A qPCR Method to Distinguish between Expression of Transgenic and Endogenous Copies of Genes.

Study of gene function in eukaryotes frequently requires data on the impact of the gene when it is expressed as a transgene, such as in ectopic or overexpression studies. Currently, the use of transgenic constructs designed to achieve these aims is often hampered by the difficulty in distinguishing between the expression levels of the endogenous gene and its transgene equivalent, which may involve either laborious microdissection to isolate specific cell types or harvesting tissue at narrow timepoints. To address this challenge, we have exploited a feature of the Golden Gate cloning method to develop a simple, restriction digest-based protocol to differentiate between expression levels of transgenic and endogenous gene copies. This method is straightforward to implement when the endogenous gene contains a Bpi1 restriction site but, importantly, can be adapted for most genes and most other cloning strategies. Key features This protocol was developed to determine the expression level of an ectopically expressed transcription factor with broad native expression in all surrounding tissues. The method described is most directly compatible with Golden Gate cloning but is, in principle, compatible with any cloning method. The protocol has been developed and validated in the model plant Arabidopsis thaliana but is applicable to most eukaryotes. Graphical overview.

SOBIR1/EVR prevents precocious initiation of fiber differentiation during wood development through a mechanism involving BP and ERECTA.

In plants, secondary growth results in radial expansion of stems and roots, generating large amounts of biomass in the form of wood. Using genome-wide association studies (GWAS)-guided reverse genetics in Arabidopsis thaliana, we discovered SOBIR1/EVR, previously known to control plant immunoresponses and abscission, as a regulator of secondary growth. We present anatomical, genetic, and molecular evidence indicating that SOBIR1/EVR prevents the precocious differentiation of xylem fiber, a key cell type for wood development. SOBIR1/EVR acts through a mechanism that involves BREVIPEDICELLUS (BP) and ERECTA (ER), 2 proteins previously known to regulate xylem fiber development. We demonstrate that BP binds SOBIR1/EVR promoter and that SOBIR1/EVR expression is enhanced in bp mutants, suggesting a direct, negative regulation of BP over SOBIR1/EVR expression. We show that SOBIR1/EVR physically interacts with ER and that defects caused by the sobir1/evr mutation are aggravated by mutating ER, indicating that SOBIR1/EVR and ERECTA act together in the control of the precocious formation of xylem fiber development.

Men with Duchenne muscular dystrophy and end of life planning.

There is very limited evidence about the views of men with Duchenne muscular dystrophy (DMD) and end of life issues including death and dying. Studies have shown the physiological and psychological benefits of talking about and planning for end of life. Despite policy documents and guidance in the UK about end of life planning, there is consensus on the need for improvement. The study reported here is a qualitative one with 15 men with DMD (aged 20-45 years). Participants could not recall any significant conversations with clinicians about end of life and assumed that clinicians were reluctant to discuss the issue. The men in the study wanted to be given proactive cues that they could bring up topics such as death and dying and wanted to have these conversations with clinicians who combined expert knowledge about the condition as well as good listening skills. Topics of interest to participants included likely nature and place of death; practical planning for funerals and wills; and sources of information and support. Emotional or psychological support to think about end of life was not routinely offered and participants found it very difficult to discuss these issues with family members. The study suggests that more could be done to encourage clinicians, men with Duchenne, family members and the wider NMD community to pay attention to end of life planning issues and the associated need for emotional support and high quality interactions between patients and clinicians.

The Mechanism Forming the Cell Surface of Tip-Growing Rooting Cells Is Conserved among Land Plants.

To discover mechanisms that controlled the growth of the rooting system in the earliest land plants, we identified genes that control the development of rhizoids in the liverwort Marchantia polymorpha. 336,000 T-DNA transformed lines were screened for mutants with defects in rhizoid growth, and a de novo genome assembly was generated to identify the mutant genes. We report the identification of 33 genes required for rhizoid growth, of which 6 had not previously been functionally characterized in green plants. We demonstrate that members of the same orthogroup are active in cell wall synthesis, cell wall integrity sensing, and vesicle trafficking during M. polymorpha rhizoid and Arabidopsis thaliana root hair growth. This indicates that the mechanism for constructing the cell surface of tip-growing rooting cells is conserved among land plants and was active in the earliest land plants that existed sometime more than 470 million years ago [1, 2].

RSL Class I Genes Controlled the Development of Epidermal Structures in the Common Ancestor of Land Plants.

The colonization of the land by plants, sometime before 470 million years ago, was accompanied by the evolution tissue systems [1-3]. Specialized structures with diverse functions-from nutrient acquisition to reproduction-derived from single cells in the outermost layer (epidermis) were important sources of morphological innovation at this time [2, 4, 5]. In extant plants, these structures may be unicellular extensions, such as root hairs or rhizoids [6-9], or multicellular structures, such as asexual propagules or secretory hairs (papillae) [10-12]. Here, we show that a ROOTHAIR DEFECTIVE SIX-LIKE (RSL) class I basic helix-loop-helix transcription factor positively regulates the development of the unicellular and multicellular structures that develop from individual cells that expand out of the epidermal plane of the liverwort Marchantia polymorpha; mutants that lack MpRSL1 function do not develop rhizoids, slime papillae, mucilage papillae, or gemmae. Furthermore, we discovered that RSL class I genes are also required for the development of multicellular axillary hairs on the gametophyte of the moss Physcomitrella patens. Because class I RSL proteins also control the development of rhizoids in mosses and root hairs in angiosperms [13, 14], these data demonstrate that the function of RSL class I genes was to control the development of structures derived from single epidermal cells in the common ancestor of the land plants. Class I RSL genes therefore controlled the generation of adaptive morphological diversity as plants colonized the land from the water.

