The frequency and duration of Salmonella–macrophage adhesion events determines infection efficiency

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.     

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt‐based anti‐cancer drugs

Although platinum‐based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume‐regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8‐dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug‐induced apoptosis independently from drug uptake, possibly by impairing VRAC‐dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D‐containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.     

Comparison of plant communities on the Ibity and Itremo massifs,Madagascar, with contrasting conservation histories and current status

Background: Deforestation processes and species extinction on islands have made them the focus of substantial efforts to create and strengthen local environmental organisations, establish new protected areas and improve natural resource management. In Madagascar, despite promising new policies, the flora and fauna are under serious threat. More than 80% of the island shows a significant loss of natural plant cover. Current levels of diversity reflect natural disturbance regimes, with fire representing one of the more important factors.

Aims: We compared two new protected areas (Ibity and Itremo) with different environmental and management contexts to identify the roles and impacts of the environmental conditions and the different types of management applied by their respective local communities on vegetation. Both areas have extensive stands of tapia (Uapaca bojeri) woodland.

Methods: Herbaceous vegetation surveys were carried out enumerating 16 1-m² quadrats in each of 10 sites on Ibity and seven on Itremo to characterise plant community composition and structure, complemented by tree and shrub surveys within three 40 × 40 m quadrats at each site.

Results: Floristic richness was higher at Ibity. Populations of U. bojeri were better conserved at Itremo.

Conclusions: While the total exclusion of fire is impracticable, careful management should enable the maintenance of natural conditions and at least lead to a reduction in the impacts on the structure of tapia woodland vegetation.     

The Yin and Yang of Wnt/Ryk axon guidance in development and regeneration

In the developing embryo,nascent axons navigate towards their specific targets to establish the intricate network of axonal connections linking neurons within the mature nervous system.Molecular navigational systems comprising repulsive and attractive guidance cues form chemotactic gradients along the pathway of the exploring growth cone.Axon-bound receptors detect these gradients and determine the trajectory of the migrating growth cone.In contrast to their benevolent role in the developing nervous system,repulsive guidance receptors are detrimental to the axon’s ability to regenerate after injury in the adult.In this review we explore the essential and beneficial role played by the chemorepulsive Wnt receptor,Ryk/Derailed in axon navigation in the embryonic nervous system(the Yin function).Specifically,we focus on the role of Wnt5a/Rykmediated guidance in the establishment of two major axon tracts in the mammalian central nervous system,the corticospinal tract and the corpus callosum.Recent studies have also identified Ryk as a major suppressor of axonal regeneration after spinal cord injury.Thus,we also discuss this opposing aspect of Ryk function in axonal regeneration where its activity is a major impediment to axon regrowth(the Yang function).     

Examining Variable Domain Orientations in Antigen Receptors Gives Insight into TCR-Like Antibody Design

The variable domains of antibodies and T-Cell receptors (TCRs) share similar structures. Both molecules act as sensors for the immune system but recognise their respective antigens in different ways. Antibodies bind to a diverse set of antigenic shapes whilst TCRs only recognise linear peptides presented by a major histocompatibility complex (MHC). The antigen specificity and affinity of both receptors is determined primarily by the sequence and structure of their complementarity determining regions (CDRs). In antibodies the binding site is also known to be affected by the relative orientation of the variable domains, VH and VL. Here, the corresponding property for TCRs, the Vβ-Vα orientation, is investigated and compared with that of antibodies. We find that TCR and antibody orientations are distinct. General antibody orientations are found to be incompatible with binding to the MHC in a canonical TCR-like mode. Finally, factors that cause the orientation of TCRs and antibodies to be different are investigated. Packing of the long Vα CDR3 in the domain-domain interface is found to be influential. In antibodies, a similar packing affect can be achieved using a bulky residue at IMGT position 50 on the VH domain. Along with IMGT VH 50, other positions are identified that may help to promote a TCR-like orientation in antibodies. These positions should provide useful considerations in the engineering of therapeutic TCR-like antibodies.     

In Vivo Ligands of MDA5 and RIG-I in Measles Virus-Infected Cells

RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5′-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling.     

Vangl2-Regulated Polarisation of Second Heart Field-Derived Cells Is Required for Outflow Tract Lengthening during Cardiac Development

Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.