Characterization and Engineering of the Type 3 Secretion System Needle Monomer from Salmonella Through the Construction and Screening of a Comprehensive Mutagenesis Library.

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here we investigated this link with the PrgI protein, the monomer of the secretory channel of the Type 3 Secretion System (T3SS) of Salmonella enterica . Despite detailed knowledge of the PrgI needle's assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape (SFL) to characterize the PrgI needle's mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS's assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes. Importance: Protein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

Raising concerns on questionable ethics approvals - a case study of 456 trials from the Institut Hospitalo-Universitaire Méditerranée Infection.

BACKGROUND: The practice of clinical research is strictly regulated by law. During submission and review processes, compliance of such research with the laws enforced in the country where it was conducted is not always correctly filled in by the authors or verified by the editors. Here, we report a case of a single institution for which one may find hundreds of publications with seemingly relevant ethical concerns, along with 10 months of follow-up through contacts with the editors of these articles. We thus argue for a stricter control of ethical authorization by scientific editors and we call on publishers to cooperate to this end. METHODS: We present an investigation of the ethics and legal aspects of 456 studies published by the IHU-MI (Institut Hospitalo-Universitaire Méditerranée Infection) in Marseille, France. RESULTS: We identified a wide range of issues with the stated research authorization and ethics of the published studies with respect to the Institutional Review Board and the approval presented. Among the studies investigated, 248 were conducted with the same ethics approval number, even though the subjects, samples, and countries of investigation were different. Thirty-nine (39) did not even contain a reference to the ethics approval number while they present research on human beings. We thus contacted the journals that published these articles and provide their responses to our concerns. It should be noted that, since our investigation and reporting to journals, PLOS has issued expressions of concerns for several publications we analyze here. CONCLUSION: This case presents an investigation of the veracity of ethical approval, and more than 10 months of follow-up by independent researchers. We call for stricter control and cooperation in handling of these cases, including editorial requirement to upload ethical approval documents, guidelines from COPE to address such ethical concerns, and transparent editorial policies and timelines to answer such concerns. All supplementary materials are available.

Scientific Integrity Requires Publishing Rebuttals and Retracting Problematic Papers.

Recently, an article by Seneff et al. entitled "Innate immunosuppression by SARS-CoV-2 mRNA vaccinations: The role of G-quadruplexes, exosomes, and MicroRNAs" was published in Food and Chemical Toxicology (FCT). Here, we describe why this article, which contains unsubstantiated claims and misunderstandings such as "billions of lives are potentially at risk" with COVID-19 mRNA vaccines, is problematic and should be retracted. We report here our request to the editor of FCT to have our rebuttal published, unfortunately rejected after three rounds of reviewing. Fighting the spread of false information requires enormous effort while receiving little or no credit for this necessary work, which often even ends up being threatened. This need for more scientific integrity is at the heart of our advocacy, and we call for large support, especially from editors and publishers, to fight more effectively against deadly disinformation.

Bond engineering of molecular ferroelectrics renders soft and high-performance piezoelectric energy harvesting materials.

Piezoelectric materials convert mechanical stress to electrical energy and thus are widely used in energy harvesting and wearable devices. However, in the piezoelectric family, there are two pairs of properties that improving one of them will generally compromises the other, which limits their applications. The first pair is piezoelectric strain and voltage constant, and the second is piezoelectric performance and mechanical softness. Here, we report a molecular bond weakening strategy to mitigate these issues in organic-inorganic hybrid piezoelectrics. By introduction of large-size halide elements, the metal-halide bonds can be effectively weakened, leading to a softening effect on bond strength and reduction in polarization switching barrier. The obtained solid solution C6H5N(CH3)3CdBr2Cl0.75I0.25 exhibits excellent piezoelectric constants (d33 = 367 pm/V, g33 = 3595 × 10-3 Vm/N), energy harvesting property (power density is 11 W/m2), and superior mechanical softness (0.8 GPa), promising this hybrid as high-performance soft piezoelectrics.

Symbiont-conferred immunity interacts with effects of parasitoid genotype and intraguild predation to affect aphid immunity in a clone-specific fashion.

