The Role of \u3ci\u3eCellulose Synthase-like D\u3c/i\u3e Genes in Tip Growth of \u3ci\u3ePhyscomitrella patens\u3c/i\u3e

Physcomitrella patens is a non-vascular plant with a relatively small genome and is amongst the few eukaryotic organisms that have a high rate of homologous recombination. This is valuable in biological research because it allows for targeted genetic modification of the organism. In vascular plants like Arabidopsis thaliana, a model organism, Cellulose Synthase-like D (CSLD) genes have been discovered to be important in tip growth. This type of growth is observed in the pollen tubes and root hairs of these plant types. The CSLD genes in Arabidopsis were found to play a crucial role in the growth of root hairs and the production of cellulose or cellulose-like β-1,4-glucan chains in root hair tips. The CSLD genes have also been recognized to be important in pollen tube growth of vascular plants. Physcomitrella patens also contains genes similar to the vascular plant CSLDs, but their functions are not yet fully understood. Within the P. patens genome there are eight genes that make up the CSLD gene family. Additionally, the life cycle of P. patens includes a stage that consists primarily of tip growing cells. This growth stage can be optimized in order to study the role of CSLD genes in tip growth of P. patens. In an effort to further study the roles of the CSLD genes in tip growth of P. patens, we constructed a plasmid that expresses the CSLD1 protein with a green fluorescent protein (GFP) tag. This allowed us to visualize the expression of CSLD1 in living cells using fluorescence microscopy. We also constructed plasmids that were designed to remove specific CSLD genes from the genome and transformed them into wild type or CLSD1 knockout tissue of P. patens. This created single or double knockout mutants that could then be compared to the wild type for changes in the phenotypic characteristics of the plant. These findings will aid in uncovering the roles of the CSLD gene family in P. patens and may provide insight into the functions of these genes in other plants

Characterization of PPAR-gamma 1 and PPAR-gamma 2 in Knockin and Knockout Mouse Models

The global epidemic of obesity and type II diabetes has led to a growing interest in the underlying mechanisms of metabolic diseases. The peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear receptor superfamily, and is vital for the transcriptional regulation of adipogenesis, insulin sensitivity and lipid metabolism. In the mouse model, it has been demonstrated that global knockout of PPARγ leads to severe metabolic disturbance, resulting in embryonic lethality. However, the specific regulatory roles of its two protein isoforms, PPARγ1 and PPARγ2, remain uncertain, due to limitations of reagents and appropriate mouse models. To investigate the hypothesis that PPARγ1 and PPARγ2 are functionally distinct, we generated PPARγ1 and PPARγ2 tagged mice using CRISPR-Cas9 technology. PPARγ1 and PPARγ2 specific knockout mice were also generated incidentally during this process, via aberrant recombination. By reverse-transcription quantitative PCR (RT-qPCR), and western blot, we confirmed the presence of the appropriate tags in our PPARγ1 and PPARγ2 tagged mice, with no significant disruption to mRNA or protein expression. Furthermore, we found that PPARγ1 mRNA and protein expression levels were reduced in our PPARγ1 knockout model, compared to the wild type. Interestingly, we found that there was a complete loss of PPARγ2 protein expression, despite an increase in PPARγ2 mRNA expression in our PPARγ2 knockout model. These data suggest that we have successfully generated PPARγ1 and PPARγ2 knockin and knockout mice. Our mouse models provide a valuable tool to study the individual roles of PPARγ1 and PPARγ2 in adipogenesis, insulin sensitivity and metabolic disease

A Complementation Assay for in Vivo Protein Structure/Function Analysis in \u3cem\u3ePhyscomitrella patens\u3c/em\u3e (Funariaceae)

Premise of Study: A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. Methods and Results: A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. Conclusions: Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants

Ground-Laboratory to In-Space Atomic Oxygen Correlation for the Polymer Erosion and Contamination Experiment (PEACE) Polymers

