Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone‐specific Pdpn knockdown on osteocyte form and function in the post‐natal mouse. Extensive skeletal phenotyping of male and female 6‐week‐old Oc‐cre;Pdpn^flox/flox (cKO) mice and their Pdpn^flox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age‐matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis.     

Proteomic analysis of amniotic membrane prepared for human transplantation: characterization of proteins and clinical implications

Amniotic membrane is commonly exploited in several surgical procedures. Despite a freeze preservation period, it is reported to retain wound healing, anti-angiogenic, antiinflammatory and anti-scarring properties; however, little is known about the active protein content. 2-DE analysis of transplant-ready amniotic membrane (TRAM) was performed. The effects of preservation and processing on amnion proteome were investigated, and the major proteins in the TRAM characterized using mass spectrometry and immunoblotting. This identified a spectrum of proteins including thrombospondin, mimecan, BIG-H3, and integrin alpha 6. Preservation compromises cellular viability resulting in selective elution of soluble cellular proteins, leaving behind extracellular matrix-associated and cell structural proteins. A number of key architectural proteins common to the architecture of the ocular surface were demonstrated in AM, which are involved in homeostasis and wound healing. Handling procedures alter the protein composition of amniotic membrane prepared for transplantation. Without standardization, there will be inter-membrane variation, which may compromise the desired therapeutic effect of transplant ready amniotic membrane.     

Thermal responses of Symbiodinium photosynthetic carbon assimilation

The symbiosis between hermatypic corals and their dinoflagellate endosymbionts, genus Symbiodinium, is based on carbon exchange. This symbiosis is disrupted by thermally induced coral bleaching, a stress response in which the coral host expels its algal symbionts as they become physiologically impaired. The disruption of the dissolved inorganic carbon (DIC) supply or the thermal inactivation of Rubisco have been proposed as sites of initial thermal damage that leads to the bleaching response. Symbiodinium possesses a highly unusual Form II ribulose bisphosphate carboxylase/oxygenase (Rubisco), which exhibits a lower CO₂:O₂ specificity and may be more thermally unstable than the Form I Rubiscos of other algae and land plants. Components of the CO₂ concentrating mechanism (CCM), which supplies inorganic carbon for photosynthesis, may also be temperature sensitive. Here, we examine the ability of four cultured Symbiodinium strains to acquire and fix DIC across a temperature gradient. Surprisingly, the half-saturation constant of photosynthesis with respect to DIC concentration (K _P), an index of CCM function, declined with increasing temperature in three of the four strains, indicating a greater potential for photosynthetic carbon acquisition at elevated temperatures. In the fourth strain, there was no effect of temperature on K _P. Finding no evidence for thermal inhibition of the CCM, we conclude that CCM components are not likely to be the primary sites of thermal damage. Reduced photosynthetic quantum yields, a hallmark of thermal bleaching, were observed at low DIC concentrations, leaving open the possibility that reduced inorganic carbon availability is involved in bleaching.     

A tree-based method for the rapid screening of chemical fingerprints

Background  

The fingerprint of a molecule is a bitstring based on its structure, constructed such that structurally similar molecules will have similar fingerprints. Molecular fingerprints can be used in an initial phase of drug development for identifying novel drug candidates by screening large databases for molecules with fingerprints similar to a query fingerprint.     

Anti-inflammatory potential of human corneal stroma-derived stem cells determined by a novel in vitro corneal epithelial injury model

BACKGROUND An in vitro injury model mimicking a corneal surface injury was optimised using human corneal epithelial cells(hCEC).AIM To investigate whether corneal-stroma derived stem cells(CSSC) seeded on an amniotic membrane(AM) construct manifests an anti-inflammatory, healing response.METHODS Treatment of hCEC with ethanol and pro-inflammatory cytokines were compared in terms of viability loss, cytotoxicity, and pro-inflammatory cytokine release, in order to generate the in vitro injury. This resulted in an optimal injury of 20%(v/v) ethanol for 30 s with 1 ng/mL interleukin-1(IL-1) beta. Co-culture experiments were performed with CSSC alone and with CSSC-AM constructs.The effect of injury and co-culture on viability, cytotoxicity, IL-6 and IL-8 production, and IL1 B, TNF, IL6, and CXCL8 mRNA expression were assessed.RESULTS Co-culture with CSSC inhibited loss of hCEC viability caused by injury. Enzyme linked immunosorbent assay and polymerase chain reaction showed a significant reduction in the production of IL-6 and IL-8 pro-inflammatory cytokines, and reduction in pro-inflammatory cytokine mRNA expression during co-culture with CSSC alone and with the AM construct. These results confirmed the therapeutic potential of the CSSC and the possible use of AM as a cell carrier for application to the ocular surface.CONCLUSION CSSC were shown to have a potentially therapeutic anti-inflammatory effectwhen treating injured hCEC, demonstrating an important role in corneal regeneration and wound healing, leading to an improved knowledge of their potential use for research and therapeutic purposes.     

