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Brazilian peppertree, Schinus terebinthifolia Raddi (Anacardiaceae) is an invasive weed of natural and agricultural areas of Florida, Hawaii, and Texas (USA). A thrips, Pseudophilothrips ichini (Hood) is being developed as a potential agent for biological control of this invasive weed. As field releases are planned, methods are needed for the mass production and to predict the impact of the thrips. Brazilian peppertree seedlings were grown in potting media amended with three fertilizer (24N-8P-16K) levels: low (0 g/l water), medium (1.8 g/l water) or high (3.6 g/l water). The greatest number of P. ichini thrips developed on the medium fertilized plants. To measure P. ichini thrips impact, we compared growth of these fertilized seedlings with or without thrips. While maintaining the number of P. ichini thrips adults constant (20 adults), we measured the impact of one or two generations of feeding. Fertilizer level had a significant effect on all plant responses. Both one and two generations of thrips significantly decreased plant height, number of branches, leaflets, leaves, and tips. Two generations of thrips feeding caused significant reductions in branch, leaf, and total biomass. Thrips feeding increased percent nitrogen in roots and decreased percent nitrogen in branches at the medium and high fertilizer levels. Percent nitrogen of field collected stems and leaves matched that of the low fertilizer level. These results suggest a medium fertilizer application is best for mass production of P. ichini thrips. Moreover, sustained damage for more than one generation of thrips feeding will have a significant impact on total biomass of S. terebinthifolia seedlings. The use of fertilized nursery sites may help in initial establishment and field mass production of P. ichini thrips. [ABSTRACT FROM AUTHOR]

Botryosphaeriaceous species are the causal agent of grapevine ( Vitis vinifera ) trunk diseases with very few options available for their control. They are also common endophytes in mānuka ( Leptospermum scoparium ), a New Zealand native medicinal plant, but there is no evidence of pathogenicity in this host. International research has demonstrated that endophytic bacteria can produce antimicrobial metabolites in planta . Thus, endophytic bacteria from mānuka may be viable options for biocontrol of botryosphaeriaceous species. This study was aimed at elucidating whether endophytic bacteria from mānuka with biocontrol activities can be transferred to grapevine as a heterologous host and express their bioactivity. Ten endophytic bacteria from a collection of 330 bacteria produced diffusible and volatile compound(s) that inhibited growth of six botryosphaeriaceous species. A combination of spontaneous rifampicin mutants and ERIC PCR was used to confirm endophytic colonization of the introduced antagonistic bacteria in planta . The results showed that two isolates, Pseudomonas sp. I2R21 and Pseudomonas sp. W1R33, were transferable from mānuka to grapevine when inoculated onto wounds, where they inhibited colonization by two botryosphaeriaceous species, Neofusicoccum luteum and N. parvum , respectively. The endophytic bacteria reduced lesion length caused by the pathogens (32–52%) compared to untreated controls. This study indicated that mānuka can provide a new source of microorganisms for use in sustainable agriculture. [ABSTRACT FROM AUTHOR]

Trichoderma atroviride LU132, the active organism in two commercialised biological control products, was grown under different in vitro culture carbon to nitrogen ratios, to assess effects on conidium production, germination and bioactivity. Sucrose as a carbon source in agar-based growth media was adjusted to 4.2, 8.4, or 16.8 g/l carbon, and soy peptone as a nitrogen source to 0.03, 0.05, 0.11, 0.21, 0.42, 0.84, 1.68, or 3.36 g/l, to give C:N ratios in the growth media ranging from 5:1 to 160:1. Conidium production was greatest at 16.8 g/l carbon and least at 4.2 g/l, and was prolific at C:N 5:1, but was sparse at C:N 160:1. Mean conidium germination was greatest (68%) at 4.2 g/l carbon and least (58%) at 16.8 g/l carbon. Germination was greatest (74%) for C:N 5:1 and least (52%) for C:N 160:1. Trichoderma inhibition activity against the diseases-causing Rhizoctonia solani was also affected slightly for colonies obtained from conidia grown on media with 4.2 g/l carbon from 67% to 70% with 16.8 g/l carbon. Inhibition activity was greatest (73%) from conidia produced at C:N 5:1 and least (63%) from C:N 160:1. Low amounts of carbohydrate in growth media at C:N ratios near 5:1 were optimum for growth of T . atroviride LU132. Large amounts of carbohydrate probably repressed carbon catabolism, resulting in decreased conidium fitness. These results provide new knowledge to assist commercial production of biocontrol agents based on T . atroviride LU132. [ABSTRACT FROM AUTHOR]

