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Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1–7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death., Nature, 618 (7967), ISSN:0028-0836, ISSN:1476-4687

Drilling low-pressure formations represent challenges. In the present paper, an operational situation became an issue when during the drilling of a deviated well, the circulation was lost, and the BHA got stuck, followed by a gas inflow observed in the return line. After a well control procedure and several failed attempts to control the well and recover the drilling BHA, another problem occurred when the drill pipe got plugged, making it impossible to continue pumping control pills. The scenario described above was presented to the Coiled Tubing and Thru Tubing companies with the request of a proposal to help solve three specific goals to achieve remove the plug in the inner of the drill pipe, getting communication between the drill pipe and the open hole to be able to pump Lost Control Material (LCM) and control de well, perform a cut of the drill pipe in case the pipe couldn't be recover after control the gas inflow. With an interdisciplinary group, the problem was analyzed, and a proposal was submitted, including three runs with Coiled Tubing and Thru Tubing tools. The first run a cleanout of the drill pipe with a downhole motor and taper mill. The second run will be with an abrasive punching BHA to establish communication with the annuls, and the third run will be for an abrasive cutting that will allow the recovery of the drill pipe above the free point. The job preparation includes the engineering program with the operational parameters and flow chart and the logistic preparation with the resources such as coiled tubing equipment, thru-tubing BHAs, chemicals, and fluids, among others. During the operation, other discussions and revisions forced a change in the plan from the initial proposal, performing two of the three runs for the cleanout and abrasive cutting, which represented a benefit by controlling the well with the slurry pumped working as a LCM that helped control the gas inflow. With a conclusion of a successful cut and drill pipe recovery, the operation data and analysis of the preparation vs. the execution are presented in the paper.

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Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation and virulence. Competent bacteria produce so called ‘fratricins’ such as CbpD, that kill non-competent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall- and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here, we present cryo–electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies). Our results provide previously unknown insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.

RESUMEN Las garrapatas son artrópodos de gran impacto sobre la economía pecuaria y la salud humana debido a las pérdidas generadas en la producción, y a su capacidad de transmitir microorganismos. Pese a la evidencia de circulación de una gran diversidad de garrapatas en Colombia, no se cuenta con compilaciones actualizadas de la variedad de especies presentes, por lo cual los autores presentan una revisión de literatura sobre las garrapatas de la familia Ixodidae reportadas en Colombia incluyendo su distribución geográfica, sus hospederos y los microorganismos reportados en aquellas especies registradas en este país. En la recopilación se evidenció la presencia en Colombia de diferentes especies de garrapatas de los géneros Amblyomma, Ixodes, Haemaphysalis, Rhipicephalus y Dermacentor. El género con mayor variedad fue Amblyomma con 29 especies, seguido de Ixodes con 11 especies, y de los géneros Dermacentor, Haemaphysalis y Rhipicephalus se reportaron dos especies para cada uno. Las especies se distribuyeron en la mayoría de los departamentos de Colombia y en una amplia diversidad de hospederos entre los cuales se incluyen animales domésticos, sinantrópicos y silvestres. Adicionalmente se encontró que algunas garrapatas reportadas podrían jugar un papel como vectores potenciales de diversos microorganismos que pueden afectar a la salud publica y veterinaria. Esperamos que este trabajo sea un aporte para apoyar la investigación en garrapatas y el estudio de los agentes transmitidos por estas en Colombia. ABSTRACT Ticks are arthropods with significant impact on the economy of livestock and public health, due to losses generated in production, and their ability to transmit microorganisms. Despite the evidence of circulation of different tick species in the country, there are no updates concerning the variety of existing species, therefore, the authors of this manuscript offer a literature review on ticks from the Ixodidae family reported in Colombia including their geographical distribution, their hosts and the microorganisms they carry and might transmit in Colombia. In this manuscript, we described the presence in the country of different species of ticks of the genera Amblyomma, Ixodes, Haemaphysalis, Rhipicephalus, and Dermacentor. The genus with the greatest variety was Amblyomma with 29 species, followed by Ixodes with 11 species, and of the genus Dermacentor, Haemaphysalis and Rhipicephalus two species were reported for each. These species are distributed in most departments and a wide variety of hosts, including domestic, synanthropic and wild animals. Additionally, it was found that some reported ticks could play a role as potential vectors of various microorganisms that could affect public and veterinary health. We expect that this work will be a contribution to support tick research and the study of tick-borne agents in Colombia.

