Dynamic changes in primexine during the tetrad stage of pollen development.

Formation of pollen wall exine is preceded by the development of several transient layers of extracellular materials deposited on the surface of developing pollen grains. One such layer is primexine (PE), a thin, ephemeral structure that is present only for a short period of time and is difficult to visualize and study. Recent genetic studies suggested that PE is a key factor in the formation of exine, making it critical to understand its composition and the dynamics of its formation. In this study, we used high-pressure frozen/freeze-substituted samples of developing Arabidopsis (Arabidopsis thaliana) pollen for a detailed transmission electron microscopy analysis of the PE ultrastructure throughout the tetrad stage of pollen development. We also analyzed anthers from wild-type Arabidopsis and three mutants defective in PE formation by immunofluorescence, carefully tracing several carbohydrate epitopes in PE and nearby anther tissues during the tetrad and the early free-microspore stages. Our analyses revealed likely sites where these carbohydrates are produced and showed that the distribution of these carbohydrates in PE changes significantly during the tetrad stage. We also identified tools for staging tetrads and demonstrate that components of PE undergo changes resembling phase separation. Our results indicate that PE behaves like a much more dynamic structure than has been previously appreciated and clearly show that Arabidopsis PE creates a scaffolding pattern for formation of reticulate exine.

Understanding the experience of the implementer: teachers' perspectives on implementing a classroom-based nutrition education program.

School-based programs are widely implemented to combat childhood obesity, but these programs have mixed results. Dissemination and implementation science approaches to evaluation using qualitative methods can provide more robust details about program functioning that may be able to help explain the variation in the impact of these programs. Fourteen in-depth interviews were conducted with classroom teachers implementing a school-based program, the Integrated Nutrition Education Program (INEP), to explore their experience. Factors related to organization, individual and intervention levels emerged as facilitators and barriers to program implementation. Key factors were school culture at the organization level, individual perception and belief in the intervention at the individual level and program content, perceived complexity and adaptability at the intervention level. Socioeconomic status of the community and family involvement were contextual factors identified across all levels. Findings from this qualitative evaluation can be used for the quality improvement of INEP, but beyond this these can also be informative for other school-based programs to promote adoption, implementation and maintenance.

Motor learning rapidly increases synaptogenesis and astrocytic structural plasticity in the rat cerebellum.

Motor-skill learning is associated with cerebellar synaptogenesis and astrocytic hypertrophy, but most of these assessments of cerebellar ultrastructure have been completed after one month of training. After one month of training, the motor-skills necessary to complete these tasks have been acquired for weeks. This experiment aimed to characterize cerebellar ultrastructure during the acquisition phase of motor-skill learning, at a point when performance is still improving. Male and female rats trained for four days on the acrobatic motor learning task, which involved traversing challenging obstacles such as narrow beams and ladders. Concurrently, rats in the motor control condition walked a flat alleyway requiring no skilled movements. After training was complete, all rats were euthanized, and tissue was prepared for electron microscopy. Unbiased stereology techniques were used to assess synaptic and astrocytic plasticity. Results indicated that during the initial days of training, female rats made fewer errors and had shorter latencies on the acrobatic course compared to male rats. However, there were no sex differences in cerebellar ultrastructure. Male and female rats that completed four days of acrobatic training displayed an increase in the density of parallel fiber-Purkinje cell synapses per Purkinje cell and an increase in astrocytic volume, relative to rats in the motor control group.

Pollen Aperture Factor INP1 Acts Late in Aperture Formation by Excluding Specific Membrane Domains from Exine Deposition.

Accurate placement of extracellular materials is a critical part of cellular development. To study how cells achieve this accuracy, we use formation of pollen apertures as a model. In Arabidopsis (Arabidopsis thaliana), three regions on the pollen surface lack deposition of pollen wall exine and develop into apertures. In developing pollen, Arabidopsis INAPERTURATE POLLEN1 (INP1) protein acts as a marker for the preaperture domains, assembling there into three punctate lines. To understand the mechanism of aperture formation, we studied the dynamics of INP1 expression and localization and its relationship with the membrane domains at which it assembles. We found that INP1 assembly occurs after meiotic cytokinesis at the interface between the plasma membrane and the overlying callose wall, and requires the normal callose wall formation. Sites of INP1 localization coincide with positions of protruding membrane ridges in proximity to the callose wall. Our data suggest that INP1 is a late-acting factor involved in keeping specific membrane domains next to the callose wall to prevent formation of exine at these sites.

