What the COVID pandemic taught us about the management of pediatric minor trauma.

The COVID-19 pandemic necessitated a change in our practice in the management of pediatric soft-tissue injuries. Patients were managed conservatively whenever possible. Our aim in this study was to see whether this more conservative approach adversely affected clinical, and patient-reported outcomes, including scarring. A prospective record of children presenting to the plastic surgery "Early Bird" clinic for pediatric trauma between 01.04.2020 and 30.06.2020 was kept. Electronic patient records were reviewed. An outpatient telephone clinic was scheduled for all patients. Parents were asked about complications and what they thought about the scar and to rate it as either: "poor," "satisfactory," "good," or "excellent." There were 240 patients, including 136 (57%) males and 104 (43%) females. The most frequent type of injury was a facial laceration in 123 patients (51.3%), followed by hand lacerations in 43 (17.9%), fingertip injuries in 31 (12.9%), and others. Ninety out of 240 (37.5%) were offered surgery. Follow-up times ranged from 17 to 20 months. Most parents (86.2%) were happy with the scarring and reported it as "good" or "excellent." The proportion rating the scar "excellent" or "good" was similar in the non-operated cohort (i.e., 85.5%) versus the operated cohort (88.5%) (p-value 0.16). The overall complication rate of patients seen during this time was 5.9%; 7.4% in the conservatively managed and 4.9% of those who went to the theater. Despite managing more wounds, including some dog bites, conservatively, patients and parents reported low complication rates and high levels of satisfaction with the final scarring.

Implementing post-normal science with or for EU policy actors: using quantitative story-telling.

There is increasing recognition of the wicked nature of the intertwined climate, biodiversity and economic crises, and the need for adaptive, multi-scale approaches to understanding the complexity of both the problems and potential responses. Most science underpinning policy responses to sustainability issues, however, remains overtly apolitical and focussed on technical innovation; at odds with a critical body of literatures insisting on the recognition of systemic problem framing when supporting policy processes. This paper documents the experience of implementing a mixed method approach called quantitative story-telling (QST) to policy analysis that explicitly recognises this normative dimension, as the methodology is part of a post-normal science (PNS) toolkit. The authors reflect on what was learnt when considering how QST fared as a tool for science-policy interaction, working with European Union (EU) level policy actors interested in sustainable agriculture and sustainable development goal 2. These goals-also known as UN Agenda 2030-are the latest institutionalisation of the pursuit of sustainable development and the EU has positioned itself as taking a lead in its implementation. Thus, the paper illustrates our experience of using PNS as an approach to science policy interfaces in a strategic policy context; and illustrates how the challenges identified in the science-policy literature are amplified when working across multiple policy domains and taking a complex systems approach. Our discussion on lessons learnt may be of interest to researchers seeking to work with policy-makers on complex sustainability issues.

BAG-1 suppresses expression of the key regulatory cytokine transforming growth factor ß (TGF-ß1) in colorectal tumour cells.

As colorectal cancer remains the second highest cause of cancer-related deaths in much of the industrialised world, identifying novel strategies to prevent colorectal tumour development remains an important challenge. BAG-1 is a multi-functional protein, the expression of which is up-regulated at relatively early stages in colorectal tumorigenesis. Importantly, BAG-1 is thought to enhance colorectal tumour progression through promoting tumour cell survival. Here, we report for the first time a novel role for BAG-1, establishing it as a suppressor of transforming growth factor ß (TGF-ß1) expression in colorectal tumour cells. Microarray analysis first highlighted the possibility that BAG-1 may regulate TGF-ß1 expression, a key cytokine in normal colonic tissue homoeostasis. Q-RT-PCR and ELISA demonstrated TGFB1 mRNA and protein expression to be significantly increased when BAG1 levels were reduced by small interfering RNA; additionally, induction of BAG-1L caused suppression of TGFB1 mRNA in colorectal tumour cells. Using reporter and chromatin immunoprecipitation assays, a direct association of BAG-1 with the TGFB1 gene regulatory region was identified. Immunohistochemistry and Weiser fraction data indicated that the levels of BAG-1 and TGF-ß1 are inversely correlated in the normal colonic epithelium in vivo, consistent with a role for BAG-1-mediated repression of TGF-ß1 production. In vitro studies showed that the change in TGF-ß1 production following manipulation of BAG-1 is functionally relevant; through induction of anchorage-independent growth in TGF-ß1-dependent normal rat kidney fibroblasts and regulation of SMAD2 phosphorylation in TGF-ß1-sensitive adenoma cells. Taken together, this study identifies the anti-apoptotic protein BAG-1 as a suppressor of the inhibitory growth factor TGF-ß1, suggesting that high expression of BAG-1 can impact on a number of the hallmarks of cancer, of potential importance in promoting the early stages of colorectal tumorigenesis. Establishing BAG-1 as a repressor of TGF-ß1 has important biological implications, and highlights a new role for BAG-1 in colorectal tumorigenesis.

