Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method

<p>Abstract</p> <p>Background</p> <p>Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using <it>Arabidopsis </it>mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and <it>in vivo </it>protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments.</p> <p>Results</p> <p>In this report, we present a new procedure that we call the Tape-<it>Arabidopsis </it>Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis) are used to make a "Tape-<it>Arabidopsis </it>Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC) to investigate protein-protein interactions <it>in vivo</it>, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr.</p> <p>Conclusion</p> <p>The protoplasts generated by this new Tape-<it>Arabidopsis </it>Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time.</p

Complete chloroplast genome of Oncidium Gower Ramsey and evaluation of molecular markers for identification and breeding in Oncidiinae

<p>Abstract</p> <p>Background</p> <p><it>Oncidium </it>spp. produce commercially important orchid cut flowers. However, they are amenable to intergeneric and inter-specific crossing making phylogenetic identification very difficult. Molecular markers derived from the chloroplast genome can provide useful tools for phylogenetic resolution.</p> <p>Results</p> <p>The complete chloroplast genome of the economically important <it>Oncidium </it>variety <it>Onc</it>. Gower Ramsey (Accession no. GQ324949) was determined using a polymerase chain reaction (PCR) and Sanger based ABI sequencing. The length of the <it>Oncidium </it>chloroplast genome is 146,484 bp. Genome structure, gene order and orientation are similar to <it>Phalaenopsis</it>, but differ from typical Poaceae, other monocots for which there are several published chloroplast (cp) genome. The <it>Onc</it>. Gower Ramsey chloroplast-encoded <it>NADH dehydrogenase </it>(<it>ndh</it>) genes, except <it>ndhE</it>, lack apparent functions. Deletion and other types of mutations were also found in the <it>ndh </it>genes of 15 other economically important Oncidiinae varieties, except <it>ndhE </it>in some species. The positions of some species in the evolution and taxonomy of Oncidiinae are difficult to identify. To identify the relationships between the 15 Oncidiinae hybrids, eight regions of the <it>Onc</it>. Gower Ramsey chloroplast genome were amplified by PCR for phylogenetic analysis. A total of 7042 bp derived from the eight regions could identify the relationships at the species level, which were supported by high bootstrap values. One particular 1846 bp region, derived from two PCR products (<it>trnH</it>^GUG-<it>psbA </it>and <it>trnF</it>^GAA-<it>ndhJ</it>) was adequate for correct phylogenetic placement of 13 of the 15 varieties (with the exception of <it>Degarmoara </it>Flying High and <it>Odontoglossum </it>Violetta von Holm). Thus the chloroplast genome provides a useful molecular marker for species identifications.</p> <p>Conclusion</p> <p>In this report, we used <it>Phalaenopsis. aphrodite </it>as a prototype for primer design to complete the <it>Onc</it>. Gower Ramsey genome sequence. Gene annotation showed that most of the <it>ndh </it>genes inOncidiinae, with the exception of <it>ndhE</it>, are non-functional. This phenomenon was observed in all of the Oncidiinae species tested. The genes and chloroplast DNA regions that would be the most useful for phylogenetic analysis were determined to be the <it>trnH</it>^GUG-<it>psbA </it>and the <it>trnF</it>^GAA-<it>ndhJ </it>regions. We conclude that complete chloroplast genome information is useful for plant phylogenetic and evolutionary studies in <it>Oncidium </it>with applications for breeding and variety identification.</p

Nutritionist’s Variation in Counseling Style and the Effect on Weight Change of Patients Attending a Community Based Lifestyle Modification Program

Information concerning the nature of nutritionist-patient relationships is very limited. This qualitative and quantitative study examined nutritionist’s skills, attributes, and beliefs towards nutrition counseling during a lifestyle modification intervention program, and whether this affected the patient’s weight outcome. 24 nutrition consultations were observed during the program and the nutritionists were interviewed for their perception on practice (n = 4). A statistically significant difference was observed between the nutritionists in regard to patient’s weight change after adjustment for age and baseline weight (p < 0.001). Key nutritionist skills identified that influenced weight outcome were meticulous investigation of the underlying obesity cause, identification of the subject’s stage of change, and psychological support

The Asian Jumping Worm (Amynthas spp.)

