Melanoma secretion of transforming growth factor-ß2 leads to loss of epidermal AMBRA1 threatening epidermal integrity and facilitating tumour ulceration.

BACKGROUND: For patients with early American Joint Committee on Cancer (AJCC)-stage melanoma the combined loss of the autophagy regulatory protein AMBRA1 and the terminal differentiation marker loricrin in the peritumoral epidermis is associated with a significantly increased risk of metastasis. OBJECTIVES: The aim of the present study was to evaluate the potential contribution of melanoma paracrine transforming growth factor (TGF)-ß signalling to the loss of AMBRA1 in the epidermis overlying the primary tumour and disruption of epidermal integrity. METHODS: Immunohistochemistry was used to analyse AMBRA1 and TGF-ß2 in a cohort of 109 AJCC all-stage melanomas, and TGF-ß2 and claudin-1 in a cohort of 30 or 42 AJCC stage I melanomas, respectively, with known AMBRA1 and loricrin (AMLo) expression. Evidence of pre-ulceration was analysed in a cohort of 42 melanomas, with TGF-ß2 signalling evaluated in primary keratinocytes. RESULTS: Increased tumoral TGF-ß2 was significantly associated with loss of peritumoral AMBRA1 (P < 0·05), ulceration (P < 0·001), AMLo high-risk status (P < 0·05) and metastasis (P < 0·01). TGF-ß2 treatment of keratinocytes resulted in downregulation of AMBRA1, loricrin and claudin-1, while knockdown of AMBRA1 was associated with decreased expression of claudin-1 and increased proliferation of keratinocytes (P < 0·05). Importantly, we show loss of AMBRA1 in the peritumoral epidermis was associated with decreased claudin-1 expression (P < 0·05), parakeratosis (P < 0·01) and cleft formation in the dermoepidermal junction (P < 0·05). CONCLUSIONS: Collectively, these data suggest a paracrine mechanism whereby TGF-ß2 causes loss of AMBRA1 overlying high-risk AJCC early-stage melanomas and reduced epidermal integrity, thereby facilitating erosion of the epidermis and tumour ulceration.

Harnessing autophagy to overcome mitogen-activated protein kinase kinase inhibitor-induced resistance in metastatic melanoma.

BACKGROUND: Patients with malignant melanoma often relapse after treatment with BRAF and/or mitogen-activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance. OBJECTIVES: To establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy. METHODS: Immunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours. Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations. Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib-induced cell death and CD271 expression. MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib-resistant melanoma cell survival and invasion, respectively. RESULTS: CD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi. In vitro studies demonstrate MEKi of BRAF-mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271-expressing subpopulations. Trametinib-induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance. Treatment of CD271-expressing melanoma subpopulations with RNA interference and small-molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents - including Δ9-tetrahydrocannabinol and Vps34 - reduced survival of MEKi-resistant melanoma cells. Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi-resistant cells in an in vivo zebrafish xenograft. CONCLUSIONS: These results highlight a novel mechanism of MEKi-induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug-resistant melanoma cells to the cytotoxic effects of MEKi.

Effects of leucine and its metabolite ß-hydroxy-ß-methylbutyrate on human skeletal muscle protein metabolism.

Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses-fed gains) in protein turnover. Of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is 'sensed' have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-(13)C2]Leu and [(2)H5]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A-V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70% vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling 90 min vs. 30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; -57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

What is a tibial pilon fracture and how should they be acutely managed? A survey of consultant British Orthopaedic Foot and Ankle Society members and non-members.

INTRODUCTION: Controversy exists around which distal tibial fractures are pilon fractures. We evaluated views to define a pilon fracture and support the development of standards of care. METHODS: Views regarding the characteristics of a pilon fracture and acute soft tissue management were determined through a questionnaire. This was trialled, approved by the British Orthopaedic Foot and Ankle Society and distributed to its members. This was also distributed nationally as part of the ENFORCE study. RESULTS: In total, 282 consultants from 27 units responded, of whom 24% (69/282) were foot and ankle specialists. Some 58% (163/282) agreed that a pilon fracture is primarily a soft tissue injury, 81% (228/282) that pilon fractures occur though high-energy transfer, 81% (228/282) that pilon fractures are sustained through an axial compression mechanism and 93% (265/282) that they are a potentially limb-threatening injury. Overall, 83% (234/282) agreed that in a length-unstable pilon fracture it is not possible to maintain the talus near anatomically under the tibial plafond without rigid fixation to control length - with 87% (246/282) agreeing that the acute first-line management should be a spanning external fixator. Opinions were that the time frame between diagnosis and intervention should be: less than 6h (63%; 154/246), 6-12h (31%; 77/246) and 12-24h (6%; 15/246). CONCLUSION: Consensus supports defining a pilon fracture as a potentially limb-threatening high-energy axial compression injury, and a spanning external fixator as the first-line management of a length-unstable injury less than 12h from diagnosis.

