The prostaglandin EP4 receptor is a master regulator of the expression of PGE2 receptors following inflammatory activation in human monocytic cells

Prostaglandin E₂ (PGE₂) is responsible for inflammatory symptoms. However, PGE₂ also suppresses pro-inflammatory cytokine production. There are at least 4 subtypes of PGE₂ receptors, EP1–EP4, but it is unclear which of these specifically control cytokine production. The aim of this study was to determine which of the different receptors, EP1R–EP4R modulate production of tumor necrosis factor-α (TNF-α) in human monocytic cells.Human blood, or the human monocytic cell line THP-1 were stimulated with LPS. The actions of PGE₂, alongside selective agonists of EP1–EP4 receptors, were assessed on LPS-induced TNF-α, IL-1β and IL-10 release. The expression profiles of EP2R and EP4R in monocytes and THP-1 cells were characterised by RT-qPCR. In addition, the production of cytokines was evaluated following knockdown of the receptors using siRNA and over-expression of the receptors by transfection with constructs.PGE₂ and also EP2 and EP4 agonists (but not EP1 or EP3 agonists) suppressed TNF-α production in blood and THP-1 cells. LPS also up regulated expression of EP2R and EP4R but not EP1 or EP3. siRNA for either EP2R or EP4R reversed the suppressive actions of PGE₂ on cytokine production and overexpression of EP2R and EP4R enhanced the suppressive actions of PGE₂.This indicates that PGE₂ suppression of TNF-α by human monocytic cells occurs via EP2R and EP4R expression. However EP4Rs also control their own expression and that of EP2 whereas the EP2R does not affect EP4R expression. This implies that EP4 receptors have an important master role in controlling inflammatory responses.     

Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Hull‐less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull‐less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull‐less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over‐express genes encoding synthases for the important dietary fiber component, (1,3;1,4)‐β‐glucan (mixed‐linkage glucan), primarily present in starchy endosperm cell walls. Over‐expression of the HvCslF6 gene, driven by an endosperm‐specific promoter, produced lines where mixed‐linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub‐aleurone cells. This work provides proof‐of‐concept evidence that mixed‐linkage glucan content in hull‐less barley grain can be increased by over‐expression of the HvCslF6 gene, but also indicates that hull‐less cultivars may be more sensitive to attempts to modify cell wall composition.     

ACTIN AND MYOSIN REGULATE PSEUDOPODIA OF PORPHYRA PULCHELLA (RHODOPHYTA) ARCHEOSPORES1

Archeospores of Porphyra pulchella Ackland, J. A. West et Zuccarello (Rhodophyta) display amoeboid and gliding motility. Time‐lapse videomicroscopy revealed that amoeboid cells extend and retract pseudopodia as they translocate through the media. We investigated the involvement of actin and myosin in generating the force for amoeboid motility using immunofluorescence, time‐lapse videomicroscopy, and cytoskeletal inhibitors. Actin filaments were seen as short and long rodlike bundles around the periphery of spores. The actin inhibitors cytochalasin D (CD) and latrunculin B (Lat B), and the myosin inhibitor butanedione monoxime (BDM) disrupted the actin filament network and reversibly inhibited pseudopodial activity, resulting in the rounding and immobilization of spores. It was uncertain whether forward translocation of archeospores resumed following drug removal. These results demonstrate that actin and myosin have a role in generating force for pseudopodial activity. This is the first report of cytoskeletal involvement in red algal cell movement. The involvement of actin and myosin in forward translocation of amoeboid archeospores can only be speculated upon.     

Coral bleaching response index: a new tool to standardize and compare susceptibility to thermal bleaching

As coral bleaching events become more frequent and intense, our ability to predict and mitigate future events depends upon our capacity to interpret patterns within previous episodes. Responses to thermal stress vary among coral species; however the diversity of coral assemblages, environmental conditions, assessment protocols, and severity criteria applied in the global effort to document bleaching patterns creates challenges for the development of a systemic metric of taxon‐specific response. Here, we describe and validate a novel framework to standardize bleaching response records and estimate their measurement uncertainties. Taxon‐specific bleaching and mortality records (2036) of 374 coral taxa (during 1982–2006) at 316 sites were standardized to average percent tissue area affected and a taxon‐specific bleaching response index (taxon‐BRI) was calculated by averaging taxon‐specific response over all sites where a taxon was present. Differential bleaching among corals was widely variable (mean taxon‐BRI = 25.06 ± 18.44%, ±SE). Coral response may differ because holobionts are biologically different (intrinsic factors), they were exposed to different environmental conditions (extrinsic factors), or inconsistencies in reporting (measurement uncertainty). We found that both extrinsic and intrinsic factors have comparable influence within a given site and event (60% and 40% of bleaching response variance of all records explained, respectively). However, when responses of individual taxa are averaged across sites to obtain taxon‐BRI, differential response was primarily driven by intrinsic differences among taxa (65% of taxon‐BRI variance explained), not conditions across sites (6% explained), nor measurement uncertainty (29% explained). Thus, taxon‐BRI is a robust metric of intrinsic susceptibility of coral taxa. Taxon‐BRI provides a broadly applicable framework for standardization and error estimation for disparate historical records and collection of novel data, allowing for unprecedented accuracy in parameterization of mechanistic and predictive models and conservation plans.     

