The One Minute Preceptor: A Vital Tool During COVID-19.

The One Minute Preceptor (OMP) model of teaching has an important role to play during the COVID-19 pandemic. It's quick and easy to learn and can be applied to any clinical setting. By responding directly to a student's needs, and building on the knowledge they already hold, the OMP is able to offer relevant and opportunistic teaching that the learner can immediately apply. Finally, the OMP can be taught in under two hours meaning medical staff not used to regularly teaching can develop the confidence to offer high quality educational interventions.

Intracellular manipulation and measurement with multipole magnetic tweezers.

The capability to directly interrogate intracellular structures inside a single cell for measurement and manipulation is important for understanding subcellular and suborganelle activities, diagnosing diseases, and developing new therapeutic approaches. Compared with measurements of single cells, physical measurement and manipulation of subcellular structures and organelles remain underexplored. To improve intracellular physical measurement and manipulation, we have developed a multipole magnetic tweezers system for micromanipulation involving submicrometer position control and piconewton force control of a submicrometer magnetic bead inside a single cell for measurement in different locations (spatial) and different time points (temporal). The bead was three-dimensionally positioned in the cell using a generalized predictive controller that addresses the control challenge caused by the low bandwidth of visual feedback from high-resolution confocal imaging. The average positioning error was quantified to be 0.4 µm, slightly larger than the Brownian motion-imposed constraint (0.31 µm). The system is also capable of applying a force up to 60 pN with a resolution of 4 pN for a period of time longer than 30 min. The measurement results revealed that significantly higher stiffness exists in the nucleus' major axis than in the minor axis. This stiffness polarity is likely attributed to the aligned actin filament. We also showed that the nucleus stiffens upon the application of an intracellularly applied force, which can be attributed to the response of structural protein lamin A/C and the intracellular stress fiber actin filaments.

An evolutionary shift in the regulation of the Hippo pathway between mice and flies.

The Hippo pathway plays a key role in controlling organ growth in many animal species and its deregulation is associated with different types of cancer. Understanding the regulation of the Hippo pathway and discovering upstream regulators is thus a major quest. Interestingly, while the core of the Hippo pathway contains a highly conserved kinase cascade, different components have been identified as upstream regulators in Drosophila and vertebrates. However, whether the regulation of the Hippo pathway is indeed different between Drosophila and vertebrates or whether these differences are due to our limited analysis of these components in different organisms is not known. Here we show that the mouse Fat4 cadherin, the ortholog of the Hippo pathway regulator Fat in Drosophila, does not apparently regulate the Hippo pathway in the murine liver. In fact, we uncovered an evolutionary shift in many of the known upstream regulators at the base of the arthropod lineage. In this evolutionary transition, Fat and the adaptor protein Expanded gained novel domains that connected them to the Hippo pathway, whereas the cell-adhesion receptor Echinoid evolved as a new protein. Subsequently, the junctional adaptor protein Angiomotin (Amot) was lost and the downstream effector Yap lost its PDZ-binding motif that interacts with cell junction proteins. We conclude that fundamental differences exist in the upstream regulatory mechanisms of Hippo signaling between Drosophila and vertebrates.

Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding.

The t(8;21) translocation fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the ETO protein. The resultant RUNX1/ETO fusion protein is a leukemia-initiating transcription factor that interferes with RUNX1 function. The result of this interference is a block in differentiation and, finally, the development of acute myeloid leukemia (AML). To obtain insights into RUNX1/ETO-dependant alterations of the epigenetic landscape, we measured genome-wide RUNX1- and RUNX1/ETO-bound regions in t(8;21) cells and assessed to what extent the effects of RUNX1/ETO on the epigenome depend on its continued expression in established leukemic cells. To this end, we determined dynamic alterations of histone acetylation, RNA Polymerase II binding and RUNX1 occupancy in the presence or absence of RUNX1/ETO using a knockdown approach. Combined global assessments of chromatin accessibility and kinetic gene expression data show that RUNX1/ETO controls the expression of important regulators of hematopoietic differentiation and self-renewal. We show that selective removal of RUNX1/ETO leads to a widespread reversal of epigenetic reprogramming and a genome-wide redistribution of RUNX1 binding, resulting in the inhibition of leukemic proliferation and self-renewal, and the induction of differentiation. This demonstrates that RUNX1/ETO represents a pivotal therapeutic target in AML.

Cystic kidney diseases and planar cell polarity signaling.

Renal cystic diseases are a major clinical concern as they are the most common genetic cause of end-stage renal disease. While many of the genes causing cystic disease have been identified in recent years, knowing the molecular nature of the mutations has not clarified the mechanisms underlying cyst formation. Recent research in model organisms has suggested that cyst formation may be because of defective planar cell polarity (PCP) and/or ciliary defects. In this review, we first outline the clinical features of renal cystic diseases and then discuss current research linking our understanding of cystic kidney disease to PCP and cilia.

Distribution of extracellular signal-regulated protein kinases 1 and 2 in the rat adrenal and their activation by angiotensin II.