Biological control activity of Trichoderma spp. is heavily influenced by abiotic factors. Effects of pH and water activity ( a w ) in culture media of Trichoderma atroviride LU132 were assessed on conidium production, germination percentage and bioactivity against a soil-borne pathogen Rhizoctonia solani in dual agar culture assays. The effects of pH and buffer capacity in media were tested on T. atroviride colonies using different proportions (50, 100, 200 and 400 mM) of phosphate buffers at pHs of 3.5, 4.5, 5.5, 6.5, 7.5 or 8.5. Water activity experiments tested different a w values (0.998, 0.995, 0.985, 0.977, 0.961 and 0.948). Conidium production was reduced with increasing phosphate buffer concentrations from 50 to 400 mM. Maximum germination, and inhibition activity against R. solani was measured for conidia obtained from medium at pH 7.5. Greatest conidium production occurred at a w 0.995 and the least at a w 0.948. Mean conidium germination was greatest (78%) from media at a w 0.961, and least (21%) from a w 0.985. Inhibition and overgrowth activities of Trichoderma colonies were greatest at a w 0.961 and least at a w 0.998. This study has demonstrated that manipulation of culture conditions as such may improve conidium fitness (quantity and quality). However, these factors may not have caused independently effects on the fungus. Effects of physical growth conditions (besides nutritional requirements) are likely to be important for optimum production of biocontrol agents based on T. atroviride LU132, and other similar biocontrol agents. [ABSTRACT FROM AUTHOR]

The four secreted R-spondin (Rspo1-4) proteins of vertebrates function as stem cell growth factors and potentiate canonical Wnt signalling. Rspo proteins act by cross-linking members of two cell surface receptor families, complexing the stem cell markers LGR4-6 with the Frizzled-specific E3 ubiquitin ligases ZNRF3/RNF43. The consequent internalisation of the ternary LGR–Rspo–E3 complex removes the E3 ligase activity, which otherwise targets the Wnt receptor Frizzled for degradation, and thus enhances Wnt signalling. Multiple combinations of LGR4-6, Rspo1-4 and ZNRF3/RNF43 are possible, implying the existence of generic interaction determinants, but also of specific differences in complex architecture and activity. We present here a high resolution crystal structure of an ectodomain variant of human LGR5 (hLGR5 ecto ) complexed with a signalling competent fragment of mouse Rspo2 (mRspo2 Fu1-Fu2 ). The structure shows that the particularly potent Rspo2 ligand engages LGR5 in a fashion almost identical to that reported for hRSPO1. Comparison of our hLGR5 ecto structure with previously published structures highlights a surprising plasticity of the LGR ectodomains, characterised by a nearly 9° or larger rotation of the N-terminal half of the horseshoe-like fold relative to the C-terminal half. We also report a low resolution hLGR5–mRspo2 Fu1-Fu2 –mZNRF3 ecto ternary complex structure. This crystal structure confirms our previously suggested hypothesis, showing that Rspo proteins cross-link LGRs and ZNRF3 into a 2:2:2 complex, whereas a 1:1:1 complex is formed with RNF43. [ABSTRACT FROM AUTHOR]

Earths were medicinal clays reported by authors of classical antiquity. Today, there are no archaeological samples thereof, at least with irrefutable proof that they were used as medicines. Therefore, their mineral identity and potential pharmacological actions have to be investigated on the basis of field work, laboratory testing and analysis as well as scrutiny of the relevant ancient texts. This paper focuses on Samian Earth, from the island of Samos in the East Aegean, used in antiquity as an eye salve and for the cleaning of cloth. Previously, Samian Earth has been equated with kaolin but we suggest its composition was probably more closely associated with the local borates to be found in the proximity of bentonitic clays. Microbiological testing of hydrated layered silicates (kaolin and bentonite) from Samos and Melos in the Aegean in association with different borates (colemanite, tincalconite) was carried out against common pathogens, like Staphylococcus aureus and Pseudomonas aeruginosa . Experimental results showed that even a few hundred ppms of boron/boric acid in the Samos bentonitic clays can have a significant effect on the reduction of bacterial colonies. This observation corroborates the assumption that the medicinal properties of the earths were integrally associated with the mineralogy of specific localities within the volcanic islands of their origin. [ABSTRACT FROM AUTHOR]