A visual guide to the invasive Asian jumping worm, including an explanation of why these worms matter, how they spread, how they may cause an increase in toxic mercury in water, and their potential impacts on the ecosystem

Clients’ Experiences of a Community Based Lifestyle Modification Program: A Qualitative Study

There is little information about how clients attending lifestyle modification programs view the outcomes. This qualitative study examined the clients’ experience of a community based lifestyle modification program in Hong Kong. Semi-structured interviews were conducted with 25 clients attending the program. Clients perceived the program had positive impacts on their health and nutrition knowledge. They experienced frustration, negative emotion, lack of motivation, and pressure from others during the program. Working environment and lack of healthy food choices in restaurants were the major perceived environmental barriers for lifestyle modification. Clients valued nutritionists’ capability of providing professional information and psychological support in the program. Our results suggest that nutritionist’s capability of providing quality consultations and patient-centered care are important for empowering clients achieve lifestyle modification

NDH Expression Marks Major Transitions in Plant Evolution and Reveals Coordinate Intracellular Gene Loss

Key innovations have facilitated novel niche utilization, such as the movement of the algal predecessors of land plants into terrestrial habitats where drastic fluctuations in light intensity, ultraviolet radiation and water limitation required a number of adaptations. The NDH (NADH dehydrogenase-like) complex of Viridiplantae plastids participates in adapting the photosynthetic response to environmental stress, suggesting its involvement in the transition to terrestrial habitats. Although relatively rare, the loss or pseudogenization of plastid NDH genes is widely distributed across diverse lineages of photoautotrophic seed plants and mutants/transgenics lacking NDH function demonstrate little difference from wild type under non-stressed conditions. This study analyzes large transcriptomic and genomic datasets to evaluate the persistence and loss of NDH expression across plants. Results: Nuclear expression profiles showed accretion of the NDH gene complement at key transitions in land plant evolution, such as the transition to land and at the base of the angiosperm lineage. While detection of transcripts for a selection of non-NDH, photosynthesis related proteins was independent of the state of NDH, coordinate, lineage-specific loss of plastid NDH genes and expression of nuclear-encoded NDH subunits was documented in Pinaceae, gnetophytes, Orchidaceae and Geraniales confirming the independent and complete loss of NDH in these diverse seed plant taxa. Conclusion: The broad phylogenetic distribution of NDH loss and the subtle phenotypes of mutants suggest that the NDH complex is of limited biological significance in contemporary plants. While NDH activity appears dispensable under favorable conditions, there were likely sufficiently frequent episodes of abiotic stress affecting terrestrial habitats to allow the retention of NDH activity. These findings reveal genetic factors influencing plant/environment interactions in a changing climate through 450 million years of land plant evolution.National Science Foundation IOS-1027259Innovative Translational Agricultural Research Administrative OfficeIntegrative Biolog

Complete Chloroplast Genome Sequence of an Orchid Model Plant Candidate: Erycina pusilla Apply in Tropical Oncidium Breeding

Oncidium is an important ornamental plant but the study of its functional genomics is difficult. Erycina pusilla is a fast-growing Oncidiinae species. Several characteristics including low chromosome number, small genome size, short growth period, and its ability to complete its life cycle in vitro make E. pusilla a good model candidate and parent for hybridization for orchids. Although genetic information remains limited, systematic molecular analysis of its chloroplast genome might provide useful genetic information. By combining bacterial artificial chromosome (BAC) clones and next-generation sequencing (NGS), the chloroplast (cp) genome of E. pusilla was sequenced accurately, efficiently and economically. The cp genome of E. pusilla shares 89 and 84% similarity with Oncidium Gower Ramsey and Phalanopsis aphrodite, respectively. Comparing these 3 cp genomes, 5 regions have been identified as showing diversity. Using PCR analysis of 19 species belonging to the Epidendroideae subfamily, a conserved deletion was found in the rps15-trnN region of the Cymbidieae tribe. Because commercial Oncidium varieties in Taiwan are limited, identification of potential parents using molecular breeding method has become very important. To demonstrate the relationship between taxonomic position and hybrid compatibility of E. pusilla, 4 DNA regions of 36 tropically adapted Oncidiinae varieties have been analyzed. The results indicated that trnF-ndhJ and trnH-psbA were suitable for phylogenetic analysis. E. pusilla proved to be phylogenetically closer to Rodriguezia and Tolumnia than Oncidium, despite its similar floral appearance to Oncidium. These results indicate the hybrid compatibility of E. pusilla, its cp genome providing important information for Oncidium breeding