The adrenal gland of the rat is continuously regenerated through proliferation of a stem cell population in the outer part of the gland. To clarify the location of proliferative events within the adrenal gland, and the factors that stimulate them, rat adrenal capsule preparations, consisting of capsule, zona glomerulosa (ZG) and the outer zona fasciculata (ZF) were maintained in vitro under different conditions of stimulation, for varying periods. Sites of proliferation were identified by 5-bromo-2'-deoxy-uridine (BrdU) staining, and the distribution of classical MAP kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) 1 and 2, immunoreactivity was determined using immunocytochemistry. BrdU staining was limited to the outer glomerulosa and the capsule, where it was enhanced by angiotensin II, but not by a high potassium ion concentration nor by ACTH. In contrast, ERK1/2 immunoreactivity was distributed throughout the ZG and in the medulla, with none detectable in the ZF and reticularis. Furthermore, angiotensin II, potassium ions and ACTH were all shown to induce ERK1 and ERK2 phosphorylation in the ZG. Treatment of adrenal capsule tissue with the specific MAPK kinase inhibitor PD98059 revealed inhibition of ERK1/2 phosphorylation, but no effect on angiotensin II-induced aldosterone secretion. Although the distribution and activation of the MAPK pathway suggest a link with proliferation, the findings clearly designated only the outer part of the glomerulosa and capsule as a potential stem cell population. Further functions should be sought for the apparently silent major part of the glomerulosa.

Postcoital vaginal cuff rupture 10 months after a total vaginal hysterectomy. A case report.

BACKGROUND: Transvaginal evisceration following total vaginal hysterectomy secondary to coitus is extremely rare. CASE: A woman presented 10 months following a total vaginal hysterectomy with complaints of progressive postcoital abdominal and shoulder pain as well as a pinkish vaginal discharge. Examination revealed a 3-cm defect at the left edge of the vaginal cuff. Corrective surgery followed overnight observation with pain management. CONCLUSION: Postcoital vaginal cuff disruption is rare, and complications can range from bowel evisceration to hemorrhage. Management should be tailored to the severity of the complications.

Biting back at poor oral hygiene.

Nurses who work in a critical care area face many challenges within their daily role of caring for very sick patients in a stressful and high-tech environment. Underpinning their advanced skills should be a broad base of fundamental nursing skills that has been formulated from research-based evidence. It is, however, the author's belief that oral hygiene--a crucial nursing skill--is not always provided using research-based practice. For this reason, a critical analysis of the issues surrounding the provision of oral hygiene to orally intubated patients within intensive care may provide useful indicators of how the problem could be addressed. This paper considers why orally intubated patients are at particular risk of developing oral health problems, the use of oral assessment tools and the various techniques employed in oral cleansing. The consequences for the patient of poor oral care will also be discussed.

Development of a nonantibiotic dominant marker for positively selecting expression plasmids in multivalent Salmonella vaccines.

We report the novel application of a herbicide-resistance-based dominant marker for the positive selection of expression plasmids in Salmonella serovar vaccines. The beta-lactamase gene of the plasmid pTETnir15, which expresses fragment C of tetanus toxin (TetC), has been replaced with the bar gene marker. The new plasmid pBAT1 can be positively selected in vitro within Salmonella serovars in the presence of the herbicide DL-phosphinothricin. The expression of TetC remains unaltered, and the Salmonella enterica serovar Typhimurium vaccine strain is stable and immunogenic in vivo.

The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation.

Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation.

Regulation of MAPK activity in response to dietary sodium in the rat adrenal gland.

Stimulation of aldosterone biosynthesis by angiotensin II (AII) is thought to be mediated via the PLC, IP3 and intracellular calcium signalling pathway. MAPK (p42/p44) is involved in cell proliferation, and is also activated by AII, but its role in the adrenal response to dietary sodium is unclear. To study the relationship between AII receptor (ATR), MAPK and PKC isoforms, PKCalpha and PKCepsilon, mature Wistar rats were maintained on low or high sodium diets for 1 week. In adrenals from animals on a sodium deplete diet, total ligand binding to both ATR subtypes decreased in the zona glomerulosa (ZG). Under these conditions, active MAPK in the ZG decreased paralleling a decrease in active PKCalpha. In the inner zones (IZ), largely reflecting medullary events, low sodium did not affect MAPK activity. However active PKCalpha decreased. In adrenals from sodium-loaded animals, type 2 ATR (AT2R) binding was reduced in the ZG, while type 1 ATR (AT1R) increased in the IZ. Active MAPK increased in ZG, as did active PKCalpha and PKCepsilon. In IZ, ERK, PKCalpha and PKCepsilon were unchanged. These results suggest that in the ZG and IZ, two different modes of MAPK regulation may exist, utilising different PKC isoforms.

Sticking together and sorting things out: adhesion as a force in development.

During development it is not sufficient for cells to differentiate properly--they must also become physically grouped into appropriate structures, to form skin on the outside, and blood and muscle on the inside. How does this three-dimensional patterning occur? One classic explanation for this resolution of cells and tissues into distinct three-dimensional structures has been that as cells differentiate, they develop differential adhesive properties, and that these affinity differences allow cells to sort out from one another. This classic hypothesis is being investigated with increasing intensity, as recent work on the Drosophila wing and the vertebrate brain has shown that signalling between tissues is essential for the establishment of differential affinities.

mirror controls planar polarity and equator formation through repression of fringe expression and through control of cell affinities.

