Genetically encoded, light activatable proteins furnish the means to probe biochemical pathways at specific sub-cellular locations with exquisite temporal control. However, engineering these systems to provide a dramatic jump in localized activity while retaining a low dark-state background remains a significant challenge. We describe herein an actin-remodelling protein cofilin that, when placed within the framework of a genetically encodable, light activatable heterodimerizer system, induces dramatic changes in the F-actin network and consequent cell motility upon illumination. We demonstrate that the use of a partially impaired mutant of cofilin is critical for maintaining low background activity in the dark. We also show that light-directed recruitment of the reduced activity cofilin mutants to the cytoskeleton is sufficient to induce F-actin remodeling, formation of filopodia, and directed cell motility.
optogenetics; cofilin; cell motility; F-actin; protein design
Despite extensive infection control measures against parasitic diseases, hydatid disease, caused by Echinococcus granulosus, still occurs in a minor group of our population. If the infection is not treated adequately, it goes on to developing life-threatening complications, one of which is hepatopulmonary fistula. These complications usually warrant early surgical intervention, or else may lead to extensive sepsis and ultimately death. We discuss the case of an elderly female suffering from pulmonary hydatid disease, further complicated by a hepatopulmonary fistula and underwent surgical treatment. This case emphasises the importance of early recognition of pulmonary hydatid disease given its atypical nature of presentation before the disease is further exacerbated by this aggressive complication. Furthermore, it is imperative to incorporate radical surgery as the first-line treatment in established hepatopulmonary fistula, in order to prevent further clinical deterioration and curative outcome.
Hydatid disease; Hepatopulmonary fistula; Thoracotomy
This study aimed to examine friendship networks and social support outcomes for mothers according to patterns of playgroup participation.
Data from the Longitudinal Study of Australian Children were used to examine the extent to which patterns of playgroup participation across the ages of 3–19 months (Wave 1) and 2–3 years (Wave 2) were associated with social support outcomes for mothers at Wave 3 (4–5 years) and four years later at Wave 5 (8–9 years). Analyses were adjusted for initial friendship attachments at Wave 1 and other socio-demographic characteristics.
Log-binomial regression models estimating relative risks showed that mothers who never participated in a playgroup, or who participated at either Wave 1 or Wave 2 only, were 1.7 and 1.8 times as likely to report having no support from friends when the child was 4–5 years, and 2.0 times as likely to have no support at age 8–9 years, compared with mothers who persistently participated in playgroup at both Wave 1 and Wave 2.
These results provide evidence that persistent playgroup participation may acts as a protective factor against poor social support outcomes. Socially isolated parents may find playgroups a useful resource to build their social support networks.
Tarsal coalitions affect up to 13% of the population and can be a cause of chronic ankle and hindfoot pain. They can be subdivided as osseous, cartilaginous, or fibrous types, each with unique radiographic, CT, and MR imaging findings. In particular, MR imaging offers the unique ability to determine the exact type of tarsal coalition that is present as well as whether any associated soft tissue abnormalities are present.
The purposes of this paper were to (1) review the anatomy of the hindfoot; (2) review the radiographic, CT, and MR imaging findings of tarsal coalitions; and (3) review the imaging appearance of the specific types of tarsal coalitions.
Online searches were performed using Google Scholar with the search criteria of “tarsal coalition,” “hindfoot anatomy,” and “subtalar coalition,” and limiting the searches to papers published in the last 10 years in major radiology journals.
The anatomy of the hindfoot is complex but essential to understand. There are various radiographic, CT, and MR imaging findings that can be consistently noted in cases of tarsal coalition. The specific types of tarsal coalition demonstrate characteristic imaging findings.
Knowledge of the normal anatomy of the foot, in particular the hindfoot, combined with the knowledge of the imaging characteristics of different histologic subtypes of coalitions (osseous, cartilaginous, and fibrous) is essential for interpreting radiographic, CT, and MR images of the ankle and foot.