DNACPR ('do not attempt cardiopulmonary resuscitation') orders in patients with a fractured neck of femur who lack capacity.

Nationally, half of all deaths occur in hospital, with 94% having a 'do not attempt cardiopulmonary resuscitation' (DNACPR) notice in place at the time of death. Recent court rulings have raised the profile of practices surrounding DNACPR orders where patients lack capacity. Failure to consult with those close to the patient in relation to DNACPR decisions is a breach of the right to respect for private and family life under article 8 of the Human Rights Act. A report from 2016 found that those close to the patient were not consulted before one out of every five DNACPR orders are placed. We advocate addressing the issue of resuscitation in patients with a fractured neck of femur who are approaching the end of their lives. Where the patient lacks capacity, there is a legal duty to consult with those close to the patient where it is practicable and appropriate to do so. There must be a convincing and well evidenced reason to proceed without consultation, and the orthopaedic surgeon should exercise extreme caution before doing so.

Delta retinoic acid receptor isoform delta 1 is distinguished by its exceptional N-terminal sequence and abundance in the limb regeneration blastema.

In amphibian limb regeneration memory for position in the proximal-distal axis can be respecified by retinoic acid. The favoured candidates to mediate this effect are the retinoic acid receptors (RARs) and of the RARs identified in the regeneration blastema, the delta receptor is the most abundant. The presence in blastemal mesenchyme of at least two delta receptor isoforms, delta 1 and delta 2, alternatively spliced at the A-B junction, was demonstrated in expression studies and by PCR cloning. The delta 1 receptor is abundant in regenerative structures such as the limb and tail, whereas the delta 2 and alpha receptors show a more uniform pattern of expression across adult newt tissues. Full-length cloning of the delta 1 receptor established the presence of an unusually long open reading frame and N-terminal sequence that appears unique among vertebrate retinoic acid receptors. Transient transfection of expression constructs into COS cells followed by Western blotting confirmed the existence of at least three potential initiation sites for delta 1 translation. The possibility that delta 1 RAR expression may specify positional memory directly was tested in RNase protection experiments. delta 1 receptor message is increased on amputation, but does not exhibit a pronounced differential distribution along the proximal-distal axis in normal and regenerating limbs, nor does it show a persistent alteration in expression levels following a dose of retinoic acid sufficient to respecify position. The possibility that the morphogenetic effects of RA may be mediated through receptor interactions is raised by the finding that single mesenchymal blastemal cells in culture can express multiple RAR subtypes (delta 1 and alpha) and isoforms (delta 1 and delta 2).

Effects of nicotinic acid therapy on plasma lipoproteins and very low density lipoprotein apoprotein C subspecies in hyperlipoproteinemia.

The effects of long term treatment with nicotinic acid on lipids, lipoproteins, and the plasma distribution of very low density lipoproteins (VLDL) apoprotein C (ApoC) subspecies were studied in 33 patients with types IIa (n = 9), IIb (n = 11), and IV (n = 13) hyperlipidemias. After 6 months of treatment, a significant decrease in triglyceride, total cholesterol, and low density lipoprotein (LDL) cholesterol levels occurred. High density lipoprotein (HDL) cholesterol increased significantly by 31.1%, 41.8%, and 32.0% in types IIa, IIb, and IV, respectively (P less than 0.01 for all). A significant negative correlation existed between changes in HDL cholesterol and triglycerides (r = -0.613; P less than 0.02) in all groups studied. Therapy also produced changes in VLDL, LDL, and HDL protein concentrations. VLDL protein decreased from 20.9 +/- 3.9 to 15.2 +/- 1.0 mg/dl (P less than 0.05) in type IIa. In types IIb and IV, mean VLDL protein decreased from 44.7 +/- 8.2 to 27.1 +/- 3.9 mg/dl (P less than 0.001) and from 46.3 +/- 7.1 to 30.6 +/- 4.9 mg/dl (P less than 0.001), respectively. LDL protein decreased significantly, and HDL protein increased in type IIa only. Gel isoelectric focusing of VLDL before and after nicotinic acid in types IIb and IV hyperlipidemia produced a significant increase in the VLDL ApoC-II component with simultaneous decreases in the total VLDL ApoC-III subspecies. This resulted in increases in the ApoC-II to ApoC-III area ratio from 0.50 +/- 0.1 to 1.02 +/- 0.2 (P less than 0.001) in type IIb and from 0.62 +/- 0.07 to 0.88 +/- 0.13 (P less than 0.01) in type IV, respectively. The ApoE subspecies and the ApoE-III to ApoE-II area ratio did not change significantly. Our results show that nicotinic acid produces a significant improvement in the lipoprotein profiles of these patients.

Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.

Four new antibiotic-resistant derivatives of the broad-host-range (bhr) cloning vector pBBR1MCS have been constructed. These new plasmids have several advantages over many of the currently available bhr vectors in that: (i) they are relatively small (< 5.3 kb), (ii) they possess an extended multiple cloning site (MCS), (iii) they allow direct selection of recombinant plasmid molecules in Escherichia coli via disruption of the LacZ alpha peptide, (iv) they are mobilizable when the RK2 transfer functions are provided in trans and (v) they are compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons.

Stability of Legionella urinary antigens over time.

Twenty-two urine samples positive for Legionella pneumophila serogroup 1 antigen by EQUATE radioimmunoassay (RIA) (Binax, Portland, ME, USA) were stored at various temperatures and the RIA repeated at 1, 7, 30, 90, and 120 days to evaluate stability of the urinary antigens. The mean ratios of patient/negative control remained stable. Although there was a 10% decrease in the mean ratios after 1 month, changes were not significant. However, individual samples with ratios close to 3 may fall to < 3.

Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains.

The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus59% similar among the strains, indicating that the biochemical pathway for pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains.

Mothers' and fathers' singing to infants.

Mothers and fathers sang a song of their choice, once to their infant and once as if to their infant (simulated). The pitch of songs was higher and the tempo slower for infant-directed than for simulated versions. Listeners varying in child-care experience, musical background, and cultural origin reliably identified which of the two versions was infant-directed (Experiment 1). Identification accuracy was enhanced by musical training, knowledge of the singers' language and culture, and by greater pitch and tempo differences between versions. Other adult listeners who rated the singer's emotional engagement assigned significantly higher ratings to infant-directed than to simulated versions (Experiment 2). Differences in pitch and tempo between both versions predicted emotional engagement ratings. Finally, rating differences between infant-directed and simulated versions were highly correlated with identification accuracy.

Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides.

Traditionally, sperms are isolated from vaginal cell mixtures by preferential extraction methods. Although these methods work well when there is a reasonable amount of DNA present, they are problematic when there are limited amounts (ca. 250 pg). In particular, the analysis of sperm from microscope slides has proven difficult. Here, we describe the use of laser capture microdissection (LM) for the isolation of spermatozoa from microscope slides containing sperms and vaginal cells. Such slides are frequently an important source of evidential material during the forensic investigation of rape and other sexual assaults. Low copy number (LCN) PCR was used to compare profiles of sperm DNA prepared using LM and preferential lysis. LM was found to outperform preferential lysis in 15 out of 16 samples. The application of LM to the processing of actual casework slides, and in particular the potential use of LM for the analysis of old cases, is discussed. Finally, 77 post-coital slides were processed in order to accurately assess the robustness of the technique. There was a significant association between the quality of the male profile recovered and time since intercourse that was independent of the number of sperms analysed, suggesting that the DNA was degraded even though the spermhead was intact.

Parents' sung performances for infants.

Naive listeners rated the style of singing in mothers' and fathers' sung performances for infants and their simulations of those performances (Experiment 1). Performances in an infant's presence were judged as more expressive--either more playful or more soothing--than were simulations. Parents' style of singing, as reflected in these ratings, differed as a function of the sex of singer and listener. Both parents sang more playfully for same-sex infants than for opposite-sex infants. Independent listeners rated the manner in which parents enunciated the lyrics of their songs (Experiment 2). Parents rendered the lyrics of songs more expressively in infant-present than in infant-absent contexts. Moreover, this expressiveness was greater for same-sex infants than for opposite-sex infants. These findings are consistent with parents' greater attachment to same-sex infants. Discrepancies between parents' choice of songs and their manner of singing lend credence to functional rather than nominal classifications of songs for infants.

A unique parotid adenocarcinoma.

A large number of different types of benign and malignant tumours of the salivary glands are recognized, and rare and unusual variants arise from time to time that can be difficult to interpret. We report an apparently unique parotid neoplasm that does not fit any of the currently recognized diagnostic groups and can best be termed lymphadenocarcinoma. Clinical and pathological features are described, and its possible relationship to recognized tumour types is examined.

A simple reconstructive procedure for radiation-induced necrosis of the external auditory canal.