Intensity of a pulse of RSL4 transcription factor synthesis determines Arabidopsis root hair cell size.

Cell size is determined by the duration and rate of growth and plays a central role in cell function. Root hairs are tip-growing cellular projections that emerge from the root epidermis and explore the soil to acquire nutrients and water(1). Previously we demonstrated that the basic helix-loop-helix transcription factor root hair defective 6-like 4 (RSL4) is necessary and sufficient for root hair growth(2). Here we show that RSL4 is synthesized in a 4-h pulse at the initiation of hair elongation and is gradually degraded by the 26S proteasome. The amount of RSL4 synthesis during this pulse is modulated as part of a root hair growth response to low phosphate. RSL4 synthesis increases in low phosphate and this increase prolongs the growth phase, resulting in the development of long root hairs. Our data demonstrate that the amount of RSL4 synthesized during the pulse directly determines the final size of the differentiated root hair cell. We propose that the modulation of growth-promoting transcription factors by external cues could be a general mechanism for the regulation of cell growth by environmental factors during development.

"That must be so hard"--examining the impact of children's palliative care services on the psychological well-being of parents.

In 2003 the New Opportunities Fund (NOF, now known as The Big Lottery) awarded £48 million to 70 home-based care teams to enable them to provide a range of services to allow children with non-malignant life-limiting conditions to be cared for at home. Four grants were made available in Bristol, North Somerset, South Gloucestershire and Bath and North East Somerset to enhance existing children's palliative care services. As there is limited evidence about the impact of palliative care services on psychological well-being, this study measured parental stress and the psychological wellbeing of parents of newly referred children with life-limiting and life-threatening conditions. Measures were administered at the point of referral and at 12 months follow-up. Statistical analysis indicated that there was no significant change at 12 month follow-up. The lack of deterioration in levels of parental stress and psychological wellbeing is viewed positively within this context. It is hypothesised that multi-agency and partnership working was a significant contributory factor in not increasing levels of parental stress and psychological wellbeing through social support.

A role for BELLRINGER in cell wall development is supported by loss-of-function phenotypes.

BACKGROUND: Homeodomain transcription factors play critical roles in metazoan development. BELLRINGER (BLR), one such transcription factor, is involved in diverse developmental processes in Arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. BLR also has a redundant role in meristem maintenance. Cell wall remodelling underpins many of these processes, and BLR has recently been shown to regulate expression of PECTIN METHYL-ESTERASE 5 (PME5), a cell wall modifying enzyme in control of phyllotaxy. We have further explored the role of BLR in plant development by analysing phenotypes and gene expression in a series of plants over-expressing BLR, and generating combinatorial mutants with blr, brevipedicellus (bp), a member of the KNOX1 family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta (rev), required for radial patterning of the stem. RESULTS: Plants over-expressing BLR exhibited a wide range of phenotypes. Some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. Other lines with more extreme phenotypes failed to generate lateral organs, consistent with BLR repressing transcription in the shoot apex. Cell wall dynamics are also affected in blr mutant plants, and BLR has previously been shown to regulate vascular development in conjunction with BP. We found that when bp and blr were combined with rev, a set of defects was observed that were distinct from those of bp blr lines. In these triple mutants xylem development was most strikingly affected, resulting in an almost complete lack of vessels and xylem parenchyma with secondary thickening. CONCLUSIONS: Our data support a role for BLR in ordering the shoot apex and, in conjunction with BP and REV, playing a part in determining the composition and organisation of the vascular system. Microarray analysis strongly indicates that the striking vascular phenotypes of blr bp rev triple mutants and plants over-expressing BLR result from the misregulation of a suite of genes, targets of BLR in wild type plants, that determine cell size and structure in the developing vasculature.

A new Stroop-like measure of inhibitory function development: typical developmental trends.

BACKGROUND: Difficulties with inhibition are increasingly regarded as central to pathological behavioural and learning disorders in childhood. However, few measures are available to assist in the assessment of young children's inhibitory competence. METHOD: A new, Stroop-like measure of inhibitory function is described which was designed to be appropriate for use with children from 3 years of age. One hundred and fifty-five school children aged between 3 and 16 years took part in a study to examine the developmental characteristics and behavioural correlates of task performance. RESULTS: The task appears to provide a robust measure of inhibitory function across the age range, 3 to 16 years. Furthermore, the animal-stroop task appears to identify those at risk of hyperactive symptomatology within a school-based sample. CONCLUSIONS: The clinical and developmental applications of this new measure are discussed.