Mbd2 enables tumourigenesis within the intestine while preventing tumour-promoting inflammation.

Epigenetic regulation plays a key role in the link between inflammation and cancer. Here we examine Mbd2, which mediates epigenetic transcriptional silencing by binding to methylated DNA. In separate studies the Mbd2-/- mouse has been shown (1) to be resistant to intestinal tumourigenesis and (2) to have an enhanced inflammatory/immune response, observations that are inconsistent with the links between inflammation and cancer. To clarify its role in tumourigenesis and inflammation, we used constitutive and conditional models of Mbd2 deletion to explore its epithelial and non-epithelial roles in the intestine. Using a conditional model, we found that suppression of intestinal tumourigenesis is due primarily to the absence of Mbd2 within the epithelia. Next, we demonstrated, using the DSS colitis model, that non-epithelial roles of Mbd2 are key in preventing the transition from acute to tumour-promoting chronic inflammation. Combining models revealed that prior to inflammation the altered Mbd2-/- immune response plays a role in intestinal tumour suppression. However, following inflammation the intestine converts from tumour suppressive to tumour promoting. To summarise, in the intestine the normal function of Mbd2 is exploited by cancer cells to enable tumourigenesis, while in the immune system it plays a key role in preventing tumour-enabling inflammation. Which role is dominant depends on the inflammation status of the intestine. As environmental interactions within the intestine can alter DNA methylation patterns, we propose that Mbd2 plays a key role in determining whether these interactions are anti- or pro-tumourigenic and this makes it a useful new epigenetic model for inflammation-associated carcinogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Control of Anther Cell Differentiation by the Small Protein Ligand TPD1 and Its Receptor EMS1 in Arabidopsis.

A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive microsporocytes (pollen mother cells) and surrounding somatic cells. The microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS MICROSPOROCYTES1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction.

Pollen wall degradation in the Brassicaceae permits cell emergence after pollination.

PREMISE OF THE STUDY: Despite attempts to degrade the sporopollenin in pollen walls, this material has withstood a hundred years of experimental treatments and thousands of years of environmental attack in insects and soil. We present evidence that sporopollenin, nonetheless, locally degrades only minutes after pollination in Arabidopsis thaliana flowers, and describe here a two-part pollen germination mechanism in A. thaliana involving both chemical weakening of the exine wall and swelling of the underlying intine. METHODS: We explored naturally occurring components from pollen and stigma surfaces and found a tripartite mix of hydrogen peroxide, peroxidase and catalase enzymes (all at high levels at the pollination interface) to be experimentally sufficient to degrade the sporopollenin of some Brassicaceae family members. KEY RESULTS: At pollination, factors carried on the pollen surface may mix with factors on the stigma surface in a reaction that locally oxidizes the exine pollen wall. Hydrogen peroxide, catalases, and peroxidases are biologically present at the right time and place and, when mixed experimentally, are sufficient to degrade the walls of susceptible pollen. CONCLUSIONS: Our work on native biochemistry for breaching sporopollenin suggests new research directions in pollen aperture evolution and could aid efforts to analyze sporopollenin's composition, needed for application of this corrosion-resistant, but long-intractable material.

Ectopic expression of TAPETUM DETERMINANT1 affects ovule development in Arabidopsis.

Plants have evolved to extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control growth, development, and defense. In Arabidopsis thaliana, the EXCESS MICROSPOROCYTES1 (EMS1) LRR-RLK and its potential small protein ligand TAPETUM DETERMINANT1 (TPD1) are specifically required for anther cell differentiation; however, TPD1 and EMS1 orthologs also control megaspore mother cell proliferation in rice and maize ovules. Here, the molecular function of TPD1 was demonstrated during ovule development in Arabidopsis using a gain-of-function approach. In ovules, the EMS1 gene was primarily expressed in nucellus epidermis and chalaza, whereas the expression of TPD1 was weakly restricted to the distal end of integuments. Ectopic expression of TPD1 caused pleiotropic defects in ovule and seed development. RNA sequencing analysis showed that ectopic expression of TPD1 altered expression of auxin signaling genes and core cell-cycle genes during ovule development. Moreover, ectopic expression of TPD1 not only affected auxin response but also enhanced expression of cyclin genes CYCD3;3 and CYCA2;3 in ovules. Thus, these results provide insight into the molecular mechanism by which TPD1-EMS1 signaling controls plant development possibly via regulation of auxin signaling and cell-cycle genes.

