FtsEX-independent control of RipA-mediated cell separation in Corynebacteriales.

The bacterial cell wall is a multi-layered mesh, whose major component is peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. During cell division, a careful balance of PG synthesis and degradation, precisely coordinated both in time and space, is necessary to prevent uncontrolled destruction of the cell wall. In Corynebacteriales, the D,L endopeptidase RipA has emerged as a major PG hydrolase for cell separation, and RipA defaults have major implications for virulence of the human pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. However, the precise mechanisms by which RipA mediates cell separation remain elusive. Here we report phylogenetic, biochemical, and structural analysis of the Corynebacterium glutamicum homologue of RipA, Cg1735. The crystal structures of full-length Cg1735 in two different crystal forms revealed the C-terminal NlpC/P60 catalytic domain obtruded by its N-terminal conserved coiled-coil domain, which locks the enzyme in an autoinhibited state. We show that this autoinhibition is relieved by the extracellular core domain of the transmembrane septal protein Cg1604. The crystal structure of Cg1604 revealed a (ß/α) protein with an overall topology similar to that of receiver domains from response regulator proteins. The atomic model of the Cg1735-Cg1604 complex, based on bioinformatical and mutational analysis, indicates that a conserved, distal-membrane helical insertion in Cg1604 is responsible for Cg1735 activation. The reported data provide important insights into how intracellular cell division signal(s), yet to be identified, control PG hydrolysis during RipA-mediated cell separation in Corynebacteriales.

The Role of Physical Therapies in Wound Healing and Assisted Scarring.

Wound healing (WH) is a complex multistep process in which a failure could lead to a chronic wound (CW). CW is a major health problem and includes leg venous ulcers, diabetic foot ulcers, and pressure ulcers. CW is difficult to treat and affects vulnerable and pluripathological patients. On the other hand, excessive scarring leads to keloids and hypertrophic scars causing disfiguration and sometimes itchiness and pain. Treatment of WH includes the cleaning and careful handling of injured tissue, early treatment and prevention of infection, and promotion of healing. Treatment of underlying conditions and the use of special dressings promote healing. The patient at risk and risk areas should avoid injury as much as possible. This review aims to summarize the role of physical therapies as complementary treatments in WH and scarring. The article proposes a translational view, opening the opportunity to develop these therapies in an optimal way in clinical management, as many of them are emerging. The role of laser, photobiomodulation, photodynamic therapy, electrical stimulation, ultrasound therapy, and others are highlighted in a practical and comprehensive approach.

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria.

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannoside, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated glycosyltransferases for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here, we determined that PimA preferentially binds to negatively charged phosphatidyl-myo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. By using a combination of single-point mutagenesis, circular dichroism, and a variety of fluorescence spectroscopy techniques, we determined that this interaction is mainly mediated by an amphipathic α-helix (α2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. Interestingly, a flexible region within the N-terminal domain, which undergoes ß-strand-to-α-helix and α-helix-to-ß-strand transitions during catalysis, interacts with anionic phospholipids; however, the effect is markedly less pronounced to that observed for the amphipathic α2, likely reflecting structural plasticity/variability. Altogether, we propose a model in which conformational transitions observed in PimA might reflect a molten globule state that confers to PimA, a higher affinity toward the dynamic and highly fluctuating lipid bilayer.

Secondary structure reshuffling modulates glycosyltransferase function at the membrane.

Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including ß-strand-to-α-helix and α-helix-to-ß-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.

Benthodytes violeta, a new species of a deep-sea holothuroid (Elasipodida: Psychropotidae) from Mar del Plata Canyon (south-western Atlantic Ocean).

A new species of elasipodid holothuroid, Benthodytes violeta sp. nov., is described from the Mar del Plata Canyon off Buenos Aires Province (around 38ºS-54ºW). It was taken at four locations at depths ranging from 1500 to 1950 m. This new species has a violet gelatinous body of up to 200 mm in length, with eight pairs of dorsal appendages, lateral festooned edges and four rows of tube feet ventrally. Body wall ossicles comprise rods and crosses with three or four arms and a central bipartite apophysis borne on the primary cross; tentacles and gonad deposits comprise rods and crosses with three and four arms. This is the first report of a holothuroid from the Mar del Plata Canyon area.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

Complete genome sequence of Bradyrhizobium sp. 62B, a native nitrogen-fixing rhizobium isolated from peanut nodules.

