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Transcriptional enhancers are critical for maintaining cell-type–specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.

Dynamic structural properties of chromatin play an essential role in defining cell identity and function. Transcription factors and chromatin modifiers establish and maintain cell states through alteration of DNA accessibility and histone modifications. This activity is focused at both gene-proximal promoter regions and distally located regulatory elements. In the three-dimensional space of the nucleus, distal elements are localized in close physical proximity to the gene-proximal regulatory sequences through the formation of chromatin loops. These looping features in the genome are highly dynamic as embryonic stem cells differentiate and commit to specific lineages, and throughout reprogramming as differentiated cells reacquire pluripotency. Identifying these functional distal regulatory regions in the genome provides insight into the regulatory processes governing early mammalian development and guidance for improving the protocols that generate induced pluripotent cells.

NADPH oxidase activity is involved in plant adaptation and development. The reactive oxygen species sourced by NADPH oxidase activity may contribute to wall strength and protoplast volume adjustment. Root hair bulge apices of the NADPH oxidase mutant rhd2/Atrbohc were more robust than the kjk cellulose synthase mutant, but burst more readily than the wild type (WT). Root epidermal wall appeared impaired in rhd2/Atrbohc, as revealed by the number of protoplasts released by wall-degrading enzymes. Root hair bulges of rhd2/Atrbohc burst more than the WT when challenged in situ with hypo-osmotic low ionic strength medium. Inhibition of NADPH oxidase activity with diphenylene iodonium caused WT to phenocopy the rhd2/Atrbohc bursting in response to hypo-osmotic shock. This implicates RHD2/AtRBOHC in softening the cell wall to permit protoplast expansion. Overall, the results point to a role for RHD2/AtRBOHC in contributing to wall strength.

Background/Objectives: Wound tension plays an integral role in both wound healing and cosmesis. The use of W-plasty has been shown to evenly distribute tension, but is difficult to use in cutaneous surgery. This pilot study aimed to review suture techniques that may give the same wound-tension distribution as W-plasty. Methods: Porcine skin mounted on a standard board was used. A standard grid was superimposed onto the tissue, and an incision was made which was digitally photographed. The defect was then sutured using three suture techniques: simple interrupted sutures, mattress sutures and mattress sutures with knots on alternate sides. Subcutaneous sutures were also used in conjunction with each of these suturing techniques. The grid points were compared and the variation from baseline recorded. These variations were then graphed. Results: The simple interrupted and mattress sutures showed bias of tension opposite to the side of the knot. The alternate mattress suture demonstrated a more even distribution of tension. Conclusion: The mattress suture with alternating knots had a tension pattern that distributed the forces equally across the wound. This is similar to the pattern of W-plasty, suggesting the technique may be used for improved wound healing and cosmesis in areas of tension.

Background/Objectives: The use of preparations such as imiquimod in the treatment of basal cell carcinoma is well accepted. Imiquimod induces interferon-α, other cytokines, antigen-presenting cells and innate immunity, against tumour cells. The current study investigated whether the inflammation induced from a surgical procedure could have a similar effect on removing residual tumour after an excision. Method: A retrospective audit was carried out on basal cell carcinoma removed in the Dermatology Clinic of the Royal Newcastle Centre in 2007. The end-point focussed on the features of those tumours which initially had a positive margin, but were found to have no remaining tumour on subsequent excision. Result: A linear regression was carried out, revealing two significant predictors of outcome. These were the location of the basal cell carcinoma excision and the excision type. Punch biopsies and excisional biopsy had a greater number of histopathologically negative wider excisions despite initial positive margins. Facial lesions had a greater number of negative wider excisions. Conclusion: The study has shown the majority of negative re-excisions were from lesions on the head which had had an initial surgical procedure. However, the evidence is not strong enough to advocate a protocol for dealing with positive margins. A larger sample size that encompassed all three factors that affect outcome, that is, the location of lesion, type of lesion and type of excision carried out, would be required in order to make a more definitive statement on protocol change for treatment of basal cell carcinoma.

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+]cyt) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+]cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+]cyt, and may function as peroxidases in vitro.

Plant annexins are ubiquitous, soluble proteins capable of Ca(2+)-dependent and Ca(2+)-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.

