary in the event of suspected infection or metal allergies, as well as in case of unexplained pain and/or explicit patient request. The aspect of removing the washer – "the Washer problem" – has already been described in the literature , 16. To A previous biomechanical investigation reported that 7.3 mm fully threaded transiliac–transsacral screw fixation would result in less fracture displacement at the S1 foramen compared to 7.3 mm partially threaded transiliac–transsacral screw fixation in vertically transforaminal sacral fractures . This reA recent study with similarly used specimens concluded that fully threaded 7.3 mm screws could withstand significantly higher axial loads to failure than partially threaded 7.3 mm screws . HoweverFurther studies should follow in order to consolidate the obtained results on real bones and, if applicable, to confirm them in a clinical study.The main limitation of this study is the chosen specimen model implicating less physiological conditions due to lack of ligaments. This makes the simulation of a ligamentous injury to the pelvis difficult. However, as this is an experimental study with an innovative approach, with no available data for this approach in the literature, and unpredictable outcomes, the authors have chosen the above-mentioned specimen model as a first step. Only after such a first step it is ethically justifiable to carry out a cadaveric study as a second step. Further, to accommodate the main limitation, a type III APC injury according to the Young and Burgess classification was deliberately chosen. According to its definition, the ligaments are ruptured and an absolutely unstable situation prevails. Therefore, the ligaments could be largely neglected in the selected test series. Nevertheless, it is known that artificial pelvises allow for a standardization of the treatment groups – thus overpowering the almost uncountable variations in bone quality seen in human cadaveric specimens – and are more cost-effective –48. SyntAdditionally, the chosen sample size was relatively small, yet comparable to similar biomechanical studies investigating posterior pelvis fixation techniques –51, 54. From a biomechanical perspective, S1-S2 SI joint fixation using two cannulated compression headless screws or two partially threaded SI screws exhibited better interfragmentary stability compared to two fully threaded SI screws. The former can therefore be considered as a valid alternative to standard SI screw fixation in posterior pelvis ring injuries. In addition, partially threaded screw fixation was associated with less bone-implant movements versus fully threaded screw fixation. Further human cadaveric biomechanical studies with larger sample size should be initiated to understand better the potential of cannulated compression headless screw fixation for the therapy of the injured posterior pelvis ring in young trauma patients.


Natronoccoccus sp. TC6 and Halorubrum tebenquichense SU10 was not previously described, being the aim of this work. The carotenoids profile was performed by UV-Vis spectrophotometry, thin-layer chromatography, nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry (UPLC-ESI-MS/MS). Antioxidant capacity was determined for DPPH, ABTS, and FRAP. In addition, MMP-9 inhibition was studied using docking simulations. The carotenoid profile of studied strains was composed of bacterioruberin, some derivatives like mono, bis, and tris anhydrobacterioruberin, and also some bacterioruberin cis isomers. The carotenoid pools showed antioxidant capacity for DPPH > ABTS > FRAP; Natronococcus sp. TC6 carotenoid pool was better for ABTS and DPPH, while Halorubrum tebenquichense SU10 carotenoid pool was better for FRAP. Additionally, docking and molecular dynamics suggest that bacterioruberin inhibits MMP-9 through hydrophobic interactions near the catalytic site. Bacterioruberin shows the higher binding energy of −8.3 . The carotenoids profile of both strains was elucidated, their antioxidant activity and singular participation of each carotenoid on MMP-9 in silico inhibition were evaluated.Natural pigments from haloarchaea are of great interest; bacterioruberin is the major pigment, it shows higher antioxidant power when compared with β-carotene. However, characterization of bacterioruberin and its isomers along with its antioxidant and the matrix metallopeptidase 9 (MMP-9) inhibition activities in extracts from  Halobacteriaceae and Haloferacaceae families), extreme microorganisms that grow in hypersaline environments, up to 4.5 M of NaCl, can produce and accumulate red pigments in their cell membrane + m/z 740.5, 741.5, 742.5 (45H64O) at m/z 620.3 and monoanhydrobacterioruberin MABR (C50H74O3) at m/z 722.47 (40H52O4) at m/z 598.39, β-carotene (C40H56) at m/z 537. 