The dlt Operon of Bacillus cereus Is Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Δdlt_Bc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Δdlt_Bc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Δdlt_Bc). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.Gram-positive bacteria are generally enclosed by cell walls consisting of macromolecular assemblies of cross-linked peptidoglycan (murein), polyanionic teichoic acids (TAs), and surface proteins (69). TAs are polymers of repeating glycerophosphate residues. They may be covalently anchored to either peptidoglycan (wall-associated TAs) or the cytoplasmic membrane via glycolipids (lipoteichoic acids [LTAs]). TAs may be involved in controlling cell shape, autolytic enzyme activity, and cation homeostasis (69). They make a significant contribution to the overall negative charge of the bacterial cell wall, attracting negatively charged compounds, including the cationic antimicrobial peptides (AMPs) of the innate humoral immune systems of higher organisms (69).Many of the gram-positive bacterial species pathogenic to humans display resistance to cationic AMPs because of a decrease in the net negative charge of bacterial cell envelopes (75). Modifications to the TAs at the bacterial surface involving the incorporation of positively charged residues, such as d-alanine, prevent cationic AMPs from reaching their target, thereby protecting the organism against these compounds. This process involves the Dlt proteins encoded by the dltABCD operon present in most of the genome sequences established to date for gram-positive bacteria (44, 58, 74). d-Alanine is incorporated into LTAs through a two-step reaction involving a d-alanine-d-alanyl carrier protein ligase (Dcl) and a d-alanyl carrier protein (Dcp), encoded by the dltA and dltC genes, respectively (18, 44, 45, 70). The dltB and dltD genes encode other proteins required for the d-alanylation of LTAs. DltD is involved in selection of the Dcp carrier protein for ligation with d-alanine (19), whereas DltB is thought to be involved in d-alanyl-Dcp secretion (69). d-Alanine may be transferred from d-alanylated LTAs to wall-associated TAs by transacylation. For many human gram-positive bacterial pathogens, dlt operon inactivation has been shown to affect bacterial resistance to cationic AMPs and virulence. Indeed, Listeria monocytogenes, Bacillus anthracis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Lactobacillus reuteri, and group B streptococci harboring mutations in dlt genes all have a higher negative charge on the cell surface and are more susceptible to cationic AMPs of various origins (1, 34, 56, 58, 59, 77, 78, 89). The inactivation of dlt genes in these pathogenic bacterial species also decreases interactions with phagocytic and nonphagocytic cells (1, 13, 34, 78).The impact of Dlt proteins on cationic AMP resistance and virulence in insect bacterial pathogens has never before been studied, despite the major role of cationic AMPs in insect humoral immunity (9, 61). Insect bacterial pathogens also termed entomopathogenic bacteria are able to multiply in the insect hemocoel from small inocula (<10,000 viable cells) and produce fatal septicemia (8, 57). Entomopathogenic bacteria entering the hemolymph are targeted by an array of immune system mediators of both cellular and humoral reactions. The cellular response results in bacterial phagocytosis or encapsulation by circulating hemocytes, whereas the humoral response generates cationic AMPs (61). These peptides are small, inducible molecules produced in large amounts in hemolymph by the fat body (9, 26). They participate to the insect antimicrobial defense in a systemic response. Many AMP have been reported to cause damage in microbial membranes, which may ultimately lead to bacterial cell lysis (94).We investigated the role of the dlt operon in cationic AMP resistance and virulence in Bacillus cereus, a human opportunistic and insect facultative bacterial pathogen. B. cereus sensu stricto is a spore-forming gram-positive bacterium. The B. cereus sensu lato group of bacteria also includes the closely related insect pathogen Bacillus thuringiensis and the human pathogen B. anthracis. Genomic data have shown that B. thuringiensis and B. cereus have almost identical chromosomal genetic backgrounds (54, 55) but that B. thuringiensis carries a plasmid encoding entomopathogenic cytoplasmic crystalline δ-endotoxins (Cry proteins) specifically active against insect larvae upon ingestion (22, 23, 83). B. cereus can cause opportunistic food-borne gastroenteritis and local/systemic infections in immunocompromised humans (85). Both B. thuringiensis (with and without Cry toxins) and B. cereus strains are highly pathogenic when injected directly into the hemocoels of insect larvae, in which they cause lethal septicemia (46, 82, 86, 96). The occurrence, structure, and glycosylation of LTAs were studied for different Bacillus species, including B. cereus strains containing LTAs (built up of polyglycerol phosphate chains and hydrophobic anchors) and d-alanine (11, 50, 51, 62). Therefore, the presence of a dlt operon in the B. cereus 14579 genome suggests that the LTAs may be alanylated.We report here that the dlt operon of B. cereus is required for resistance to cationic AMPs of bacterial or insect origin. The dlt operon is required for full B. cereus virulence following bacterial injection into two lepidopteran insects, the caterpillar Spodoptera littoralis and the wax moth Galleria mellonella. We also detected the dlt operon in three gram-negative bacterial genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01).     