The retinoblastoma protein (Rb) as an anti-apoptotic factor: expression of Rb is required for the anti-apoptotic function of BAG-1 protein in colorectal tumour cells.

Although the retinoblastoma-susceptibility gene RB1 is inactivated in a wide range of human tumours, in colorectal cancer, the retinoblastoma protein (Rb) function is often preserved and the RB locus even amplified. Importantly, we have previously shown that Rb interacts with the anti-apoptotic Bcl-2 associated athanogene 1 (BAG-1) protein, which is highly expressed in colorectal carcinogenesis. Here we show for the first time that Rb expression is critical for BAG-1 anti-apoptotic activity in colorectal tumour cells. We demonstrate that Rb expression not only increases the nuclear localisation of the anti-apoptotic BAG-1 protein, but that expression of Rb is required for inhibition of apoptosis by BAG-1 both in a γ-irradiated Saos-2 osteosarcoma cell line and colorectal adenoma and carcinoma cell lines. Further, consistent with the fact that nuclear BAG-1 has previously been shown to promote cell survival through increasing nuclear factor (NF)-κB activity, we demonstrate that the ability of BAG-1 to promote NF-κB activity is significantly inhibited by repression of Rb expression. Taken together, data presented suggest a novel function for Rb, promoting cell survival through regulating the function of BAG-1. As BAG-1 is highly expressed in the majority of colorectal tumours, targeting the Rb-BAG-1 complex to promote apoptosis has exciting potential for future therapeutic development.

BAG-1 interacts with the p50-p50 homodimeric NF-κB complex: implications for colorectal carcinogenesis.

Understanding the mechanisms that promote aberrant tumour cell survival is critical for the determination of novel strategies to combat colorectal cancer (CRC). We have recently shown that the anti-apoptotic protein BAG-1, highly expressed in pre-malignant and CRC tissue, can potentiate cell survival through regulating NF-κB transcriptional activity. In this study, we identify a novel complex between BAG-1 and the p50-p50 NF-κB homodimers, implicating BAG-1 as a co-regulator of an atypical NF-κB pathway. Importantly, the BAG-1-p50 complex was detected at gene regulatory sequences including the epidermal growth factor receptor (EGFR) and COX-2 (PTGS2) genes. Suppression of BAG-1 expression using small interfering RNA was shown to increase EGFR and suppress COX-2 expression in CRC cells. Furthermore, mouse embryonic fibroblasts derived from the NF-κB1 (p105/p50) knock-out mouse were used to demonstrate that p50 expression was required for BAG-1 to suppress EGFR expression. This was shown to be functionally relevant as attenuation of BAG-1 expression increased ligand activated phosphorylation of EGFR in CRC cells. In summary, this paper identifies a novel role for BAG-1 in modulating gene expression through interaction with the p50-p50 NF-κB complexes. Data presented led us to propose that BAG-1 can act as a selective regulator of p50-p50 NF-κB responsive genes in colorectal tumour cells, potentially important for the promotion of cell survival in the context of the fluctuating tumour microenvironment. As BAG-1 expression is increased in the developing adenoma through to metastatic lesions, understanding the function of the BAG-1-p50 NF-κB complexes may aid in identifying strategies for both the prevention and treatment of CRC.

BAG-1L promotes keratinocyte differentiation in organotypic culture models and changes in relative BAG-1 isoform abundance may lead to defective stratification.