BACKGROUND: Host-parasite interactions represent complex co-evolving systems in which genetic and associated phenotypic variation within a species can significantly affect selective pressures on traits, such as host immunity, in the other. While often modelled as a two-species interaction between host and parasite, some systems are more complex due to effects of host enemies, intraguild predation, and endosymbionts, all of which affect host immunity. However, it remains unclear how these factors, combined with genetic variation in the host and the parasitoid, affect host immunity. We address this question in an important agricultural pest system, the pea aphid Acyrthosiphon pisum, which shows significant intraspecific variability in immunity to the parasitoid wasp Aphidius ervi. In a complex experiment, we use a quantitative genetic design in the parasitoid, two ecologically different aphid lineages and the aphid lion Chrysoperla carnea as an intraguild predator to unravel the complex interdependencies. RESULTS: We demonstrate that aphid immunity as a key trait of this complex host-parasite system is affected by intraspecific genetic variation in the parasitoid and the aphid, the interaction of intraspecific genetic variation with intraguild predation, and differences in defensive endosymbionts between aphid lineages. Further, aphid lineages differ in their altruistic behaviour whereby infested aphids move away from the clonal colony to facilitate predation. CONCLUSIONS: Our findings provide new insights into the influence of endosymbiosis and genetic variability in an important host-parasitoid system which is influenced by natural enemies of the parasitoid and the aphid, including its endosymbiont communities. We show that endosymbiosis can mediate or influence the evolutionary arms race between aphids and their natural enemies. The outcome of these complex interactions between species has significant implications for understanding the evolution of multitrophic systems, including eco-agricultural settings.

Ferroelastic-switching-driven large shear strain and piezoelectricity in a hybrid ferroelectric.

Materials that can produce large controllable strains are widely used in shape memory devices, actuators and sensors1,2, and great efforts have been made to improve the strain output3-6. Among them, ferroelastic transitions underpin giant reversible strains in electrically driven ferroelectrics or piezoelectrics and thermally or magnetically driven shape memory alloys7,8. However, large-strain ferroelastic switching in conventional ferroelectrics is very challenging, while magnetic and thermal controls are not desirable for practical applications. Here we demonstrate a large shear strain of up to 21.5% in a hybrid ferroelectric, C6H5N(CH3)3CdCl3, which is two orders of magnitude greater than that in conventional ferroelectric polymers and oxides. It is achieved by inorganic bond switching and facilitated by structural confinement of the large organic moieties, which prevents undesired 180° polarization switching. Furthermore, Br substitution can soften the bonds, allowing a sizable shear piezoelectric coefficient (d35 ≈ 4,830 pm V-1) at the Br-rich end of the solid solution, C6H5N(CH3)3CdBr3xCl3(1-x). The electromechanical properties of these compounds suggest their potential in lightweight and high-energy-density devices, and the strategy described here could inspire the development of next-generation piezoelectrics and electroactive materials based on hybrid ferroelectrics.

Rotation of a helical coordination polymer by mechanical grinding.

Anisotropic cell volume expansion by mechanical grinding of the solid facilitates the concerted rotation of the photo-inert helical coordination polymer, which causes the misaligned arms containing olefin functional groups in the neighbouring strands to align to undergo [2+2] cycloaddition reaction in 83% yield.

Synthesis through 3D printing: formation of 3D coordination polymers.

Coordination polymers (CPs) and coordination network solids such as metal-organic frameworks (MOFs) have gained increasing interest during recent years due to their unique properties and potential applications. Preparing 3D printed structures using CP would provide many advantages towards utilization in fields such as catalysis and sensing. So far, functional 3D structures were printed mostly by dispersing pre-synthesized particles of CPs and MOFs within a polymerizable carrier. This resulted in a CP active material dispersed within a 3D polymeric object, which may obstruct or impede the intrinsic properties of the CP. Here, we present a new concept for obtaining 3D free-standing objects solely composed of CP material, starting from coordination metal complexes as the monomeric building blocks, and utilizing the 3D printer itself as a tool to in situ synthesize a coordination polymer during printing, and to shape it into a 3D object, simultaneously. To demonstrate this, a 3D-shaped nickel tetra-acrylamide monomeric complex composed solely of the CP without a binder was successfully prepared using our direct print-and-form approach. We expect that this work will open new directions and unlimited potential in additive manufacturing and utilization of CPs.