Host selection and parasitism behavior of Lysiphlebus testaceipes: Role of plant,aphid species and instar

The aphid parasitoid Lysiphlebus testaceipes is a potentially valuable biological control agent of Aphis gossypii a major worldwide pest of cotton. One means of increasing the abundance of a biological control agent is to provide an alternative host habitat adjacent to cropping, from which they can provide pest control services in the crop. Host selection and parasitism rate of an alternative host aphid, Aphis craccivora by L. testaceipes were studied in a series of experiments that tested its host suitability relative to A. gossypii on cotton, hibiscus and mungbean. Host acceptance, as measured by number of ovipositions was much greater in A. craccivora compared to A. gossypii, while more host aphids were accepted on mungbean than cotton. When given a choice L. testaceipes attacks more 4th instar and adult stages (63% and 70%, respectively) of both hosts than 2nd instar nymphs (47%). In a switching (host choice) experiment, L. testaceipes preferentially attacked A. craccivora on mungbean over A. gossypii on cotton. Observations of parasitoid contact with A. gossypii cornicle secretion suggest it provides a useful deterrent against parasitoid attack. From these experiments it appears L. testaceipes has a preference for A. craccivora and mungbean compared to A. gossypii and cotton, in this respect using A. craccivora and mungbean as alternative habitat may not work as the parasitoid is unlikely to switch away from its preferred host.     

A phylogenetic approach to the identification of phosphoglucomutase genes

The expanding molecular database provides unparalleled opportunities for characterizing genes and for studying groups of related genes. We use sequences drawn from the database to construct an evolutionary framework for examining the important glycolytic enzyme phosphoglucomutase (PGM). Phosphoglucomutase plays a pivotal role in the synthesis and utilization of glycogen and is present in all organisms. In humans, there are three well-described isozymes, PGMI, PGM2, and PGM3. PGM1 was cloned 5 years ago; however, repeated attempts using both immunological approaches and molecular probes designed from PGM1 have failed to isolate either PGM2 or PGM3. Using a phylogenetic strategy, we first identified 47 highly divergent prokaryotic and eukaryotic PGM-like sequences from the database. Although overall amino acid identity often fell below 20%, the relative order, position, and sequence of three structural motifs, the active site and the magnesium-- and sugar-binding sites, were conserved in all 47 sequences. The phylogenetic history of these sequences was complex and marked by duplications and translocations; two instances of transkingdom horizontal gene transfer were identified. Nonetheless, the sequences fell within six well-defined evolutionary lineages, three of which contained only prokaryotes. Of the two prokaryotic/eukaryotic lineages, one contained bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human PGM1 and the second contained likely homologs to human PGM2. Indeed, an amino acid sequence, derived from a partial human cDNA, that fell within the second cross-kingdom lineage bears several characteristics expected for PGM2. A third lineage may contain homologs to human PGM3. On a general level, our phylogenetic-based approach shows promise for the further utilization of the extensive molecular database.      

Effective control of a field population of Helicoverpa armigera by using the small RNA virus Helicoverpa armigera stunt virus (Tetraviridae: Omegatetravirus)

The first comprehensive field trial using an insect small RNA virus as a control agent on a cropping system was conducted with the Helicoverpa armigera stunt virus (family Tetraviridae, genus Omegatetravirus, HaSV). The virus was semipurified, quantified, and applied at two rates, 4 x 10(15) and 4 x 10(14) virus particles/ha, with minimal formulation on sorghum against the bollworm Helicoverpa armigera (Hübner). For comparison, a commercial preparation of Helicoverpa zea single-nucleopolyhedrovirus (HzSNPV, Gemstar) was applied at the same time at 9.27 x 10(11) polyhedral inclusion bodies/ha. The HaSV application rates were determined by a novel procedure using laboratory LC50 bioassay data for HaSV and HzSNPV and calibration to the known field application rate of the HzSNPV. The baculovirus and the higher rate of HaSV produced statistically equivalent reductions in the larval populations of around 50% at both 3 and 6 d postapplication (dpa) compared with untreated plots. The 10-fold lower rate of HaSV reduced the larval population by 50% at 3 dpa and approximately 30% at 6 dpa. Persistence of HaSV over a 72-h period was found to be similar to that of HzSNPV, although the amount of HaSV available on the sorghum heads increased at 130 h postapplication, due most likely to dispersal of newly produced virus from cadavers and frass. The results from this trial indicate that HaSV could be used as an effective biopesticide for the control of H. armigera in sorghum and the ramifications for its broader use are discussed.