Identification of 4 novel human ocular coloboma genes ANK3, BMPR1B, PDGFRA, and CDH4 through evolutionary conserved vertebrate gene analysis

Purpose: Ocular coloboma arises from genetic or environmental perturbations that inhibit optic fissure (OF) fusion during early eye development. Despite high genetic heterogeneity, 70% to 85% of patients remain molecularly undiagnosed. In this study, we have identified new potential causative genes using cross-species comparative meta-analysis. Methods: Evolutionarily conserved differentially expressed genes were identified through in silico analysis, with in situ hybridization, gene knockdown, and rescue performed to confirm spatiotemporal gene expression and phenotype. Interrogation of the 100,000 Genomes Project for putative pathogenic variants was performed. Results: Nine conserved differentially expressed genes between zebrafish and mouse were identified. Expression of zebrafish ank3a, bmpr1ba/b, cdh4, and pdgfaa was localized to the OF, periocular mesenchyme cells, or ciliary marginal zone, regions traversed by the OF. Knockdown of ank3, bmpr1b, and pdgfaa revealed a coloboma and/or microphthalmia phenotype. Novel pathogenic variants in ANK3, BMPR1B, PDGFRA, and CDH4 were identified in 8 unrelated coloboma families. We showed BMPR1B rescued the knockdown phenotype but variant messenger RNAs failed, providing evidence of pathogenicity. Conclusion: We show the utility of cross-species meta-analysis to identify several novel coloboma disease-causing genes. There is a potential to increase the diagnostic yield for new and unsolved patients while adding to our understanding of the genetic basis of OF morphogenesis

Assessing the digenic model in rare disorders using population sequencing data

An important fraction of patients with rare disorders remains with no clear genetic diagnostic, even after whole-exome or whole-genome sequencing, posing a difficulty in giving adequate treatment and genetic counseling. The analysis of genomic data in rare disorders mostly considers the presence of single gene variants in coding regions that follow a concrete monogenic mode of inheritance. A digenic inheritance, with variants in two functionally-related genes in the same individual, is a plausible alternative that might explain the genetic basis of the disease in some cases. In this case, digenic disease combinations should be absent or underrepresented in healthy individuals. We develop a framework to evaluate the significance of digenic combinations and test its statistical power in different scenarios. We suggest that this approach will be relevant with the advent of new sequencing efforts including hundreds of thousands of samples

Clinical, genetic, epidemiologic, evolutionary, and functional delineation of TSPEAR-related autosomal recessive ectodermal dysplasia 14

TSPEAR variants cause autosomal recessive ectodermal dysplasia (ARED) 14. The function of TSPEAR is unknown. The clinical features, the mutation spectrum, and the underlying mechanisms of ARED14 are poorly understood. Combining data from new and previously published individuals established that ARED14 is primarily characterized by dental anomalies such as conical tooth cusps and hypodontia, like those seen in individuals with WNT10A-related odontoonychodermal dysplasia. AlphaFold-predicted structure-based analysis showed that most of the pathogenic TSPEAR missense variants likely destabilize the β-propeller of the protein. Analysis of 100000 Genomes Project (100KGP) data revealed multiple founder TSPEAR variants across different populations. Mutational and recombination clock analyses demonstrated that non-Finnish European founder variants likely originated around the end of the last ice age, a period of major climatic transition. Analysis of gnomAD data showed that the non-Finnish European population TSPEAR gene-carrier rate is ∼1/140, making it one of the commonest AREDs. Phylogenetic and AlphaFold structural analyses showed that TSPEAR is an ortholog of drosophila Closca, an extracellular matrix-dependent signaling regulator. We, therefore, hypothesized that TSPEAR could have a role in enamel knot, a structure that coordinates patterning of developing tooth cusps. Analysis of mouse single-cell RNA sequencing (scRNA-seq) data revealed highly restricted expression of Tspear in clusters representing enamel knots. A tspeara−/−;tspearb−/− double-knockout zebrafish model recapitulated the clinical features of ARED14 and fin regeneration abnormalities of wnt10a knockout fish, thus suggesting interaction between tspear and wnt10a. In summary, we provide insights into the role of TSPEAR in ectodermal development and the evolutionary history, epidemiology, mechanisms, and consequences of its loss of function variants