In keratinocytes the human Bag-1 gene produces three different protein isoforms from a single messenger RNA, BAG-1L, BAG-1M and BAG-1S. In this study we questioned whether BAG-1L or the shorter isoforms would promote keratinocyte differentiation in organotypic cultures of HaCaT. HaCaT parental and vector cells showed stratification, but terminal differentiation was not complete. Cultures overexpressing BAG-1L isoform-specifically were of increased thickness, demonstrated pronounced expression of basal cytokeratin 5 and ß1-integrin, suprabasal involucrin, cytokeratin 1 and plasma membrane-localised filaggrin, and a marked keratinized layer of cells at the surface. We were unable to overexpress BAG-1S and BAG-1M isoform-specifically. Overexpression of BAG-1M gave rise to organotypic cultures intermediate in differentiation to controls and those overexpressing BAG-1L. Cells overexpressing BAG-1S also exhibited elevated endogenous BAG-1. These produced slow growing cultures with high levels of basal cytokeratin 5, but little involucrin or cytokeratin 1. Suprabasal ß1-integrin and Ki67 positive cells indicated defective stratification. The results suggest that BAG-1L potentiates epidermal differentiation, but disruption in the relative balance of isoforms towards overexpression of BAG-1S can lead to defective tissue patterning. Hence, a delicate balance of BAG-1 isoforms may be required to regulate normal epidermal stratification and differentiation, with important implications for aberrant differentiation in cancer.

Co-overexpression of Bag-1 and heat shock protein 70 in human epidermal squamous cell carcinoma: Bag-1-mediated resistance to 5-fluorouracil-induced apoptosis.

BACKGROUND: The aim was to determine whether Bcl-2-associated athanogene-1 (Bag-1) and/or its binding protein heat shock protein-70 (Hsp70) exhibit deregulated expression in epidermal squamous cell carcinoma (SCC) and whether Bag-1 confers apoptosis resistance. METHOD: Immunohistochemistry for Bag-1 and Hsp70 was performed on 60 epidermal SCC and 10 normal skin samples. The epidermal SCC cell line SCC-13 was treated with 5-fluorouracil (5-FU) after Bag-1 knockdown to determine whether high Bag-1 levels contribute to growth and/or apoptosis resistance. RESULTS: Normal epithelium expressed primarily nuclear Bag-1. Most tumours showed reduced nuclear Bag-1 staining, but a subset exhibited strong Bag-1 staining, with cytoplasmic Bag-1 staining intensity correlating with cytoplasmic Hsp70 staining intensity (r(s)=0.462; P<0.001) and less differentiation (P<0.001). Bag-1 knockdown resulted in markedly reduced SCC-13 cell yield, increased spontaneous apoptosis and enhanced sensitivity to 5-FU-induced apoptosis. Apoptosis induced by 5-FU in the Bag-1-knockdown cells was significantly greater than the additive apoptotic effect of 5-FU or Bag-1 knockdown alone. CONCLUSIONS: Overexpression of Bag-1 and Hsp70 in poorly differentiated SCC may confer both enhanced tumour cell growth and apoptosis resistance. Bag-1 may contribute to the resistance of more advanced epidermal SCC to chemotherapy.

BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration.

Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

Nuclear BAG-1 expression inhibits apoptosis in colorectal adenoma-derived epithelial cells.

BAG-1 is an anti-apoptotic protein that is frequently deregulated in a variety of malignancies including colorectal cancer. There are three isoforms: BAG-1L is located in the nucleus, BAG-1M and BAG-1S are located both in the nucleus and the cytoplasm. In colon cancer, the expression of nuclear BAG-1 is associated with poorer prognosis and is potentially a useful predictive factor for distant metastasis. However, the function of BAG-1 in colonic epithelial cells has not been studied. Having previously shown a predominant nuclear localisation of BAG-1 in adenoma-derived cell lines, we wanted to determine the function of nuclear BAG-1 in these non-tumourigenic cells, to identify whether nuclear BAG-1 was implicated in tumour progression in the colon. In the current report we established that nuclear BAG-1 inhibits apoptosis in a colorectal adenoma-derived cell line. We demonstrate that apoptosis induced by gamma-radiation or the vitamin D analogue EB1089 in the non-tumourigenic human colorectal adenoma-derived S/RG/C2 cell line, was preceded by a decrease in nuclear and an increase in cytoplasmic BAG-1 expression. This change in subcellular localisation of BAG-1 was due to the redistribution of the BAG-1M isoform. In addition, we have shown that the maintenance of high nuclear BAG-1 through enforced expression of the nuclear localised BAG-1L isoform enhanced cellular survival after gamma-radiation or exposure to EB1089. Furthermore the expression of cytoplasmic BAG-1S isoform fused with a nuclear localisation signal protected against gamma-radiation induced apoptosis. This demonstrates that nuclear localisation of the BAG-1 protein confers a survival advantage in colorectal adenoma-derived cells and that nuclear BAG-1 could potentially be an important survival factor in colorectal carcinogenesis.