Electronic supplementary material
The online version of this article (doi:10.1007/s11420-013-9379-z) contains supplementary material, which is available to authorized users.
tarsal coalition; subtalar coalition; calcaneonavicular coalition
p53 is a master tumour repressor that participates in vast regulatory networks, including feedback loops involving microRNAs (miRNAs) that regulate p53 and that themselves are direct p53 transcriptional targets. We show here that a group of polycistronic miRNA-like non-coding RNAs derived from small nucleolar RNAs (sno-miRNAs) are transcriptionally repressed by p53 through their host gene, SNHG1. The most abundant of these, sno-miR-28, directly targets the p53-stabilizing gene, TAF9B. Collectively, p53, SNHG1, sno-miR-28 and TAF9B form a regulatory loop which affects p53 stability and downstream p53-regulated pathways. In addition, SNHG1, SNORD28 and sno-miR-28 are all significantly upregulated in breast tumours and the overexpression of sno-miR-28 promotes breast epithelial cell proliferation. This research has broadened our knowledge of the crosstalk between small non-coding RNA pathways and roles of sno-miRNAs in p53 regulation.
Surface composition information from Vesta is reported using fast neutron data collected by the gamma ray and neutron detector on the Dawn spacecraft. After correcting for variations due to hydrogen, fast neutrons show a compositional dynamic range and spatial variability that is consistent with variations in average atomic mass from howardite, eucrite, and diogenite (HED) meteorites. These data provide additional compositional evidence that Vesta is the parent body to HED meteorites. A subset of fast neutron data having lower statistical precision show spatial variations that are consistent with a 400 ppm variability in hydrogen concentrations across Vesta and supports the idea that Vesta's hydrogen is due to long-term delivery of carbonaceous chondrite material.
In this paper, we address points raised by Stephanie Dancer's article in The BMJ in which she claimed that by ‘dressing down’, physicians fail to adhere to the dignitas of the medical profession, and damage its reputation. At the beginning of this paper, we distinguish between two different senses in which a person can be, as she terms it, ‘scruffy’; and then we address Dancer's three main claims. First, we argue that in regard to the medical profession it is fallacious to assume, as she appears to do, that someone is incompetent or irresponsible when such a judgement is grounded in the fact that a physician is not dressed in a formal way. Second, we argue, contrary to her claim, that the dignified nature of the medical profession is in no coherent way linked to sartorial elegance or lack thereof, but rather, that such dignity is bound to the value of the medical practice in itself, to patients, and to society at large. Third, we examine two ways in which doctors can ‘dress down’ and show that ‘scruffiness’ does not necessarily intimates a lack of personal hygiene. Finally, we show that pointing to mere statistical correlation without causation, cannot be used as an argument against scruffiness. We conclude by suggesting that in the medical context, it is more appropriate to educate patients than to chastise practitioners for not following arbitrary cultural mores.
Applied and Professional Ethics; Autonomy; Clinical Ethics; Education; Ethics
The aims of this study were to assess participatory methods for obtaining community views on child health research.
Community participation in research is recognised as an important part of the research process; however, there has been inconsistency in its implementation and application in Australia. The Western Australian Telethon Kids Institute Participation Program employs a range of methods for fostering active involvement of community members in its research. These include public discussion forums, called Community Conversations. While participation levels are good, the attendees represent only a sub-section of the Western Australian population. Therefore, we conducted a telephone survey of randomly selected households to evaluate its effectiveness in eliciting views from a broader cross-section of the community about our research agenda and community participation in research, and whether the participants would be representative of the general population. We also conducted two Conversations, comparing the survey as a recruitment tool and normal methods using the Participation Program.
While the telephone survey was a good method for eliciting community views about research, there were marked differences in the profile of study participants compared to the general population (e.g. 78% vs 50% females). With a 26% response rate, the telephone survey was also more expensive than a Community Conversation. The cold calling approach proved an unsuccessful recruitment method, with only two out of a possible 816 telephone respondents attending a Conversation.
While the results showed that both of the methods produced useful input for our research program, we could not conclude that either method gained input that was representative of the entire community. The Conversations were relatively low-cost and provided more in-depth information about one subject, whereas the telephone survey provided information across a greater range of subjects, and allowed more quantitative analysis.