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation

The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.     

Isolation and characterization of marsupial IL5 genes

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.     

An intervention to improve teacher well-being support and training to support students in UK high schools (the WISE study): A cluster randomised controlled trial

BackgroundTeachers are at heightened risk of poor mental health and well-being, which is likely to impact on the support they provide to students, and student outcomes. We conducted a cluster randomised controlled trial, to test whether an intervention to improve mental health support and training for high school teachers led to improved mental health and well-being for teachers and students, compared to usual practice. We also conducted a cost evaluation of the intervention.Methods and findingsThe intervention comprised (i) Mental Health First Aid training for teachers to support students; (ii) a mental health awareness session; and (iii) a confidential staff peer support service. In total 25 mainstream, non-fee-paying secondary schools stratified by geographical area and free school meal entitlement were randomly allocated to intervention (n = 12) or control group (n = 13) after collection of baseline measures. We analysed data using mixed-effects repeated measures models in the intention-to-treat population, adjusted for stratification variables, sex, and years of experience. The primary outcome was teacher well-being (Warwick-Edinburgh Mental Well-being Scale). Secondary outcomes were teacher depression, absence, and presenteeism, and student well-being, mental health difficulties, attendance, and attainment. Follow-up was at months 12 (T1) and 24 (T2). We collected process data to test the logic model underpinning the intervention, to aid interpretation of the findings. A total of 1,722 teachers were included in the primary analysis. Teacher well-being did not differ between groups at T2 (intervention mean well-being score 47.5, control group mean well-being score 48.4, adjusted mean difference −0.90, 95% CI –2.07 to 0.27, p = 0.130). The only effect on secondary outcomes was higher teacher-reported absence among the intervention group at T2 (intervention group median number of days absent 0, control group median number of days absent 0, ratio of geometric means 1.04, 95% CI 1.00 to 1.09, p = 0.042). Process measures indicated little change in perceived mental health support, quality of relationships, and work-related stress. The average cost of the intervention was £9,103 per school. The study’s main limitations were a lack of blinding of research participants and the self-report nature of the outcome measures.ConclusionsIn this study, we observed no improvements to teacher or student mental health following the intervention, possibly due to a lack of impact on key drivers of poor mental health within the school environment. Future research should focus on structural and cultural changes to the school environment, which may be more effective at improving teacher and student mental health and well-being.Trial registrationwww.isrctn.com ISRCTN95909211.

Using a cluster randomized study, Judi Kidger and colleagues study an intervention to improve teacher wellbeing support and training to support students in UK high schools (the WISE study).     

Direct Microbial Reduction and Subsequent Preservation of Uranium in Natural Near-Surface Sediment

The fate of uranium in natural systems is of great environmental importance. X-ray absorption near-edge spectroscopy (XANES) revealed that U(VI) was reduced to U(IV) in shallow freshwater sediment at an open pit in an inactive uranium mine. Geochemical characterization of the sediment showed that nitrate, Fe(III), and sulfate had also been reduced in the sediment. Observations of the sediment particles and microbial cells by scanning and transmission electron microscopy, coupled with elemental analysis by energy dispersive spectroscopy, revealed that uranium was concentrated at microbial cell surfaces. U(IV) was not associated with framboidal pyrite or nanometer-scale iron sulfides, which are presumed to be of microbial origin. Uranium concentrations were not detected in association with algal cells. Phylogenetic analyses of microbial populations in the sediment by the use of 16S rRNA and dissimilatory sulfite reductase gene sequences detected organisms belonging to the families Geobacteraceae and Desulfovibrionaceae. Cultivated members of these lineages reduce U(VI) and precipitate iron sulfides. The association of uranium with cells, but not with sulfide surfaces, suggests that U(VI) is reduced by the enzymatic activities of microorganisms. Uranium was highly enriched (760 ppm) in a subsurface black layer in unsaturated sediment sampled from a pit which was exposed to seasonal fluctuations in the pond level. XANES analysis showed that the majority of uranium in this layer was U(IV), indicating that uranium is preserved in its reduced form after burial.