2, phytoene (C40H64) at m/z 543.50, and lycopersene (C40H66) at 545.96, some of these found in TLC analysis . In the case of ferric ion-reducing antioxidant power FRAP  in the inhibition between the strains from which the pigments were extracted, Natronococcus sp. TC6 was better in ABTS and DPPH radicals and Halorubrum tebenquichense SU10 was better in FRAP. In in the case of FRAP, there are significant differences (p < 0.05) for extract pigment concentration   E–G.cis-26-bacterioruberin fragment has the lowest binding energy value, and its affinity is not in the catalytic cavity of MMP-9, but near Glu402  derivatives found in the pigment extracts like monoanhydrobacterioruberin (MABR), bisanhydrobacterioruberin (BABR), and 2-isopentenyl-3,4 dihydrorhodopin (DIR) are reported in strains like ismortis ,38. BAR p. TBZ16 . A majorrotenoid ,40. In toxanthin ,42.Natronococcus sp. TC6 and Halorubrum tebenquichense SU10) were higher (above 90%) compared with pigments extracted from Aquisalibacillus elongatus MB592, Haloarcula japonica HM1, and Halobacterium salinarum HM2 that were able to inhibit 20, 30, and 80% percent, respectively [Halorubrum tebenquichense, Haloarcula japonica HM1, and Halobacterium salinarum HM2 have been reported as good FRAP inhibitors (above 70%) [Natronococcus sp. showed the best results when compared with Halorubrum tebenquichense SU10 and other strains of the same genus [Natronococcus sp. TC6 and Halorubrum tebenquichense SU10 have a major antioxidant capacity of DPPH > ABTS > FRAP dominating the capacity of hydrogen and single electron transfer  as the typical “free radical scavenger” mechanism [Halorubrum tebenquichence SU10 on FRAP that shows more antioxidant capacity than Natronococcus sp. TC6, related to the presence of astaxanthin in Halorubrum pigment extract and its participation in a synergy with the other pigments.It has been shown that carotenoid extracts from several haloarchaea have antioxidant activities and are dose- and profile-dependent, particularly of isomers of bacterioruberin and other carotenoids . DPPH scectively . Other pove 70%) ,43. Contme genus . Accordiechanism ,45. In aechanism . Additioechanism . This ca2+) and calcium (Ca2+) dependent, present intracellularly and membrane-bound. Their importance is their participation in physiological processes including development, wound, healing, remodeling of tissues, angiogenesis, and particularly participation in the tumor invasion, metastasis, and angiogenesis acting in the degradation of gelatin and type IV, V, XI, and XII collagens. The overexpression of MMP-9 is associated with different cancers, such as lung, prostate, gastric, pancreatic, and colon and with other diseases, such as autoimmune and cardiovascular diseases [The MMP-9 is an enzyme of metalloproteinase group (MMP’s) particularly zinc  [Natronococcus sp. TC6 and Halorubrum tebenquichense SU10 extracts showed the highest binding energies.Docking studies point towards the importance of the archaeal carotenoids for MMP-9 inhibition because of their interaction in the catalytic pocket or around of active site. The inhibitory effect of archaeal carotenoids on MMP-9 has been studied in vitro using a carotenoid extract of 5 µg/mL) . HoweverThere are few reports about the molecular dynamics of bacterioruberin and other related carotenoids. The potential with molecular dynamics studies for other enzymes has been reported, like acetylcholinesterase (ACE) and butyrylcholinesterase using fragments of the carotenoids and showing binding energies in a range of ΔG  −7.42 to −10.35 ΔG . MoleculMolecular dynamics results suggest that bacterioruberin blocks the entry to both Binding Site 1 and Binding Site 2. The selection of the Allosteric Binding Site was based on bacterioruberin large size. Previous crystallographic studies have confirmed MMP-9 interactions with large molecules, such as antiMMP-9 antibodies, making it plausible for bacterioruberin to interact in that area as well. As mentioned earlier, most of the residues involved in these interactions are primarily hydrophobic, indicating hydrophobic interactions as the primary mode, with some hydrogen bond formation see D. AdditiThe carotenoids profile of both strains was elucidated, and their antioxidant activity and MMP-9 inhibition in silico were evaluated, future research must be focused on elucidating the mechanism to enhance bacterioruberin and derivates production at a large scale and their incorporation in functional foods and ingredients.Halorubrum tebenquichense SU10, isolated form Uyuni saltern in Bolivia and Natronococcus sp. isolated from El Golea, a salt lake in Argelia were characterized in their carotenoids pigments. Bacterioruberin was identified in both Halorubrum tebenquichense SU10 and Natronococcus sp. TC6 pigment extracts, also, bacterioruberin derivates were identified, 2-isopentenyl-3,4 dihydrorhodopin, and monoanhydrobacterioruberin. Other pigments and intermediaries were identified, astaxanthin, β-carotene, phytoene, and lycopersene. The pigment extracts showed considerable antioxidant activity on DPPH, ABTS, and FRAP. The inhibitory effect of the haloarchaeal pigments over MMP-9 was suggested by docking and molecular dynamics assays, monoahydrobacterioruberin and trisanhydrobacterioruberin display the best binding energy values. Molecular docking suggests that bacterioruberin has hydrophobic interactions with the tested MMP-9 sites, and the ligand remains stable during molecular dynamics simulations. Future research must be focused on large scale bacterioruberin production, also in the purification of pigments on the pool and their study on health benefits and also to verify their effect on MMP-9 inhibition on in vitro assays.