Increased sensitivity to TRAIL-induced apoptosis occurs during the adenoma to carcinoma transition of colorectal carcinogenesis.

The death ligand TRAIL (Apo2L) has potential for cancer therapy, since tumour cells are thought to be more sensitive than normal cells. We investigated whether sensitivity to TRAIL increases during the adenoma to carcinoma transition of colorectal carcinogenesis. Under the same culture conditions, we compared the extent of TRAIL-induced apoptosis in four premalignant adenoma and three carcinoma cell lines. Although TRAIL induced some apoptosis in adenoma cultures, the carcinoma cell lines were significantly more sensitive (P<0.001). This finding was recapitulated in an in vitro model of tumour progression in which conversion of the adenoma cell line AA/C1 to a tumorigenic phenotype was associated with increased TRAIL sensitivity (P<0.001). Increased TRAIL sensitivity during colorectal carcinogenesis has been previously attributed to changes in the balance between TRAIL receptors TRAIL-R1 and -R2 and "decoy" receptors TRAIL-R3 and -R4 during malignant progression. To address this, cell surface receptor expression was measured by flow cytometry. In summary, during colorectal carcinogenesis, there is a marked increase in sensitivity to TRAIL-induced apoptosis associated with progression from benign to malignant tumour that could be exploited for colon cancer therapy, but alterations in cell surface TRAIL receptor expression may not be the primary reason for this change.

BAG-1 expression and function in human cancer.

BAG-1 is a multifunctional protein that interacts with a wide range of target molecules to regulate apoptosis, proliferation, transcription, metastasis and motility. Interaction with chaperone molecules may mediate many of the effects of BAG-1. The pathways regulated by BAG-1 play key roles in the development and progression of cancer and determining response to therapy, and there has been considerable interest in determining the clinical significance of BAG-1 expression in malignant cells. There is an emerging picture that BAG-1 expression is frequently altered in a range of human cancers relative to normal cells and a recent report suggests the exciting possibility that BAG-1 expression may have clinical utility as a prognostic marker in early breast cancer. However, other studies of BAG-1 expression in breast cancer and other cancer types have yielded differing results. It is important to view these findings in the context of current knowledge of BAG-1 expression and function. This review summarises recent progress in understanding the clinical significance of BAG-1 expression in cancer in light of our understanding of BAG-1 function.

Critical care. Only connect.

The establishment of a network for critical care services in five hospitals has led to a decrease in transfers of patients for non-clinical reasons. There have been no transfers outside the network's area. The introduction of common admission policies has led to more openness about bed availability. The introduction of the network has standardised data collection. The availability of extra funds and facilities was a big incentive to staff involvement.

Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy.

Vitamin D3 is believed to reduce the risk of colon cancer, and serum levels inversely correlate with colorectal cancer incidence. The active metabolite, 1alpha,25-dihydroxyvitamin D3, has previously been shown to inhibit growth and promote differentiation of colon cancer cells. The vitamin D analogue, EB1089, is currently under clinical trial in a variety of cancers because of its growth-inhibitory effects in vitro and reduced hypercalcemic effects in vivo. The mechanism of growth inhibition by EB1089, however, remained to be determined. In this study we examined the effects of alpha,25-dihydroxyvitamin D3 and EB1089 on five colorectal tumor cell lines (two adenoma and three carcinoma) to determine the mechanism of growth inhibition and to ascertain whether premalignant adenoma cells were responsive to these agents. 1alpha,25-Dihydroxyvitamin D3 and EB1089 induced p53-independent apoptosis in adenoma and carcinoma cell lines in a dose-dependent manner between 10(-10) and 10(-6) M. EB1089, as well as inducing apoptosis, increased the proportion of cells in the G1 phase, particularly in the adenoma cell lines. In two of the three carcinoma cell lines (SW620 and PC/JW), levels of apoptosis induced by EB1089 were similar or greater than those induced by 1alpha,25-dihydroxyvitamin D3. Although the carcinoma cell line HT29 was relatively resistant to apoptosis induced by EB1089 compared with 1alpha,25-dihydroxyvitamin D3, EB1089 markedly inhibited cell yields. These observations offer promise for the clinical use of EB1089. To determine whether the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 was potentially via a differentiation pathway, alkaline phosphatase activity was measured as a marker of differentiation. Induction of alkaline phosphatase was observed in the floating apoptotic cells as well as in the adherent population. A link between the induction of differentiation and apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 is suggested by the occurrence of apoptosis subsequent to the induction of differentiation. To investigate the molecular pathway to apoptosis induction, members of the Bcl-2 family of proteins were examined (Bcl-2, Bcl-x, Bax, and Bak). Decreased Bcl-2 was observed in some cell lines, particularly in response to EB1089, but was not essential for apoptosis. Levels of the proapoptotic protein Bak, however, were consistently increased in all of the five cell lines in association with apoptosis induced by either agent. The results implicate Bak protein in the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 or its analogue EB1089. The ability of EB1089 to induce apoptosis in colorectal carcinoma cells suggests that this or other vitamin D analogues may prove clinically effective for the treatment of colorectal cancer. Furthermore, the fact that it induces cell cycle arrest and apoptosis in the premalignant adenoma cells may suggest an application in colorectal cancer chemoprevention.