We present the complete genome sequence of Bradyrhizobium sp. 62B, a strain isolated from the root nodules of peanut plants that grow in central Argentina. The genome consists of 8.15 Mbp, distributed into a chromosome of 7.29 Mbp and a plasmid of 0.86 Mbp.

Insights from structure-activity relationships and the binding mode of peptidic α-ketoamide inhibitors of the malaria drug target subtilisin-like SUB1.

Plasmodium multi-resistance, including against artemisinin, seriously threatens malaria treatment and control. Hence, new drugs are urgently needed, ideally targeting different parasitic stages, which are not yet targeted by current drugs. The SUB1 protease is involved in both hepatic and blood stages due to its essential role in the egress of parasites from host cells, and, as potential new target, it would meet the above criteria. We report here the synthesis as well as the biological and structural evaluation of substrate-based α-ketoamide SUB1 pseudopeptidic inhibitors encompassing positions P4-P2'. By individually substituting each position of the reference compound 1 (MAM-117, Ac-Ile-Thr-Ala-AlaCO-Asp-Glu (Oall)-NH2), we better characterized the structural determinants for SUB1 binding. We first identified compound 8 with IC50 values of 50 and 570 nM against Pv- and PfSUB1, respectively (about 3.5-fold higher potency compared to 1). Compound 8 inhibited P. falciparum merozoite egress in culture by 37% at 100 µM. By increasing the overall hydrophobicity of the compounds, we could improve the PfSUB1 inhibition level and antiparasitic activity, as shown with compound 40 (IC50 values of 12 and 10 nM against Pv- and PfSUB1, respectively, IC50 value of 23 µM on P. falciparum merozoite egress). We also found that 8 was highly selective towards SUB1 over three mammalian serine peptidases, supporting the promising value of this compound. Finally, several crystal 3D-structures of SUB1-inhibitor complexes, including with 8, were solved at high resolution to decipher the binding mode of these compounds.

A new species of Havelockia Pearson, 1903 from the Argentine Sea (Holothuroidea: Dendrochirotida: Sclerodactylidae).

Havelockia pegi sp. nov., is here described from shallow waters of the Argentine Sea. This new species is distinctive in the purple colouration of its tentacles, scarcity of body wall ossicles and the presence of rosette-shaped ossicles in both the introvert and the tentacles. It is not closely related to any of its congenors. This is the first record of a true sclerodactylid from Argentina. Thandarum hernandezi Martinez & Brogger, 2012, described in the family Sclerodactylidae, is now classified in the family Sclerothyonidae.

A molecular perspective on the invasibility of the southern ocean benthos: The impact of hypoxia and temperature on gene expression in South American and Antarctic Aequiyoldia bivalves.

When an organism makes a long-distance transition to a new habitat, the associated environmental change is often marked and requires physiological plasticity of larvae, juveniles, or other migrant stages. Exposing shallow-water marine bivalves (Aequiyoldia cf. eightsii) from southern South America (SSA) and the West Antarctic Peninsula (WAP) to changes in temperature and oxygen availability, we investigated changes in gene expression in a simulated colonization experiment of the shores of a new continent after crossing of the Drake Passage, and in a warming scenario in the WAP. Bivalves from SSA were cooled from 7°C (in situ) to 4°C and 2°C (future warmed WAP conditions), WAP bivalves were warmed from 1.5°C (current summer in situ) to 4°C (warmed WAP), gene expression patterns in response to thermal stress by itself and in combination with hypoxia were measured after 10 days. Our results confirm that molecular plasticity may play a vital role for local adaptation. Hypoxia had a greater effect on the transcriptome than temperature alone. The effect was further amplified when hypoxia and temperature acted as combined stressors. The WAP bivalves showed a remarkable ability to cope with short-term exposure to hypoxia by switching to a metabolic rate depression strategy and activating the alternative oxidation pathway, whilst the SSA population showed no comparable response. In SSA, the high prevalence of apoptosis-related differentially expressed genes especially under combined higher temperatures and hypoxia indicated that the SSA Aequiyoldia are operating near their physiological limits already. While the effect of temperature per se may not represent the single most effective barrier to Antarctic colonization by South American bivalves, the current distribution patterns as well as their resilience to future conditions can be better understood by looking at the synergistic effects of temperature in conjunction with short-term exposure to hypoxia.