The receptor protein tyrosine phosphatases (RPTPs) exhibit a wide repertoire of cellular signalling functions. In particular, type IIa RPTP family members have recently been highlighted as hubs for extracellular interactions in neurons, regulating neuronal extension and guidance, as well as synaptic organisation. In this review, we will discuss the recent progress of structural biology investigations into the architecture of type IIa RPTP ectodomains and their interactions with extracellular ligands. Structural insights, in combination with biophysical and cellular studies, allow us to begin to piece together molecular mechanisms for the transduction and integration of type IIa RPTP signals and to propose hypotheses for future experimental validation. [ABSTRACT FROM AUTHOR]

A fast growing field, the study of infants at risk because of having an older sibling with autism (i.e. infant sibs) aims to identify the earliest signs of this disorder, which would allow for earlier diagnosis and intervention. More importantly, we argue, these studies offer the opportunity to validate existing neuro-developmental models of autism against experimental evidence. Although autism is mainly seen as a disorder of social interaction and communication, emerging early markers do not exclusively reflect impairments of the "social brain". Evidence for atypical development of sensory and attentional systems highlight the need to move away from localized deficits to models suggesting brain-wide involvement in autism pathology. We discuss the implications infant sibs findings have for future work into the biology of autism and the development of interventions. [ABSTRACT FROM AUTHOR]

Abstract: Ten years ago nothing was known of the three-dimensional structure of members of the semaphorin family of cell guidance cues, nor of their major receptors, the plexins. The structural biology of this cell surface ligand–receptor system has now come of age. Detailed atomic level information is available on the architecture of semaphorin and plexin ectodomains and their recognition complexes. Similarly the structure of the plexin cytoplasmic region, and its interactions with members of the Rho family of small GTPases have been unveiled. These structural analyses, in combination with biochemical, biophysical and cellular studies, have progressed our understanding of this signalling system into the realm of molecular mechanism. [Copyright &y& Elsevier]

Abstract: As obligate parasites, viruses are required to enter and replicate within their host, a process which employs many of their proteins to hijack natural cellular processes. High resolution X-ray crystallographic analysis has proven to be an ideal method to visualize the mechanisms by which such virus-host interactions occur and has revealed the innovative capacity of viruses to adapt efficiently to their hosts. In this review, we draw upon recently elucidated paramyxovirus-, arenavirus-, and poxvirus-host protein complex crystal structures to reveal both the capacity of viruses to appropriate one component of a physiological protein–protein binding event (often modifying it to out-compete the host-protein), and the ability to utilize novel binding sites on host cell surface receptors. The structures discussed shed light on a number of biological processes ranging from viral entry to virulence and host antagonism. Drawn together they reveal the common strategies which viruses have evolved to interact with their natural host. The structures also support molecular level rationales for how viruses can be transmitted to unrelated organisms and thus pose severe health risks. [Copyright &y& Elsevier]

Abstract: Epidemiological and genetic data have consistently identified associations with HLA class II alleles in many autoimmune diseases. In multiple sclerosis (MS), an autoimmune disease targeting central nervous system (CNS) myelin, the DR2 haplotype (DRB1*1501, DRB5*0101 and DQB1*0602) remains the strongest identified genetic risk factor in Caucasians. However, it is hard to tease apart the precise contributions of its constituent individual alleles and their modes of action remain poorly understood, due in part to the strong linkage disequilibrium in this region. Recent work in humanized mice indicates functional epistatic interactions whereby DRB5*0101 directly modulates the severity of the ensuing disease through activation-induced cell death (AICD) of encephalitogenic T cells which are restricted by DRB1*1501. Complementary structural studies help to explain how these alleles may facilitate thymic escape of autoreactive T cells and contribute to peripheral T cell activation via suboptimal binding interactions and mechanisms of molecular mimicry. Here we discuss the emerging role of the constituent alleles of the DR2 haplotype and our ongoing efforts to uncover the mechanisms by which they influence MS pathogenesis. [Copyright &y& Elsevier]