Cardiotonic steroids stimulate glycogen synthesis in human skeletal muscle cells via a Src- and ERK1/2-dependent mechanism

The cardiotonic steroid, ouabain, a specific inhibitor of Na(+),K(+)-ATPase, initiates protein-protein interactions that lead to an increase in growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in human skeletal muscle cells (HSMC) and clarified the mechanisms of ouabain signal transduction. In HSMC, ouabain increased glycogen synthesis in a concentration-dependent manner reaching the maximum at 100 nM. The effect of ouabain was additive to the effect of insulin and was independent of phosphatidylinositol 3-kinase inhibitor LY294002 but was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a Src inhibitor (PP2). Ouabain increased Src-dependent tyrosine phosphorylation of alpha(1)- and alpha(2)-subunits of Na(+),K(+)-ATPase and promoted interaction of alpha(1)- and alpha(2)-subunits with Src, as assessed by co-immunoprecipitation with Src. Phosphorylation of ERK1/2 and GSK3alpha/beta, as well as p90rsk activity, was increased in response to ouabain in HSMC, and these responses were prevented in the presence of PD98059 and PP2. Incubation of HSMC with 100 nM ouabain increased phosphorylation of the alpha-subunits of the Na-pump at a MAPK-specific Thr-Pro motif. Ouabain treatment decreased the surface abundance of alpha(2)-subunit, whereas abundance of the alpha(1)-subunit was unchanged. Marinobufagenin, an endogenous vertebrate bufadienolide cardiotonic steroid, increased glycogen synthesis in HSMC at 10 nM concentration, similarly to 100 nM ouabain. In conclusion, ouabain and marinobufagenin stimulate glycogen synthesis in skeletal muscle. This effect is mediated by activation of a Src-, ERK1/2-, p90rsk-, and GSK3-dependent signaling pathway.     

Epistemic artefacts: on the uses of complexity in anthropology

Distinctions between the ‘simple’ and the ‘complex’ have enjoyed a long and varied career in anthropology. Simplicity was once part of a collective fantasy about what life was like elsewhere, tingeing studies of tribal life with human longing for simpler ways of being. With the reflexive turn and the rise of cultural critique, simplicity has been all but excommunicated in favour of widespread diagnoses of complexity. In this article, I tease out some transformations in the uses of complexity in anthropology, and weave in some critical responses to these uses, spanning many decades, from within the discipline. I pay special attention to recent critiques by anthropologists who are beginning to grow weary of complexity as both an end‐in‐itself for scholarship and an empirical diagnosis. For these critics, complexity is deeply entwined with anthropological methods and knowledge practices. Drawing on these critical views, I suggest that complexity may be an epistemological artefact, rather than something that can be diagnosed ‘out there’, and offer a way of reframing complexity as a ‘dominant problematic’ in anthropology and beyond.     