Decreased levels of p26-Bcl-2, but not p30 phosphorylated Bcl-2, precede TGFbeta1-induced apoptosis in colorectal adenoma cells.

Bcl-2 expression is confined to the base of the colonic crypt, whereas transforming growth factor beta (TGFbeta) is expressed in the upper crypt, as are the apoptotic death promoters, Bak and Bax. In colonic adenoma cells, TGFbeta induces a growth arrest. In some adenoma cell lines, this is accompanied by apoptosis and in others it is not. In this study, we used two human colonic adenoma cell lines: RG/C2, in which TGFbeta induces a G1 arrest without apoptosis, and BH/C1, in which TGFbeta induces both a G1 arrest and apoptosis. TGFbeta does not induce apoptosis in RG/C2 cells even if hydrocortisone and insulin are removed from the culture medium. In BH/C1 cells, TGFbeta induces apoptosis in the presence of insulin and hydrocortisone. Apoptosis induced by TGFbeta is preceded by a reduction in p26-Bcl-2 protein levels. There was no change in the levels of the p30 phosphorylated form of Bcl-2 or in levels of the proapoptotic proteins Bax or Bak. RG/C2 cells did not show decreased Bcl-2 levels in response to TGFbeta-induced growth inhibition. Therefore, TGFbeta regulates Bcl-2 expression in colonic adenoma cells which undergo apoptosis in response to TGFbeta, but not in those which are growth inhibited, but resistant to TGFbeta-induced apoptosis. TGFbeta may play an important role in the colonic epithelium, not only in the inhibition of cell proliferation, but also in the regulation of apoptosis.

bcl-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells; however overexpression of bcl-2 does not protect against bak-mediated apoptosis.

Butyrate, a short chain fatty acid produced in the colon as a result of fermentation of dietary fibre by symbiotic bacteria, induces apoptosis in colonic tumour cell lines. Three human colonic adenoma cell lines (AA/C1, RG/C2 and BH/C1) and one carcinoma cell line (S/KS/FI) were used to determine the effects of butyrate on the expression of bcl-2, bax and bak to examine the possible role of these proteins in the induction of apoptosis. RG/C2 and BH/C1 cells express p-26-bcl-2 and butyrate treatment decreased p26-bcl-2 levels in association with apoptosis, whereas bax and bak levels remained constant. AA/C1 and S/KS/FI cells have no detectable p26-bcl-2. In S/KS/FI cells, bax or bak levels did not change in response to butyrate. However, in AA/C1 cells, butyrate-induced apoptosis was associated with increased bak levels. Therefore, in AA/C1 cells butyrate-induced apoptosis appears to be mediated through bak. Furthermore, butyrate also induced apoptosis and increased bak levels in AA/C1 cells transfected with a bcl-2 expression vector which expressed high levels of p26-bcl-2. For S/KS/FI cells, two bcl-2 transfectants gave different results. bcl-2 protected against apoptosis in one transfectant in which bak levels were not elevated in response to butyrate, whereas it did not protect in the other transfectant in which bak levels were increased after butyrate treatment. The results suggest that expression of constitutively high levels of p26-bcl-2 only conferred protection against apoptosis when bak levels were not elevated in response to butyrate and that expression of constitutively high levels of p26-bcl-2 does not counter the effects of bak. Different mechanisms appear to be involved in cell death signalling in different tumours since butyrate may induce apoptosis via elevated levels of bak or reduced levels of p26-bcl-2.

Primary care. Who's sorry now?

Almost two-thirds of health authorities believe their relationships with GPs have improved since the merger. Forty-four percent of LMCs think there has been no change, and 42 per cent report a deterioration. More than half the LMCs believe knowledge and understanding of GPs' contracts, and payments, have declined since the merger.