A rhamnose-deficient lipopolysaccharide mutant of Rhizobium sp. IRBG74 is defective in root colonization and beneficial interactions with its flooding-tolerant hosts Sesbania cannabina and wetland rice.

Rhizobium sp. IRBG74 develops a classical nitrogen-fixing symbiosis with the aquatic legume Sesbania cannabina (Retz.). It also promotes the growth of wetland rice (Oryza sativa L.), but little is known about the rhizobial determinants important for these interactions. In this study, we analyzed the colonization of S. cannabina and rice using a strain of Rhizobium sp. IRBG74 dually marked with ß-glucuronidase and the green fluorescent protein. This bacterium colonized S. cannabina by crack entry and through root hair infection under flooded and non-flooded conditions, respectively. Rhizobium sp. IRBG74 colonized the surfaces of wetland rice roots, but also entered them at the base of lateral roots. It became endophytically established within intercellular spaces in the rice cortex, and intracellularly within epidermal and hypodermal cells. A mutant of Rhizobium sp. IRBG74 altered in the synthesis of the rhamnose-containing O-antigen exhibited significant defects, not only in nodulation and symbiotic nitrogen fixation with S. cannabina, but also in rice colonization and plant growth promotion. Supplementation with purified lipopolysaccharides from the wild-type strain, but not from the mutant, restored the beneficial colonization of rice roots, but not fully effective nodulation of S. cannabina Commonalities and differences in the rhizobial colonization of the roots of wetland legume and rice hosts are discussed.

Arabidopsis ETHE1 encodes a sulfur dioxygenase that is essential for embryo and endosperm development.

Mutations in human (Homo sapiens) ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized in part by brain lesions, lactic acidemia, excretion of ethylmalonic acid, and ultimately death. ETHE1-like genes are found in a wide range of organisms; however, the biochemical and physiological role(s) of ETHE1 have not been examined outside the context of ethylmalonic encephalopathy. In this study we characterized Arabidopsis (Arabidopsis thaliana) ETHE1 and determined the effect of an ETHE1 loss-of-function mutation to investigate the role(s) of ETHE1 in plants. Arabidopsis ETHE1 is localized in the mitochondrion and exhibits sulfur dioxygenase activity. Seeds homozygous for a DNA insertion in ETHE1 exhibit alterations in endosperm development that are accompanied by a delay in embryo development followed by embryo arrest by early heart stage. Strong ETHE1 labeling was observed in the peripheral and chalazal endosperm of wild-type seeds prior to cellularization. Therefore, ETHE1 appears to play an essential role in regulating sulfide levels in seeds.

Case study: Treating infraspinatus and supraspinatus trigger points and supraspinatus tendinopathy utilizing piezoelectric shockwave.

Two individual case studies demonstrate piezoelectric shockwave treatment for the resolution of a supraspinatus tendinopathy and supraspinatus and infraspinatus myofascial trigger points (MTPs) via musculoskeletal ultrasound. This is the first documentation of improvement of both tendon and muscle fiber patterns in canine patients treated with piezoelectric shockwave. These cases validate the use of piezoelectric shockwave during the rehabilitation of common canine shoulder injuries.

Specification of female germline by microRNA orchestrated auxin signaling in Arabidopsis.

Germline determination is essential for species survival and evolution in multicellular organisms. In most flowering plants, formation of the female germline is initiated with specification of one megaspore mother cell (MMC) in each ovule; however, the molecular mechanism underlying this key event remains unclear. Here we report that spatially restricted auxin signaling promotes MMC fate in Arabidopsis. Our results show that the microRNA160 (miR160) targeted gene ARF17 (AUXIN RESPONSE FACTOR17) is required for promoting MMC specification by genetically interacting with the SPL/NZZ (SPOROCYTELESS/NOZZLE) gene. Alterations of auxin signaling cause formation of supernumerary MMCs in an ARF17- and SPL/NZZ-dependent manner. Furthermore, miR160 and ARF17 are indispensable for attaining a normal auxin maximum at the ovule apex via modulating the expression domain of PIN1 (PIN-FORMED1) auxin transporter. Our findings elucidate the mechanism by which auxin signaling promotes the acquisition of female germline cell fate in plants.