UK resilience to a chemical incident.

Following the deliberate chemical attacks in Salisbury last year, a review of UK resilience to chemical incidence was undertaken. The scope was not limited to deliberate attacks, related to terrorism, but was to include non-deliberate and accidental events. Chemical incidents have wide-reaching consequences irrespective of whether they are deliberate or not. The effects of these incidents manifest themselves in terms of immediate health consequences and will also include economic, political and public health effects that may have a much longer impact than the initial disruption. The economic consequences of the Novichok attack were devastating to the local population and businesses in both the short term and the long term, being felt over a year later. This review discusses the effects of these incidents on infrastructure, healthcare provision, law and order, economics and government at a local, regional and national level. The NHS Emergency Preparedness, Resilience and Response Framework was reviewed, and this provides a basic outline of advice for local government to follow in the case of chemical incidents. However, the wider issues of interdepartmental co-operation and how to maintain a response in the long term require further thought. Moreover, the methods by which interagency and regional resilience is maintained in preparing for such an event require a clearer guideline.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus).

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Caracterización de la atención farmacéutica en pacientes poli medicados con enfermedades crónicas no transmisibles del Hospital Nacional Especializado Rosales, atendidos en Farmacia Especializada del MINSAL. Agosto 2017 - Agosto 2018 / Characterization of pharmaceutical care in polymedicated patients with chronic noncommunicable diseases of the Rosales Specialized National Hospital, treated in the specialized pharmacy of MINSAL. August 2017 - August 2018

OBJETIVO: Caracterizar la atención farmacéutica en pacientes poli medicados con enfermedades crónicas no transmisibles, del Hospital Nacional Especializado Rosales, atendidos en la farmacia especializada del MINSAL. Agosto 2017 - Agosto 2018. METODOLOGÍA: Se realizó un estudio descriptivo de corte transversal; La muestra fue 378 expedientes de pacientes diagnosticados con Enfermedades Crónicas no Transmisibles, con seguimiento en el área de atención farmacéutica; Se diseñó una matriz para recolectar la información y facilitar su análisis haciendo uso de frecuencias de variables múltiples de SPSS. RESULTADOS: el 40% de las edades estaban en el rango de 66 años o más, el sexo femenino predominó con el 69%, más del 50% de los participantes estaban solteros, la ocupación más frecuente del estudio, fue ama de casa con un 43%, el 56% de las personas sabían leer, pero no habían realizado estudios de nivel básico, Las características personales fueron el PRM que predominó en el estudio con un 39% de los casos, la educación en medidas no farmacológicas fue la intervención que más se realizó de parte de los profesionales farmacéuticos con un 50% del total de casos, y el 71% de los casos reportados logró una mejoría en el control de su presión arterial con el seguimiento farmacoterapéutico. CONCLUSIONES: Se encontró que las características personales son el problema relacionado con la medicación más frecuente en pacientes con Enfermedades Crónicas no Transmisibles, la educación en medidas no farmacológicas es la intervención que más se realiza en atención farmacéutica y los logros en pacientes se evidenciaron en un mejor control de la presión arterial

Developing Gram-negative bacteria for the secretion of heterologous proteins.

Gram-negative bacteria are attractive hosts for recombinant protein production because they are fast growing, easy to manipulate, and genetically stable in large cultures. However, the utility of these microbes would expand if they also could secrete the product at commercial scales. Secretion of biotechnologically relevant proteins into the extracellular medium increases product purity from cell culture, decreases downstream processing requirements, and reduces overall cost. Thus, researchers are devoting significant attention to engineering Gram-negative bacteria to secrete recombinant proteins to the extracellular medium. Secretion from these bacteria operates through highly specialized systems, which are able to translocate proteins from the cytosol to the extracellular medium in either one or two steps. Building on past successes, researchers continue to increase the secretion efficiency and titer through these systems in an effort to make them viable for industrial production. Efforts include modifying the secretion tags required for recombinant protein secretion, developing methods to screen or select rapidly for clones with higher titer or efficiency, and improving reliability and robustness of high titer secretion through genetic manipulations. An additional focus is the expression of secretion machineries from pathogenic bacteria in the "workhorse" of biotechnology, Escherichia coli, to reduce handling of pathogenic strains. This review will cover recent advances toward the development of high-expressing, high-secreting Gram-negative production strains.