LtaA catalyzes glycolipid translocation by a 'trap-and-flip' mechanism, pointing to a shared mechanistic model among MFS lipid transporters. Asymmetric lateral openings allow access of the entire lipid substrate to the amphipathic central cavity. Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters take part in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acid synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a 'trap-and-flip' mechanism that might be shared among MFS lipid transporters.

Nucleotide-based signaling molecules (NSMs) are widespread in bacteria and eukaryotes, where they control important physiological and behavioral processes. In bacteria, NSM-based regulatory networks are highly complex, entailing large numbers of enzymes involved in the synthesis and degradation of active signaling molecules. How the converging input from multiple enzymes is transformed into robust and unambiguous cellular responses has remained unclear. Here we show that Escherichia coli converts dynamic changes of c-di-GMP into discrete binary signaling states, thereby generating heterogeneous populations with either high or low c-di-GMP. This is mediated by an ultrasensitive switch protein, PdeL, which senses the prevailing cellular concentration of the signaling molecule and couples this information to c-di-GMP degradation and transcription feedback boosting its own expression. We demonstrate that PdeL acts as a digital filter that facilitates precise developmental transitions, confers cellular memory, and generates functional heterogeneity in bacterial populations to evade phage predation. Based on our findings, we propose that bacteria apply ultrasensitive regulatory switches to convert dynamic changes of NSMs into binary signaling modes to allow robust decision-making and bet-hedging for improved overall population fitness.

Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters participate in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acids synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a ‘trap-and-flip’ mechanism that might be shared among MFS lipid transporters.

Photopolymer flexographic printing plate is a new photopolymeric material used for microdevices fabrication. This work demonstrates that a photopolymer flexographic master mold can be used for the fabrication of PDMS (polydimethylsiloxane) microdevices by a multi-step manufacturing process. The methodology entails three main fabrication steps: (1) a photopolymer flexographic printing plate mold (FMold) is generated by UV exposure through a transparent film, (2) an epoxy resin mold (ERmold) is fabricated by transferring the features of the photopolymer mold and (3) a PDMS microdevice is manufactured from the epoxy resin mold. The characterization of the manufactured PDMS microdevices was performed using scanning electron microscopy (SEM) and profilometry. Results showed high accuracy in the replication of the profiles. To show the feasibility of the fabrication process a microdevice for microdroplet generation was designed, manufactured and tested. Hence, the manufacturing process described in this work provides an easy, robust, and low-cost strategy that facilitates the scaling-up of microfluidic devices without requiring any sophisticated equipment. Fil: Olmos Carreno, Carol Maritza. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vaca Mora, Andrea Vanessa. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rosero, Gustavo. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina Fil: Peñaherrera Pazmiño, Ana Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina Fil: Pérez Sosa, Camilo José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina Fil: de Sá Carneiro, Igor. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina Fil: Vizuete, Karla. Universidad de Las Fuerzas Armadas Espe; Ecuador Fil: Arroyo, Carlos R.. Universidad de Las Fuerzas Armadas; Ecuador Fil: Debut, Alexis. Universidad de Las Fuerzas Armadas; Ecuador Fil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Ingeniería Biomédica; Argentina Fil: Cumbal, Luis. Universidad de Las Fuerzas Armadas; Ecuador Fil: Lerner, Betiana. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Ingeniería Biomédica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.