Abstract: Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative. [Copyright &y& Elsevier]

Abstract: αβ T-cell receptors (TcRs) play a central role in cellular immune response. They are members of the Ig superfamily, with extracellular regions of the α and β chains each comprising a V-type domain and a C-type domain. We have determined the ectodomain structure of an αβ TcR, which recognizes the autoantigen myelin basic protein. The 2.0-Å-resolution structure reveals canonical main-chain conformations for the Vα, Vβ, and Cβ domains, but the Cα domain exhibits a main-chain conformation remarkably different from those previously reported for TcR crystal structures. The global IgC-like fold is maintained, but a piston-like rearrangement between BC and DE β-turns results in β-strand slippage. This substantial conformational change may represent a signaling intermediate. Our structure is the first example for the Ig fold of the increasingly recognized concept of “metamorphic proteins.” [Copyright &y& Elsevier]

Abstract: Ionotropic glutamate receptors are functionally diverse but have a common architecture, including the 400-residue amino-terminal domain (ATD). We report a 1.8-Å resolution crystal structure of human GluR2-ATD. This dimeric structure provides a mechanism for how the ATDs can drive receptor assembly and subtype-restricted composition. Lattice contacts in a 4.1-Å resolution crystal form reveal a tetrameric (dimer–dimer) arrangement consistent with previous cellular and cryo-electron microscopic data for full-length AMPA receptors. [Copyright &y& Elsevier]

Abstract: Antibodies contain a conserved glycosylation site that has emerged as a target for the modulation of antibody effector functions. The crystal structure of a biosynthetic intermediate of human IgG1, bearing immature oligomannose-type glycans and reported to display increased antibody-dependent cellular cytotoxicity, demonstrates that glycan engineering can bias the Fc to an open conformation primed for receptor binding. [Copyright &y& Elsevier]

Abstract: The Siglec family of receptors mediates cell-surface interactions through recognition of sialylated glycoconjugates. Previously reported structures of the N-terminal domain of the Siglec sialoadhesin (SnD1) in complex with various sialic acid analogs revealed the structural template for sialic acid binding. To characterize further the carbohydrate-binding properties, we have determined the crystal structures of SnD1 in the absence of ligand, and in complex with 2-benzyl-Neu5NPro and 2-benzyl-Neu5NAc. These structures reveal that SnD1 undergoes very few structural changes on ligand binding and detail how two novel classes of sialic acid analogs bind, one of which unexpectedly can induce Siglec dimerization. In conjunction with in silico analysis, this set of structures informs us about the design of putative ligands with enhanced binding affinities and specificities to different Siglecs, and provides data with which to test the effectiveness of different computational drug design protocols. [Copyright &y& Elsevier]

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1–3 and 11 are known to have a conserved β-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R. [Copyright &y& Elsevier]

Background: National data on providers' colorectal cancer (CRC) screening knowledge, attitudes, and practices are sparse. This study assessed primary care physicians' (PCPs') beliefs about the effectiveness of CRC screening, their recommendations for screening, their perceptions of the influence of published guidelines on their CRC screening recommendations, and how they conduct CRC screening in their clinical practices.Methods: A questionnaire was administered to a nationally representative sample of practicing PCPs. Of 1718 eligible physicians, 1235 (72%) responded.Results: Only 2% of PCPs said they did not recommend CRC screening. Over 80% indicated that they most often recommend CRC screening with fecal occult blood testing and/or flexible sigmoidoscopy, although colonoscopy was perceived as the more effective screening modality. Nearly two-thirds of obstetrician/gynecologists and one-fourth of other practitioners reported conducting fecal occult blood testing exclusively by digital rectal exam. Only 29% of PCPs said they perform sigmoidoscopy. Estimated volumes of ordering, performing, or referring for CRC screening were low, and <20% reported that three-fourths or more of their older patients were up to date with CRC screening as recommended by the physician. Many PCPs reported recommending CRC screening at nonstandard starting ages or too-frequent intervals.Conclusions: Awareness of CRC screening among PCPs in the United States is high. However, knowledge gaps about the timing and frequency of screening and suboptimal screening delivery were evident. [ABSTRACT FROM AUTHOR]