Indirect ecological effects interact with community genetic effects in a host-parasite system and dramatically reduce parasite burden.

Community genetic (CG) effects and ecological factors create a complex set of interactions that are key drivers of evolutionary dynamics in ecological systems. To date, most studies investigating trait variation have focused on either effects of intraspecific genetic variation or on genotype by environment (GxE) interactions in isolation. Poorly investigated but very important are the interactions between CGs and indirect ecological effects (IEEs) that are caused by plant-soil interactions. Here, we tested how CGs in a cabbage host and its aphid parasite depended on the ecological conditions under which the host was grown. We established microcosms of different cabbage cultivars and aphid genotypes on soils inoculated with samples of other soils previously trained with onion. We hypothesized that such IEEs will have significantly different outcomes for ecosystems than predicted from simpler CG or GxE studies. Our analysis demonstrated a large IEE that differed by context and aphid genotype causing reduced parasite population sizes by up to 90%. The IEE is induced by insect-repellent properties and the microbiome of the onion. Our results highlight the importance of interacting IEEs and CGs for ecosystems dynamics showing that IEEs offer sustainable solutions by dramatically reducing parasite burden on cash crops.

Novel resilience in response to revitalisation after exposure to lethal salinity causes differential reproductive success in an extremely plastic organism.

Phenotypic plasticity is central to an organism's ability to adapt to variable environmental conditions. For aquatic organisms, exposure to elevated salt levels poses a challenge and organisms may fail to tolerate or survive much higher levels short-term. Here we demonstrate, for the first time, in a laboratory study of Daphnia magna that exposure to levels of salinity higher than those previously shown to lead to apparent death (paralysis) can be reversed following a transfer to optimal conditions. We established experimental populations from one clone of D. magna, each with five replicates, that were exposed to different short periods of three different lethal levels of salinity (12.27 PSU [45, 60, 90 and 120 min], 18.24 PSU [45, 60 and 90 min] and 24.22 PSU [45, 60 and 90 min]). In all populations, all individuals were paralysed at the end of their exposure, usually classified in the literature as dead. Subsequently, all individuals were transferred to optimal conditions. However, after the transfer, a proportion of the individuals not only came back from the verge of death (i.e. were revitalised), but also showed afterwards differential reproductive success over a period of 20 days, depending on the level and the length of exposure before revitalisation. Both exposure level and time had an overall negative effect on population size that differed across all treatments. Revitalisation occurred within an hour after the transfer to optimal conditions for 18.24 PSU but took 14-16 h for 12.27 PSU. There was no instantaneous revitalisation nor was there any revitalisation after 16 h no matter how long the paralysed Daphnia individuals were left in the optimal conditions. Our findings cast new light on resilience in cladocerans and suggest that abrupt environmental change can reveal novel plastic responses to extreme conditions.

Templated Chromophore Assembly on Peptide Scaffolds: A Structural Evolution.

The use of a template that bears pre-programmed receptor sites for selectively accommodating chromophores at given positions is an attractive approach for engineering artificial-light-harvesting systems. Indulging this line of thought, this work tackles the creation of tailored antenna architectures with yellow, red and blue chromophores, exploiting three dynamic covalent reactions simultaneously, namely disulfide exchange, acyl hydrazone, and boronic ester formations. The effect of various structural modifications, such as the chromophores as well as their spatial organization (distance, orientation, order) on the energy transfer within the antennas was studied by means of steady-state UV/Vis absorption and fluorescence spectroscopies. This systematic study allowed for a significant improvement of the energy-transfer efficiencies to a noticeable 22 and 15 % for the yellow and red donors, respectively, across the chromophores to the blue acceptor. Metadynamics simulations suggested that the conformational properties of the antennas are driven by intramolecular chromophoric stacking interactions that, upon forcing the α-helix to fold on itself, annul any effects deriving from the programming of the spatial arrangement of the receptor sides in the peptide backbone.