In the present study, the effects of 10- or 100-nm silica oxide (SiO2) NPs on human peripheral blood mononuclear cells (PBMC) were examined. Cytotoxic effects and oxidative stress effects, including glutathione (GSH) depletion, the formation of protein radical species, and pro-inflammatory cytokine responses, were measured. PBMC exposed to 10-nm NP concentrations from 50 to 4,000 ppm showed concentration-response increases in cell death; whereas, for 100-nm NPs, PBMC viability was not lost at <500 ppm. Interestingly, 10-nm NPs were more cytotoxic and induced more oxidative stress than 100-nm NPs. Immunoelectron micrographs show the cellular distribution of GSH and NPs. As expected based on the viability data, the 10-nm NPs disturbed cell morphology to a greater extent than did the 100-nm NPs. Antibody to the radical scavenger, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was used for Western blot analysis of proteins with radicals; more DMPO proteins were found after exposure to 10-nm NPs than 100-nm NPs. Examination of cytokines (TNF-α, IL-1ra, IL-6, IL-8, IL-1β, and IFN-γ) indicated that different ratios of cytokines were expressed and released after exposure to 10- and 100-nm NPs. IL-1β production was enhanced by 10- and 100-nm NPs;, the cytotoxicity of the NPs was associated with an increase in the IL-1β/IL-6 ratio and 100-nm NPs at concentrations that did not induce loss of cell viability enhanced IL-1β and IL-6 to an extent similar to phytohemagglutinin (PHA), a T cell mitogen. In conclusion, our results indicate that SiO2 NPs trigger a cytokine inflammatory response and induce oxidative stress in vitro, and NPs of the same chemistry, but of different sizes, demonstrate differences in their intracellular distribution and immunomodulatory properties, especially with regard to IL-1β and IL-6 expression.
Silica (SiO2) nanoparticles; PBMCs; Oxidative stress; Glutathione (GSH); Radicals; 5,5-dimethyl-1-pyrroline N-oxide (DMPO); Inflammatory cytokines
Inflammatory myofibroblastic tumour (IMT) of the lung represents an extremely rare type of inflammatory pseudo tumor that appears most commonly in children and young individuals. There has been an ongoing controversy whether an IMT is a reactive lesion or a true neoplasm making the further management extremely challenging. Purpose of the paper is through a literature review to highlight the existence of this rare tumour along with its key features and the management options available.
Lung neoplasms; inflammatory myofibroblastic tumors (IMTs)
An optimized peptide substrate was
used to measure protein kinase
B (PKB) activity in single cells. The peptide substrate was introduced
into single cells, and capillary electrophoresis was used to separate
and quantify nonphosphorylated and phosphorylated peptide. The system
was validated in three model pancreatic cancer cell lines before being
applied to primary cells from human pancreatic adenocarcinomas propagated
in nude mice. As measured by phosphorylation of peptide substrate,
each tumor cell line exhibited statistically different median levels
of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human
pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic
adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma),
respectively) with CFPAC-1 cells demonstrating two populations of
cells or bimodal behavior in PKB activation levels. The primary cells
exhibited highly variable PKB activity at the single cell level, with
some cells displaying little to no activity and others possessing
very high levels of activity. This system also enabled simultaneous
characterization of peptidase action in single cells by measuring
the amount of cleaved peptide substrate in each cell. The tumor cell
lines displayed degradation rates statistically similar to one another
(0.02, 0.06, and 0.1 zmol pg–1 s–1, for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the
degradation rate in primary cells was 10-fold slower. The peptide
cleavage sites also varied between tissue-cultured and primary cells,
with 5- and 8-residue fragments formed in tumor cell lines and only
the 8-residue fragment formed in primary cells. These results demonstrate
the ability of chemical cytometry to identify important differences
in enzymatic behavior between primary cells and tissue-cultured cell
A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter.
Biosensors; Peptides; Florescent Probes; Dyes/Pigments; Signal Transduction
Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins that have a wide range of functions in cellular homeostasis and immunity. MTs can be induced by a variety of conditions including metals, glucocorticoids, endotoxin, acute phase cytokines, stress, and irradiation. In addition to their important immunomodulatory functions, MTs can protect essential cellular compartments from toxicants, serve as a reservoir of essential heavy metals, and regulate cellular redox potential. Many of the roles of MTs in the neuroinflammation, intestinal inflammation, and stress response have been investigated and were the subject of a session at the 6th International Congress on Stress Proteins in Biology and Medicine in Sheffield, UK. Like the rest of the cell stress response, there are therapeutic opportunities that arise from an understanding of MTs, and these proteins also provide potential insights into the world of the heat shock protein.
Metallothionein; Stress; Immunity; Inflammation
Spontaneous haemothorax (SH) is a subcategory of haemothorax that involves the accumulation of blood within the pleural space in the abscence of trauma or other causes. The clinical presentation is variable and includes a rapid progression of symptoms of chest pain and dyspnea that can be life threatening when hemodynamic instability and hypovolemic shock occurs. Despite haemothorax, SH is much less common with data limited to case reports and case series. A literature review has been performed to identify and summarise all potentials causes leading to this clinical entity.