The Materials International Space Station Experiment 2 (MISSE 2) Polymer Erosion and Contamination Experiment (PEACE) polymers were exposed to the environment of low Earth orbit (LEO) for 3.95 years from 2001 to 2005. There were 41 different PEACE polymers, which were flown on the exterior of the International Space Station (ISS) in order to determine their atomic oxygen erosion yields. In LEO, atomic oxygen is an environmental durability threat, particularly for long duration mission exposures. Although spaceflight experiments, such as the MISSE 2 PEACE experiment, are ideal for determining LEO environmental durability of spacecraft materials, ground-laboratory testing is often relied upon for durability evaluation and prediction. Unfortunately, significant differences exist between LEO atomic oxygen exposure and atomic oxygen exposure in ground-laboratory facilities. These differences include variations in species, energies, thermal exposures and radiation exposures, all of which may result in different reactions and erosion rates. In an effort to improve the accuracy of ground-based durability testing, ground-laboratory to in-space atomic oxygen correlation experiments have been conducted. In these tests, the atomic oxygen erosion yields of the PEACE polymers were determined relative to Kapton H using a radio-frequency (RF) plasma asher (operated on air). The asher erosion yields were compared to the MISSE 2 PEACE erosion yields to determine the correlation between erosion rates in the two environments. This paper provides a summary of the MISSE 2 PEACE experiment; it reviews the specific polymers tested as well as the techniques used to determine erosion yield in the asher, and it provides a correlation between the space and ground laboratory erosion yield values. Using the PEACE polymers asher to in-space erosion yield ratios will allow more accurate in-space materials performance predictions to be made based on plasma asher durability evaluation

Rx Activité physique : Développement et implantation d’objectifs d’apprentissage en matière de counseling et de prescription d’activité physique dans les cursus des facultés de médecine canadiennes

Physical activity is an important component of health and well-being, and is effective in the prevention, management, and treatment of numerous non-communicable chronic diseases. Despite the known health benefits of physical activity in all populations, most Canadians do not meet physical activity recommendations. Physicians play a key role in assessing, counselling, and prescribing physical activity. Unfortunately, many barriers, including the lack of adequate education and training, prevent physicians from promoting this essential health behaviour. To support Canadian medical schools in physical activity curriculum development, a team of researchers, physicians, and exercise physiologists collaborated to develop a key set of learning objectives deemed essential to physican education in physical activity counselling and prescription. This commentary will review the newly developed Canadian Physical Activity Counselling Learning Objectives and give case examples of three Canadian medical schools that have implemented these learning objectives.L’activité physique est une composante importante de la santé et du bien-être, et elle est efficace dans la prévention, la prise en charge et le traitement de nombreuses maladies chroniques non transmissibles. Malgré les bienfaits qu’on lui reconnaît pour la santé des populations, la plupart des Canadiens ne suivent pas les recommandations en matière d’exercice. Les médecins jouent un rôle clé dans l’évaluation, le counseling et la prescription de l’activité physique, mais de nombreux obstacles, dont le manque de formation adéquate, les empêchent de promouvoir cette habitude de vie essentielle pour la santé. Afin d’aider les facultés de médecine canadiennes dans l’élaboration de leur cursus sur l’activité physique, une équipe composée de chercheurs, de médecins et de physiologistes de l’exercice a collaboré à la définition d’un ensemble d’objectifs d’apprentissage jugés indispensables à la formation des médecins pour qu’ils puissent offrir des conseils sur l’activité physique et la prescrire. Ce commentaire passe en revue les nouveaux objectifs d’apprentissage en matière de counseling en activité physique et donne des exemples de cas de trois facultés de médecine canadiennes qui ont mis en œuvre ces objectifs d’apprentissage

Cellulose synthase ‘class specific regions’ are intrinsically disordered and functionally undifferentiated