Mitochondrial Heteroplasmy and PCR Amplification Bias Lead to Wrong Species Delimitation with High Confidence in the South American and Antarctic Marine Bivalve Aequiyoldia eightsii Species Complex.

The species delimitation of the marine bivalve species complex Aequiyoldia eightsii in South America and Antarctica is complicated by mitochondrial heteroplasmy and amplification bias in molecular barcoding. In this study, we compare different data sources (mitochondrial cytochrome c oxidase subunit I (COI) sequences; nuclear and mitochondrial SNPs). Whilst all the data suggest that populations on either side of the Drake Passage belong to different species, the picture is less clear within Antarctic populations, which harbor three distinct mitochondrial lineages (p-dist ≈ 6%) that coexist in populations and in a subset of individuals with heteroplasmy. Standard barcoding procedures lead to amplification bias favoring either haplotype unpredictably and thus overestimate the species richness with high confidence. However, nuclear SNPs show no differentiation akin to the trans-Drake comparison, suggesting that the Antarctic populations represent a single species. Their distinct haplotypes likely evolved during periods of temporary allopatry, whereas recombination eroded similar differentiation patterns in the nuclear genome after secondary contact. Our study highlights the importance of using multiple data sources and careful quality control measures to avoid bias and increase the accuracy of molecular species delimitation. We recommend an active search for mitochondrial heteroplasmy and haplotype-specific primers for amplification in DNA-barcoding studies.

3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor.

The constant selection and propagation of multi-resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as-yet untargeted metabolic pathways. Subtilisin-like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro-region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme-inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full-length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1Cat) without a pro-region. High-resolution 3D structures of PvS1Cat, alone and in complex with an α-ketoamide substrate-derived inhibitor (MAM-117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α-keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1' and P2' positions of the inhibitor, although P' residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate-derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1-specific inhibitors that may define a novel class of antimalarial candidates.

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic Actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. These bacteria have multi-layered cell walls composed of the mycolyl-arabinogalactan-peptidoglycan complex and a polar growth mode, thus requiring tight coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the coiled-coil protein Wag31. Here, using C. glutamicum, we report the discovery of two divisome members: a gephyrin-like repurposed molybdotransferase (Glp) and its membrane receptor (GlpR). Our results show how cell cycle progression requires interplay between Glp/GlpR, FtsZ and Wag31, showcasing a crucial crosstalk between the divisome and elongasome machineries that might be targeted for anti-mycobacterial drug discovery. Further, our work reveals that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis, similar to the gephyrin/GlyR system that mediates synaptic signalling in higher eukaryotes through network organization of membrane receptors and the microtubule cytoskeleton.

Whole-Genome Sequence of Burkholderia ambifaria Strain Q53, a Potential Plant Growth Promoter Isolated from the Rhizosphere of Peanut.

We report the complete genome sequence of Burkholderia ambifaria strain Q53, an environmental rhizobacterium isolated from the rhizosphere of peanut plants. The genome consists of 7.4 Mbp distributed into three circular chromosomes and was determined using a hybrid long- and short-read assembly approach.

Genome sequence of Mesorhizobium mediterraneum strain R31, a nitrogen-fixing rhizobium used as an inoculant for chickpea in Argentina.

Here, we report the complete genome sequence of Mesorhizobium mediterraneum R31, a rhizobial strain recommended and used as a commercial inoculant for chickpea in Argentina. The genome consists of 7.25 Mb, distributed into four circular replicons: a chromosome of 6.72 Mbp and three plasmids of 0.29, 0.17, and 0.07 Mbp.

Extracellular vesicles from Mycobacterium tuberculosis-infected neutrophils induce maturation of monocyte-derived dendritic cells and activation of antigen-specific Th1 cells.

Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.

[Clinical cases about the therapeutic use of debriding dressing hidrodetersive polyacrylate fibers with TLC and foam dressings TLC-NOSF polyurethane in chronic wounds]. / Casos clínicos sobre el uso terapéutico en heridas crónicas de: Apósitos desbridantes de fibras hidrodetersivas de poliacrilato con TLC. Apósitos de espuma de poliuretano con TLC-NOSF.