The Drosophila eye is divided into dorsal and ventral mirror image fields that are separated by a sharp boundary known as the equator. We have previously demonstrated that Mirror, a homeodomain-containing putative transcription factor with a dorsal-specific expression pattern in the eye, induces the formation of the equator at the boundary between mirror-expressing and non-expressing cells. Here, we provide evidence that suggests mirror regulates equator formation by two mechanisms. First, mirror defines the location of the equator by creating a boundary of fringe expression at the mid-point of the eye. We show that mirror creates this boundary by repressing fringe expression in the dorsal half of the eye. Significantly, a boundary of mirror expression cannot induce the formation of an equator unless a boundary of fringe expression is formed simultaneously. Second, mirror acts to sharpen the equator by reducing the mixing of dorsal and ventral cells at the equator. In support of this model, we show that clones of cells lacking mirror function tend not to mix with surrounding mirror-expressing cells. The tendency of mirror-expressing and non-expressing cells to avoid mixing with each other is not determined by their differences in fringe expression. Thus mirror acts to regulate equator formation by both physically separating the dorsal cells from ventral cells, and restricting the formation of a fng expression boundary to the border where the dorsal and ventral cells meet.

Apoptosis: Ras to the rescue in the fly eye.

Programmed cell death has seemed to be regulated in quite different ways in mammals and Drosophila. Recent results on the way Ras and downstream pathways can influence cell-death induction suggest the regulatory pathways in these distinct organisms might be more similar than was at first sight apparent.

MAP Kinase in the rat adrenal gland.

The role of MAP Kinase (MAPK/ERK) in adrenal growth and steroidogenesis is unclear, though in other tissues it is known to act as an integrator of mitogenic signals originating from receptor tyrosine kinases and G-protein coupled receptors. Angiotensin II (AngII) is a major regulator of tissue differentiation and function in the adrenal, acting mainly through the AT1 receptor. Immunocytochemical and enzyme assay methods were used to study the distribution of MAPK and the action of AngII and associated antagonists saralasin and losartan(DuP753) in the rat adrenal gland. MAPK is localised in the zona glomerulosa (ZG) and the medulla, but absent from the zonae fasiculata and reticularis (ZF/ZR). Stimulation with AngII led to decreases in cytosolic and increases in nuclear MAPK activity, and its redistribution from the cytoplasm in unstimulated cells to its localisation around the nucleus, which was confirmed by immunocytochemistry. This translocation was inhibited in the presence of the AngII antagonist saralasin. Therefore, MAPK is located in the glomerulosa, where the AT1 receptor is localised and concerned with aldosterone biosynthesis, and in the medulla where MAPK activation results from AT2R activation. The results indicate the importance of the glomerulosa as the main site of cell proliferation in the adrenal cortex, and that MAPK may represent new signalling pathways related to zone function in the adrenal gland.

mirror encodes a novel PBX-class homeoprotein that functions in the definition of the dorsal-ventral border in the Drosophila eye.

The Drosophila eye is composed of dorsal and ventral mirror-image fields of opposite chiral forms of ommatidia. The boundary between these fields is known as the equator. We describe a novel gene, mirror (mrr), which is expressed in the dorsal half of the eye and plays a key role in forming the equator. Ectopic equators can be generated by juxtaposing mrr expressing and nonexpressing cells, and the path of the normal equator can be altered by changing the domain of mrr expression. These observations suggest that mrr is a key component in defining the dorsal-ventral boundary of tissue polarity in the eye. In addition, loss of mrr function leads to embryonic lethality and segmental defects, and its expression pattern suggests that it may also act to define segmental borders. Mirror is a member of the class of homeoproteins defined by the human proto-oncogene PBX1. mrr is similar to the Iroquois genes ara and caup and is located adjacent to them in this recently described homeotic cluster.

The SH2-containing tyrosine phosphatase corkscrew is required during signaling by sevenless, Ras1 and Raf.

The sevenless gene encodes a receptor tyrosine kinase which is required for the development of the R7 photoreceptor cell in each ommatidium of the Drosophila eye. We have previously used a sensitized genetic screen to identify mutations, designated Enhancers of sevenless (E(sev)), which affect genes that encode components of the sevenless signaling pathway. Here, we report that one of these mutations, E(sev)1Ae0P is a dominantly inhibiting allele of corkscrew, which encodes an SH2 domain-containing protein tyrosine phosphatase (Perkins et al., 1992). We show that corkscrew function is essential for sevenless signaling and that expression of a membrane-targeted form of corkscrew can drive R7 photoreceptor development in the absence of sevenless function. Furthermore, we have used the dominantly inhibiting corkscrew allele to examine the role of corkscrew during signaling by activated forms of Ras1 and Raf. Our analysis indicates that corkscrew function is still required during signaling by activated forms Ras1 and Raf proteins. These results define a function for corkscrew that is either downstream of Ras1 activation or in a parallel pathway that acts with activated Ras1/Raf to specify R7 photoreceptor development.