BRAF Mutation Analysis of Fine-Needle Aspiration Biopsies of Papillary Thyroid Carcinoma: Impact on Diagnosis and Prognosis

Objective: The BRAF V600E mutation has been associated with aggressive disease in papillary thyroid carcinoma (PTC). Molecular testing has been proposed as a useful adjunct to cytology in the diagnosis of malignancy and for tailoring clinical management. The aims of our study were to evaluate the BRAF mutational status using archived fine-needle aspiration biopsy (FNAB) material from patients with long-term follow-up and to correlate it with the original cytology diagnosis, clinicopathological stage at surgery, and prognosis. Study Design: FNAB material from 52 cases of PTC, with a mean follow-up of 8.4 years, was used in this study. DNA was extracted from archival cytology slides. Mutation analysis was performed by standard sequencing and locked nucleic acid-PCR/sequencing. Results: The BRAF V600E mutation was present in 46% of cases, but it was absent in all FNABs diagnosed originally as atypical and in 14 of 17 suspicious cases. Recurrence was significantly more frequent (p = 0.006) in cases with BRAF mutations and 54% of these cases presented with stage 2 or higher. Conclusion: The BRAF V600E mutation is associated with a higher pathological stage at surgery and a higher rate of recurrence. BRAF mutation analysis did not provide a significant increase in the accuracy of thyroid FNABs diagnosed as suspicious or atypical in our institution.     

Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions.     

Development of respiratory trees in the holothurian <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Aspidochirotida: Holothuroidea)

The development of respiratory trees in the holothurian Apostichopus japonicus has been studied using light and electron microscopy. Primordial respiratory trees emerge in 2–3-mm-long animals (2 months after fertilization). They arise as two independent outgrowths from the dorsal wall of the anterior part of the cloaca. Upon first emerging and throughout the course of development, the left respiratory tree is longer than the right one. A common base develops in 4-mm-long animals (2–3 months after fertilization). In yearlings, the left respiratory tree grows into gaps between the loops of the intestinal tube interlaced with intestinal hemal vessels. The developing coelomic and luminal epithelia have ultrastructural peculiarities. The luminal epithelium of respiratory trees has been shown for the first time to comprise nerve cells and their processes. Characteristic structural features of the epithelia are shown to begin developing as early as in 4-mm-long animals (2–3 months after fertilization). In yearlings, the respiratory trees demonstrate definitive structural patterns and are entirely functional.     

Remote opto‐acoustic probing of single‐cell adhesion on metallic surfaces

The reflection of picosecond ultrasonic pulses from a cell‐substrate interface is used to probe cell‐biomaterial adhesion with a subcell resolution. We culture monocytes on top of a thin biocompatible Ti metal film, supported by a transparent sapphire substrate. Low‐energy femtosecond pump laser pulses are focused at the bottom of the Ti film to a micron spot. The subsequent ultrafast thermal expansion launches a longitudinal acoustic pulse in Ti, with a broad spectrum extending up to 100 GHz. We measure the acoustic echoes reflected from the Ti‐cell interface through the transient optical reflectance changes. The time‐frequency analysis of the reflected acoustic pulses gives access to a map of the cell acoustic impedance Z_c and to a map of the film‐cell interfacial stiffness K simultaneously. Variations in Z_c across the cell are attributed to rigidity and density fluctuations within the cell, whereas variations in K are related to interfacial intermolecular forces and to the nano‐architecture of the transmembrane bonds. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Germination of foliar viviparous buds in Bolbitis semicordata (Lomariopsidaceae); its ontogeny, vasculature and ecological significance

Ontogeny and vasculature of viviparous buds in Bolbitis semicordata (Baker) Ching was studied. The bud showed a superficial origin, later developing in to a rhizomatous structure. The bud developed root primordia from a meristematic cell having four cutting faces. Simultaneous with leaf primordial development, the mid-rib of the leaf produces a lateral bundle which later on supplies vasculature to the developing bud. Roots and fronds develop in an alternate manner, the increasing weight causes the bud to hang down and thus roots get established in the soil. Since no sexual reproduction was noticed during viviparous germination, this method may be an alternative in unfavorable conditions to replace the same.