Spontaneous haemothorax (SH); coagulopathy; haemopneumothorax
Photochemistry; Energy transfer; Drug delivery; Vitamins; Fluorescence
Protein folding by the endoplasmic reticulum (ER) is physiologically critical, while its disruption causes ER stress and augments disease. ER stress activates the unfolded protein response (UPR) to restore homeostasis. If stress persists, the UPR induces apoptotic cell death, but the mechanisms remain elusive. Here we find that unmitigated ER stress promotes apoptosis through cell-autonomous, UPR-controlled activation of death receptor 5 (DR5). ER stressors induced DR5 transcription via the UPR mediator CHOP; however, the UPR sensor IRE1α transiently catalyzed DR5 mRNA decay, allowing time for adaptation. Persistent ER stress built up intracellular DR5 protein, driving ligand-independent DR5 activation and apoptosis engagement via caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress and apoptotic cell fate.
Retrograde perfusion into coronary sinus during coronary artery bypass graft (CABG) surgery reduces the need for cardioplegic interruptions and ensures the distribution of cardioplegia to stenosed vessel territories, therefore enhancing the delivery of cardioplegia to the subendocardium. Peri-operative myocardial injury (PMI), as measured by the rise of serum level of cardiac biomarkers, has been associated with short and long-term clinical outcomes. We conducted a retrospective analysis to investigate whether the combination of antegrade and retrograde techniques of cardioplegia delivery is associated with a reduced PMI than that observed with the traditional methods of myocardial preservation.
Fifty-four consecutive patients underwent CABG surgery using either antegrade cold blood cardioplegia (group 1, n = 28) or cross-clamp fibrillation (group 2, n = 16) or antegrade retrograde warm blood cardioplegia (group 3, n = 10). The study primary end-point was PMI, evaluated with total area under the curve (AUC) of high-sensitivity Troponin-T (hsTnT), measured pre-operatively and at 6, 12, 24, 48 and 72 hours post-surgery. Secondary endpoints were acute kidney injury (AKI) and inotrope scores, length of intensive care unit (ICU) and hospital stay, new onset atrial fibrillation (AF) and clinical outcomes at 6 weeks (death, non-fatal myocardial infarction, coronary artery revascularization, stroke).
There was evidence that mean total AUC of hsTnT was different among the three groups (P = 0.050). In particular mean total AUC of hsTnT was significantly lower in group 3 compared to both group 1 (-16.55; 95% CI: -30.08, -3.01; P = 0.018) with slightly weaker evidence of a lower mean hsTnT in group 3 when compared to group 2 (-15.13; 95% CI -29.87, -0.39; P = 0.044). There was no evidence of a difference when comparing group 2 to group 1 (-1.42,; 95% CI: -12.95, 10.12, P = 0.806).
Our retrospective analysis suggests that, compared to traditional methods of myocardial preservation, antegrade retrograde cardioplegia may reduce PMI in patients undergoing first time CABG surgery.
Coronary artery bypass graft surgery; Peri-operative myocardial injury; Antegrade cardioplegia; Retrograde cardioplegia; Cross-clamp fibrillation
Primary pulmonary synovial sarcomas represent a rare clinical entity and account for approximately 0.5% of lung malignancies. We report the case of a 30-year-old male who presented clinically with haemothorax. Imaging revealed a complex collection obscuring a multi-lobulated mass in the right lower lobe of the lung. He underwent a right thoracotomy for evacuation of collection and surgical resection of his pulmonary mass. Histological analysis confirmed a grade 3 monophasic fibrous synovial sarcoma of the lung with infiltration to adjacent pleura, causing his initial haemothorax. Postoperative period was uneventful and patient was referred to the oncology team for further management. Primary pulmonary synovial sarcoma, though rare, should remain an important differential when considering lung malignancies, as complete surgical resection is the mainstay of treatment.