The role of floral organs in carpels, an Arabidopsis loss-of-function mutation in MicroRNA160a, in organogenesis and the mechanism regulating its expression.

MicroRNAs (miRNAs) have emerged as key regulators of gene expression at the post-transcriptional level in both plants and animals. However, the specific functions of MIRNAs (MIRs) and the mechanisms regulating their expression are not fully understood. Previous studies showed that miR160 negatively regulates three genes that encode AUXIN RESPONSE FACTORs (ARF10, -16, and -17). Here, we characterized floral organs in carpels (foc), an Arabidopsis mutant with a Ds transposon insertion in the 3' regulatory region of MIR160a. foc plants exhibit a variety of intriguing phenotypes, including serrated rosette leaves, irregular flowers, floral organs inside siliques, reduced fertility, aberrant seeds, and viviparous seedlings. Detailed phenotypic analysis showed that abnormal cell divisions in the basal embryo domain and suspensor led to diverse defects during embryogenesis in foc plants. Further analysis showed that the 3' region was required for the expression of MIR160a. The accumulation of mature miR160 was greatly reduced in foc inflorescences. In addition, the expression pattern of ARF16 and -17 was altered during embryo development in foc plants. foc plants were also deficient in auxin responses. Moreover, auxin was involved in regulating the expression of MIR160a through its 3' regulatory region. Our study not only provides insight into the molecular mechanism of embryo development via MIR160a-regulated ARFs, but also reveals the mechanism regulating MIR160a expression.

Role of Mrx fimbriae of Xenorhabdus nematophila in competitive colonization of the nematode host.

Xenorhabdus nematophila engages in mutualistic associations with the infective juvenile (IJ) stage of specific entomopathogenic nematodes. Mannose-resistant (Mrx) chaperone-usher-type fimbriae are produced when the bacteria are grown on nutrient broth agar (NB agar). The role of Mrx fimbriae in the colonization of the nematode host has remained unresolved. We show that X. nematophila grown on LB agar produced flagella rather than fimbriae. IJs propagated on X. nematophila grown on LB agar were colonized to the same extent as those propagated on NB agar. Further, progeny IJs were normally colonized by mrx mutant strains that lacked fimbriae both when bacteria were grown on NB agar and when coinjected into the insect host with aposymbiotic nematodes. The mrx strains were not competitively defective for colonization when grown in the presence of wild-type cells on NB agar. In addition, a phenotypic variant strain that lacked fimbriae colonized as well as the wild-type strain. In contrast, the mrx strains displayed a competitive colonization defect in vivo. IJ progeny obtained from insects injected with comixtures of nematodes carrying either the wild-type or the mrx strain were colonized almost exclusively with the wild-type strain. Likewise, when insects were coinjected with aposymbiotic IJs together with a comixture of the wild-type and mrx strains, the resulting IJ progeny were predominantly colonized with the wild-type strain. These results revealed that Mrx fimbriae confer a competitive advantage during colonization in vivo and provide new insights into the role of chaperone-usher fimbriae in the life cycle of X. nematophila.

Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor kinase.

Sexual reproduction requires the specification of cells with distinct fates in plants and animals. The EMS1 (also known as EXS) leucine-rich repeat receptor-like kinase (LRR-RLK) and TPD1 small protein play key roles in regulating somatic and reproductive cell fate determination in Arabidopsis anthers. Here, we show that ectopic expression of TPD1 causes abnormal differentiation of somatic and reproductive cells in anthers. In addition, ectopic TPD1 activity requires functional EMS1. Yeast two-hybrid, pull-down, and coimmunoprecipitation analyses further demonstrate that TPD1 interacts with EMS1 in vitro and in vivo. Moreover, TPD1 induces EMS1 phosphorylation in planta. Thus, our results suggest that TPD1 serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction.

The challenge of integrating new online education packages into existing curricula: a new model.