The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Two gene-proximal enhancers, Sox2 regulatory region 1 (SRR1) and SRR2, display activity in reporter assays, but deleting SRR1 has no effect on pluripotency. We identified and functionally validated the sequences required for Sox2 transcription based on a computational model that predicted transcriptional enhancer elements within 130 kb of Sox2. Our reporter assays revealed three novel enhancers—SRR18, SRR107, and SRR111—that, through the formation of chromatin loops, form a chromatin complex with the Sox2 promoter in ES cells. Using the CRISPR/Cas9 system and F1 ES cells (Mus musculus129 × Mus castaneus), we generated heterozygous deletions of each enhancer region, revealing that only the distal cluster containing SRR107 and SRR111, located >100 kb downstream from Sox2, is required for cis-regulation of Sox2 in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression, and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together, these data identify a distal control region essential for Sox2 transcription in ES cells.

Chronic copper toxicity was diagnosed in a Jersey herd in the Waikato region of New Zealand following an investigation into the deaths of six cattle from a herd of 250 dry cows. Clinical signs and post-mortem examination results were consistent with a hepatopathy, and high concentrations of copper in liver and blood samples of clinically affected animals confirmed copper toxicity. Liver copper concentrations and serum gamma-glutamyl transferase activities were both raised in a group of healthy animals sampled at random from the affected herd, indicating an ongoing risk to the remaining cattle; these animals all had serum copper concentrations within normal limits. Serum samples and liver biopsies were also collected and assayed for copper from animals within two other dairy herds on the same farm; combined results from all three herds showed poor correlation between serum and liver copper concentrations. To reduce liver copper concentrations the affected herd was drenched with 0.5 g ammonium molybdate and 1 g sodium sulphate per cow for five days, and the herd was given no supplementary feed or mineral supplements. Liver biopsies were repeated 44 days after the initial biopsies (approximately 1 month after the end of the drenching program); these showed a significant 37.3% decrease in liver copper concentrations (P

In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle. Although SBF is required for cell viability, SWI6 is not an essential gene. We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10. We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Δ and slm8-1 swi6Δ double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6. Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes. Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex. Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region. The cell cycle and transcriptional defects caused by taf17slm7-1 are consistent with the role of TAFIIs as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF.

Transcription occurs at distinct nuclear compartments termed transcription factories that are specialized for transcription by 1 of the 3 polymerase complexes (I, II, or III). Protein-coding genes appear to move in and out of RNA polymerase II (RNAPII) compartments as they are expressed and silenced. In addition, transcription factories are sites where several transcription units, either from the same chromosome or different chromosomes, are transcribed. Chromosomes occupy distinct territories in the interphase nucleus with active genes preferentially positioned on the periphery or even looped out of the territory. These chromosome territories have been observed to intermingle in the nucleus, and multiple interactions among different chromosomes have been identified in genome-wide studies. Deep sequencing of the transcriptome and RNAPII associated on DNA obtained by chromatin immunoprecipitation have revealed a plethora of noncoding transcription and intergenic accumulations of RNAPII that must also be considered in models of genome function. The organization of transcription into distinct regions of the nucleus has changed the way we view transcription with the evolving model for silencing or activation of gene expression involving physical relocation of the transcription unit to a silencing or activation compartment, thus, highlighting the need to consider the process of transcription in the 3-dimensional nuclear space.

Plant cell growth and stress signaling require Ca2+ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca2+-permeable conductance that permits Ca2+ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca2+-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca2+- and K+-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca2+ in response to OH•. An OH•-activated Ca2+ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca2+-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca2+ in plants.

Wound tension plays an integral role in both wound healing and cosmesis. The use of W-plasty has been shown to evenly distribute tension, but is difficult to use in cutaneous surgery. This pilot study aimed to review suture techniques that may give the same wound-tension distribution as W-plasty.Porcine skin mounted on a standard board was used. A standard grid was superimposed onto the tissue, and an incision was made which was digitally photographed. The defect was then sutured using three suture techniques: simple interrupted sutures, mattress sutures and mattress sutures with knots on alternate sides. Subcutaneous sutures were also used in conjunction with each of these suturing techniques. The grid points were compared and the variation from baseline recorded. These variations were then graphed.The simple interrupted and mattress sutures showed bias of tension opposite to the side of the knot. The alternate mattress suture demonstrated a more even distribution of tension.The mattress suture with alternating knots had a tension pattern that distributed the forces equally across the wound. This is similar to the pattern of W-plasty, suggesting the technique may be used for improved wound healing and cosmesis in areas of tension.