The treatment of chronic wounds requires the use of highly specific products for different phases of the healing process. This article raises a number of clinical cases with chronic wounds of vascular origin and pressure ulcers. Such cases required a initial debridement because of the large content of fibrin covering the wound bed at this stage was used dressing hidrodetersive polyacrylate fibers with TLC. Once the debridement is continued treatment with a polyurethane foam dressing with TLC-NOSF.

[Cost-effectiveness of a TLC-NOSF polyurethane foam dressing]. / Coste-Efectividad de un apósito de espuma de poliuretano con TLC-NOSF.

Chronic wounds represent a drain on the Spanish health system, nowdays is necessary an optimization of the resources used and that is for this that is necessary justify the use of the products over others through cost-effective studies for to show the economic benefit to professionals and the life quality of patient. This article compares the use of a new technology for format polyurethane foam, TLC-NOSF, with the most commonly used products for treating wounds. This comparison is made using a cost-effectiveness model (Markov Model). The results demonstrate that treatment with polyurethane foam dressing with TLC-NOSF are cost-effective versus treatments with polyurethane foams most commonly used in Spain.

Performance evaluation of a point of care cartridge of the new GEM Premier ChemSTAT analyzer.

Background and aims: GEM Premier ChemSTAT is a new point-of-care system providing rapid creatinine, BUN and tCO2 measurements together with electrolytes, metabolites, hematocrit, pH and pCO2 from a single whole blood specimen in acute care settings such as emergency departments and intensive care units. Accurate measurements of whole blood creatinine can aid in the diagnosis and treatment of renal diseases. Materials and methods: Heparinized whole blood samples from different clinical locations were evaluated on the GEM Premier ChemSTAT and results compared to plasma from the same samples on the Beckman AU5800 or whole blood on the GEM Premier 4000. Precision studies were conducted with whole blood and quality control material. Results: ChemSTAT correlated well with plasma samples on the AU5800 (regression slopes (S): 0.957-1.159, correlation coefficients (r)≥0.952) and with whole blood specimens on the GEM Premier 4000 (S: 0.9646-1.124, r ≥ 0.974). The repeatability was 0.1%-3.1% and QC precision were within lab and manufacturers' specifications. Conclusion: ChemSTAT demonstrated strong correlation to the comparative methods and excellent precision. Combining with its continuous quality management, ChemSTAT is suitable for acute care settings to provide rapid, reliable results, which could minimize time-to-treatment and improve patient outcome.

Gp29 LysA of mycobacteriophage TM4 can hydrolyze peptidoglycan through an N-acetyl-muramoyl-L-alanine amidase activity.

Bacteriophage endolysins are crucial for progeny release at the end of the lytic cycle. Mycobacteriophage's genomes carry a lysin A essential gene, whose product cleaves the peptidoglycan (PG) layer and a lysin B, coding for an esterase, that cleaves the linkage between the mycolic acids and the arabinogalactan-PG complex. Lysin A mycobacteriophage proteins are highly modular and in gp29 (LysA) of phage TM4 three distinctive domains were identified. By bioinformatics analysis the central module was previously found to be similar to an amidase-2 domain family with an N-acetylmuramoyl -L-alanine amidase activity. We demonstrated experimentally that purified LysA is able to lyse a suspension of Micrococcus lysodeikticus and can promote cell lysis when expressed in E. coli and Mycobacterium smegmatis. After incubation of LysA with MDP (Muramyl dipeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine) we detected the presence of N-acetylmuramic acid (NAcMur) and L-Ala- D- isoGlutamine (L-Ala-D-isoGln) corroborating the proposed muramidase activity of this enzyme. This protein was stabilized at acidic pH in the presence of Zn consistent with the increase of the enzymatic activity under these conditions. By homology modeling, we predicted that the Zn ion is coordinated by His 226, His 335, and Asp 347 and we also identified the amino acid Glu 290 as the catalytic residue. LysA activity was completely abolished in derived mutants on these key residues, suggesting that the PG hydrolysis solely relies on the central domain of the protein.