BACKGROUND: In 2009, The National Institute for Health and Clinical Excellence (NICE) developed an undergraduate online learning package on the practical application of evidence-based medicine with the intention that it would be integrated into existing medical curricula. METHODS: Complementary methodologies were used to yield a diversity of quantitative and qualitative data on how the online learning package was integrated. RESULTS: The modules of the online learning package received an overall positive reaction from the users but uptake of the modules was lower than expected. Even though some curriculum integration occurred, several students were unaware that the package existed, some lacked the time to use the package and others would have preferred to have had the package earlier in their course. CONCLUSIONS: A new model for the effective integration of online education packages into existing undergraduate medical curricula is proposed, especially when developed by external organisations. This new model should enable educationalists to better reveal and overcome the contextual and process challenges, barriers and solutions to implementing effective flexible learning approaches. When introducing new learning resources into a curriculum, many factors are important, especially the learners' perceived needs and how these vary at different stages of their course.

The SPOROCYTELESS/NOZZLE gene is involved in controlling stamen identity in Arabidopsis.

The stamen, which consists of an anther and a filament, is the male reproductive organ in a flower. The specification of stamen identity in Arabidopsis (Arabidopsis thaliana) is controlled by a combination of the B genes APETALA3 (AP3) and PISTILLATA, the C gene AGAMOUS (AG), and the E genes SEPALLATA1 (SEP1) to SEP4. The "floral organ-building" gene SPOROCYTELESS/NOZZLE (SPL/NZZ) plays a central role in regulating anther cell differentiation. However, much less is known about how "floral organ identity" and floral organ-building genes interact to control floral organ development. In this study, we report that ectopic expression of SPL/NZZ not only affects flower development in the wild-type background but also leads to the transformation of petal-like organs into stamen-like organs in flowers of ap2-1, a weak ap2 mutant allele. Moreover, our loss-of-function analysis indicates that the spl/nzz mutant enhances the phenotype of the ag weak allele ag-4. Furthermore, ectopic expression and overexpression of SPL/NZZ altered expression of AG, SEP3, and AP2 in rosette leaves and flowers, while ectopic expression of SPL/NZZ resulted in ectopic expression of AG and SEP3 in the outer whorls of flowers. Our results indicate that the SPL/NZZ gene is engaged in controlling stamen identity via interacting with genes required for stamen identity in Arabidopsis.

A new system for heterologous expression of membrane proteins: Rhodospirillum rubrum.

Heterologous expression of membrane proteins has met with only limited success. This work presents a new host/vector system for the production of heterologous membrane proteins based on a mutant of the facultatively phototrophic bacterium Rhodospirillum rubrum. Under certain growth conditions, R. rubrum forms an intracytoplasmic membrane (ICM) that houses the photosynthetic apparatus, the structural proteins of which are encoded by puhA and pufBALM. The mutant R. rubrum H2, which was constructed by allelic exchange deleting puhA and pufBALM, does not form ICM. This strain was used as a host for a plasmid expressing the Pseudomonas aeruginosa membrane protein MscL from the Rhodobacter capsulatus puc promoter. ICM was formed in the H2 strain producing MscL but not in the vector control strain. These results suggest that a heterologous membrane protein stimulates ICM formation in R. rubrum and indicate that the capacity to form an ICM that can accommodate heterologous proteins makes R. rubrum a host that will be useful for membrane protein production. P. aeruginosa MscL, which forms inclusion bodies when produced in Escherichia coli, was expressed in R. rubrum H2 and purified from membranes with a yield of 22.8-23.4 mg/L culture (5.53-5.60 mg/g cell paste). Additionally Streptomyces lividans KcsA and P. aeruginosa CycB were produced and purified from R. rubrum H2 with yields of 13.7-14.4 mg/L culture (2.19-2.55 mg/g cell paste) and 6.6-7.4 mg/L culture (1.1-1.2mg/g cell paste), respectively.

Functional relevance of novel p300-mediated lysine 314 and 315 acetylation of RelA/p65.

Nuclear factor kappaB (NF-kappaB) plays an important role in the transcriptional regulation of genes involved in immunity and cell survival. We show here in vitro and in vivo acetylation of RelA/p65 by p300 on lysine 314 and 315, two novel acetylation sites. Additionally, we confirmed the acetylation on lysine 310 shown previously. Genetic complementation of RelA/p65-/- cells with wild type and non-acetylatable mutants of RelA/p65 (K314R and K315R) revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by tumor necrosis factor alpha was affected by acetylation on these residues. Microarray analysis of these cells treated with TNFalpha identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of RelA/p65. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-kappaB dependent gene expression.