The use of preparations such as imiquimod in the treatment of basal cell carcinoma is well accepted. Imiquimod induces interferon-alpha, other cytokines, antigen-presenting cells and innate immunity, against tumour cells. The current study investigated whether the inflammation induced from a surgical procedure could have a similar effect on removing residual tumour after an excision.A retrospective audit was carried out on basal cell carcinoma removed in the Dermatology Clinic of the Royal Newcastle Centre in 2007. The end-point focussed on the features of those tumours which initially had a positive margin, but were found to have no remaining tumour on subsequent excision.A linear regression was carried out, revealing two significant predictors of outcome. These were the location of the basal cell carcinoma excision and the excision type. Punch biopsies and excisional biopsy had a greater number of histopathologically negative wider excisions despite initial positive margins. Facial lesions had a greater number of negative wider excisions.The study has shown the majority of negative re-excisions were from lesions on the head which had had an initial surgical procedure. However, the evidence is not strong enough to advocate a protocol for dealing with positive margins. A larger sample size that encompassed all three factors that affect outcome, that is, the location of lesion, type of lesion and type of excision carried out, would be required in order to make a more definitive statement on protocol change for treatment of basal cell carcinoma.

The food spoilage yeastsZygosaccharomyces bailiiandSaccharomyces cerevisiaehave been proposed to resist weak-acid preservative stress by different means;Z. bailiiby limiting influx of preservative combined with its catabolism,S. cerevisiaeby active extrusion of the preservative weak-acid anion and H+. Measurement of H+extrusion by exponential-phaseZ. bailiicells suggest that, in common withS. cerevisiae, this yeast uses a plasma membrane H+-ATPase to expel H+when challenged by weak-acid preservative (benzoic acid). Simultaneous measurement ofZ. bailiinet H+and K+fluxes showed that net K+influx accompanies net H+efflux during acute benzoic acid stress. Such ionic coupling is known forS. cerevisiaein short-term preservative stress. Both yeasts significantly accumulated K+on long-term exposure to benzoic acid. Analysis ofS. cerevisiaeK+transporter mutants revealed that loss of the high affinity K+uptake systemTrk1confers sensitivity to growth in preservative. The results suggest that cation accumulation is an important factor in adaptation to weak-acid preservatives by spoilage yeasts and thatZ. bailiiandS. cerevisiaeshare hitherto unsuspected adaptive responses at the level of plasma membrane ion transport.

Zygosaccharomyces bailii is a commercially important spoilage yeast capable of growth at low pH in the presence of weak organic acid preservatives, such as benzoic acid. A patch-clamp electrophysiological analysis of plasma membrane K+ transport revealed a high conductance pathway for low-affinity K+ uptake. In contrast to the equivalent K+ transporter in Saccharomyces cerevisiae, this system remained operative at low extracellular pH and may therefore facilitate K+ uptake in K(+)-rich and acidic beverages. Benzoate inhibited growth, increased intracellular K+ content, yet decreased the magnitude of the K+ uptake conductance; specifically, the hyperpolarization-activated inwardly-rectifying component was reduced. It is proposed that this adaptation helps maintain a hyperpolarized membrane voltage to effect continued ATPase-mediated H+ extrusion and so combat preservative-induced cytosolic acidosis. Again in contrast to S. cerevisiae, the K+ conductance was relatively insensitive to increased extracellular Ca2+. Paradoxically (and unlike S. cerevisiae) increasing extracellular Ca2+ inhibited growth, suggesting a simple expedient to limit spoilage by Z. bailii.

Scientific programmes in Antarctica have depended for their fulfilment on small groups of men working in conditions of prolonged social isolation in an alien physical environment. This is true of both early and recent phases of exploration: despite massive advances in technology and, hence, in material comforts and communications over the period, the element of prolonged social isolation remains as a dominant psychological problem. Contrary to the popular image of lonely living conditions and heroic tasks, the Antarctic worker has to cope with the very opposite of loneliness, and his tasks are likely to be mundane and routine. In his work he must adapt to a close, and closed, interpersonal world, and it is with this adaptation that the present article is concerned.