Cellulose synthases (CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls. Seed plant CESA isoforms cluster in six phylogenetic clades, whose non‐interchangeable members play distinct roles within cellulose synthesis complexes (CSCs). A ‘class specific region’ (CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms. We investigated CESA isoform specificity in the moss, Physcomitrella patens (Hedw.) B. S. G. to gain evolutionary insights into CESA structure/function relationships. Like seed plants, P. patens has oligomeric rosette‐type CSCs, but the PpCESAs diverged independently and form a separate CESA clade. We showed that P. patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore‐negative phenotype of a ppcesa5 knockout line. Thus, non‐interchangeable CESA classes evolved separately in mosses and seed plants. However, testing of chimeric moss CESA genes for complementation demonstrated that functional class‐specificity is not determined by the CSR. Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class‐specific sequences without selection for class‐specific function

The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases

Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial cellulose synthase, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure/function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

The secondary cell walls of tracheary elements and fibers are rich in cellulose microfibrils that are helically oriented and laterally aggregated. Support cells within the leaf midribs of mosses deposit cellulose-rich secondary cell walls, but their biosynthesis and microfibril organization have not been examined. Although the Cellulose Synthase (CESA) gene families of mosses and seed plants diversified independently, CESA knockout analysis in the moss Physcomitrella patens revealed parallels with Arabidopsis (Arabidopsis thaliana) in CESA functional specialization, with roles for both subfunctionalization and neofunctionalization. The similarities include regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls, a requirement for two or more functionally distinct CESA isoforms for secondary cell wall synthesis, interchangeability of some primary and secondary CESAs, and some CESA redundancy. The cellulose-deficient midribs of ppcesa3/8 knockouts provided negative controls for the structural characterization of stereid secondary cell walls in wild type P. patens. Sum frequency generation spectra collected from midribs were consistent with cellulose microfibril aggregation, and polarization microscopy revealed helical microfibril orientation only in wild type leaves. Thus, stereid secondary walls are structurally distinct from primary cell walls, and they share structural characteristics with the secondary walls of tracheary elements and fibers. We propose a mechanism for the convergent evolution of secondary walls in which the deposition of aggregated and helically oriented microfibrils is coupled to rapid and highly localized cellulose synthesis enabled by regulatory uncoupling from primary wall synthesis

Comparing composition and structure in old-growth and harvested (selection and diameter-limit cuts) northern hardwood stands in Quebec

Single-tree selection cutting is sometimes believed to be similar to the natural gap disturbance regime of hardwood forests, but few studies have specifically compared the compositional and structural characteristics of old-growth hardwood stands, undergoing natural gap dynamics and hardwood stands previously subjected to partial cuts. This study characterized and compared the composition (saplings and trees) and structure (gaps, foliage distribution, tree diameter and density, snags and coarse woody debris) of old-growth stands (OG), 12-year-old selection cuts (SC), and 28-33-year-old diameter-limit cuts (DLC) in sugar maple (Acer saccharum)-dominated northern hardwood stands. Results showed marked structural differences between OG and harvested stands, with stronger differences between DLC and OG than between SC and OG. The synchronized formation of numerous canopy openings in harvested stands induced a massive post-harvest recruitment of advance regeneration in both SC and DLC that created a dense foliage layer in the understory. Large living trees (dbh > 39.1 cm) and defective trees were less numerous in SC than OG, which can have a detrimental impact on species dependent on these structural elements, and on the future availability and characteristics of coarse woody debris. Relatively few compositional differences were noticed among stand types, although a greater proportion of mid-tolerant species was found in the post-harvest recruitment cohorts of harvested stands compared to OG, and a lower proportion of beech (Fagus grandifolia Ehrh.) saplings was observed in DLC compared to OG and SC. We argue that even if selection cutting is closer to the natural disturbance regime of hardwood forests than diameter-limit cutting, and therefore representing progress toward the development and implementation of a natural-disturbance-based management, a recurring application of selection cutting might lead to a homogenization of forest structure and composition, a reduction of key structural features and a reduction in biological diversity at both the stand and landscape scales. Some management recommendations are proposed