Primary pulmonary synovial sarcoma; haemothorax
High-throughput sequencing reveals an abundance of microRNA-sized fragments derived from larger non-coding RNAs. Roles for these small RNAs in gene silencing are suggested by their co-precipitation with Argonaute, the microRNA effector protein, though the extent to which they suppress gene expression endogenously remains unclear. To address this, we used luciferase reporters to determine the endogenous functionality of small RNAs from a diverse range of sources. We demonstrate small RNAs derived from snoRNAs have the capacity to act in a microRNA-like manner, though we note the vast majority of these are bound to Argonaute at levels below that required for detectable silencing activity. We show Argonaute exhibits a high degree of selectivity for the small RNAs with which it interacts and note that measuring Argonaute-associated levels is a better indicator of function than measuring total expression. Although binding to Argonaute at sufficient levels is necessary for demonstrating microRNA functionality in our reporter assay, this alone is not enough as some small RNAs derived from other non-coding RNAs (tRNAs, rRNAs, Y-RNAs) are associated with Argonaute at very high levels yet do not serve microRNA-like roles.
CYP2A13, a human P450 enzyme preferentially expressed in the respiratory tract, is highly efficient in the metabolic activation of tobacco-specific nitrosamines. The aim of this study was to test the hypothesis that inflammation suppresses CYP2A13 expression in the lung, thus explaining the large interindividual differences in CYP2A13 levels previously found in human lung biopsy samples. We first demonstrated that the bacterial endotoxin lipopolysaccharide (LPS) and the proinflammatory cytokine IL-6 can suppress CYP2A13 messenger RNA (mRNA) expression in the NCI-H441 human lung cell line. We then report that an ip injection of LPS (1mg/kg), which induces systemic and lung inflammation, caused substantial reductions in CYP2A13 mRNA (~50%) and protein levels (~80%) in the lungs of a newly generated CYP2A13-humanized mouse model. We further identified two critical CYP2A13 promoter regions, one (major) between −484 and −1008bp and the other (minor) between −134 and −216bp, for the response to LPS, through reporter gene assays in H441 cells. The potential involvement of the nuclear factor NF-κB in LPS-induced CYP2A13 downregulation was suggested by identification of putative NF-κB binding sites within the LPS response regions and effects of an NF-κB inhibitor (pyrrolidine dithiocarbamate) on CYP2A13 expression in H441 cells. Results from gel shift assays further confirmed binding of NF-κB-like nuclear proteins of H441 cells to the major LPS response region of the CYP2A13 promoter. Thus, our findings strongly support the hypothesis that CYP2A13 levels in human lung can be suppressed by inflammation associated with disease status in tissue donors, causing underestimation of CYP2A13 levels in healthy lung.
CYP2A; LPS; lung; inflammation; chemical carcinogenesis.
The purpose of this paper is to highlight the existence and the management of lung choriocarcinoma (CCA), a rare category of lung tumors. We present a 42-year-old female that presented to our department with a PET positive lesion in the left upper lobe and a history of pregnancy 6 months prior to onset of symptoms. CT guided biopsy was inconclusive for diagnosis and the patient underwent a left thoracotomy and lingula sparing upper lobectomy. Histology revealed CCA of the lung and subsequently blood results confirmed the elevated b-HCG. CCA of the lung is a clinical entity that should be considered in the differential diagnosis of lung lesions in women after pregnancy.
Choriocarcinoma (CCA); lung metastases; pregnancy
Mucoepidermoid carcinoma (MEC) of the thymus is a rare malignant neoplasm of the anterior mediastinum. There are less than 30 cases described in the English literature. We report a case of a 47-year-old lady who presented with myasthenia gravis and was found to have a well-circumscribed anterior mediastinal mass in her medical work-up. This mass was surgically resected and subsequently found to be a primary MEC of the thymus. This is the first reported case of thymic MEC with concurrent myasthenia gravis. Her myasthenia symptoms have persisted following complete surgical resection of her tumour.
Mucoepidermoid carcinoma (MEC); thymus; myasthenia gravis
An anthraquinone-based fluorescent quencher is described that is applicable to fluorophores throughout the visible spectrum and into the near IR. This species has been used to construct a palate of multicolour sensors of proteolysis and photolysis.
Lipidated light-responsive constructs that sequester bioagents to the membranes of organelles and cells have been constructed. When membrane-bound, the bioagent is not susceptible to processing by its biological target. Photolysis releases the bioagent from its membrane anchor and thereby renders it biologically active.
Photolysis; Phosphorylation; Biosensors; Fluorescent probes; Cell adhesion
The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.
Structural genomics; Phosphatase; NYSGXRC; X-ray crystallography