Localized necrosis of the bone, cartilage, and soft tissue of the external auditory canal is an uncommon side effect of radiotherapy to the parotid region. Five patients developed late onset skin necrosis of a quadrant of the ear canal secondary to an underlying osteoradionecrosis of the tympanic ring. We report a one-stage procedure to excise the necrotic tissue and replace it with a local rotational flap derived from the post-auricular skin. Otological side effects of radiotherapy are discussed.

Coordinating a multidisciplinary discharge for the technology-dependent child based on parental needs.

Primary nurses, case managers, and clinical nurse specialists in pediatric settings are continually providing care for chronically ill children. With our advancing medical knowledge and technology, the concept of chronically ill children has taken on an added dimension: technology-assisted living. Technology-dependent children pose the greatest challenge for discharge into the home environment. This article identifies the chronically ill technology-dependent child and family and their needs in order to develop a chronologic plan for a successful multidisciplinary discharge.

Developing effective labour relations skills: understanding the grievance process.

The grievance process provides a formal mechanism for dealing with worker-management disputes arising from the application of negotiated collective agreements. This paper examines the literature describing the direct and indirect functions of the grievance process, grievance procedure language, and arbitration. Some of the unique features of labour relations and the grievance process in the public sector are also reviewed. Pharmacy Managers are encouraged to improve their personnel management skills by developing a greater understanding of the grievance process. A number of recommendations are provided to assist the Pharmacy Manager in the handling of grievances.

Diffusion of innovation I: Formulary acceptance rates of new drugs in teaching and non-teaching British Columbia hospitals--a hospital pharmacy perspective.

Lag times in the diffusion of new drugs in the hospital setting have both patient care and pharmaceutical industry implications. This two-part series uses diffusion theory to examine differences in the adoption rates of new drugs in British Columbia teaching and non-teaching hospitals. Formulary addition of a new drug by a hospital's Pharmacy and Therapeutics Committee was considered the adoption indicator. Time for adoption was defined as the difference between a drug's Canadian market approval date and the date of formulary addition. Surveys were mailed in September 1990 to 41 hospital pharmacies (response rate = 88%), asking respondents to provide formulary inclusion dates of 29 drugs marketed between July 1987 and March 1990. A significant difference (Mann-Whitney U Test, p < 0.0358) in median adoption time was observed between the six teaching and 25 non-teaching study hospitals, with the former adopting a new drug in 7.5 months versus the latter adopting a new drug in 12.1 months.

Diffusion of innovation II: Formulary acceptance rates of new drugs in teaching and non-teaching British Columbia hospitals--a drug development perspective.

Diffusion theory was used to examine differences in adoption rates of new drugs by British Columbia teaching and non-teaching hospitals. Surveys were mailed in September 1990 to 41 hospital pharmacies (response rate = 88%), requesting hospital pharmacy directors to provide formulary inclusion dates of 29 study drugs marketed between July 1987 and March 1990. Of the 36 initial responses, 31 were suitable for further analysis and these were surveyed again in April 1993 (response rate = 100%) as to the formulary status of drugs not initially approved. The second survey ensured that all study drugs would have at least 36 months on the Canadian market when determining formulary acceptance times. Of the 29 study drugs, six were not approved for use in any of the 31 study hospitals. The six teaching hospitals had a median formulary approval time of 8.0 months compared to 12.8 months in the 25 non-teaching hospitals for the 23 study drugs. Although 21 of 23 study drugs were approved for use earlier in teaching hospitals than non-teaching hospitals, only alfentanil was found to be adopted significantly earlier (U = 11, n1 = 5, n2 = 19, alpha = 0.05). Variations in formulary approval times for new drugs have a bearing on patient care, Pharmacy and Therapeutics Committees, hospital budgets, and pharmaceutical firm revenues.

Surgical smoke - a health hazard in the operating theatre: a study to quantify exposure and a survey of the use of smoke extractor systems in UK plastic surgery units.

Surgeons and operating theatre personnel are routinely exposed to the surgical smoke plume generated through thermal tissue destruction. This represents a significant chemical and biological hazard and has been shown to be as mutagenic as cigarette smoke. It has previously been reported that ablation of 1 g of tissue produces a smoke plume with an equivalent mutagenicity to six unfiltered cigarettes. We studied six human and 78 porcine tissue samples to find the mass of tissue ablated during 5 min of monopolar diathermy. The total daily duration of diathermy use in a plastic surgery theatre was electronically recorded over a two-month period. On average the smoke produced daily was equivalent to 27-30 cigarettes. Our survey of smoke extractor use in UK plastic surgery units revealed that only 66% of units had these devices available. The Health and Safety Executive recommend specialist smoke extractor use, however they are not universally utilised. Surgical smoke inhalation is an occupational hazard in the operating department. Our study provides data to quantify this exposure. We hope this evidence can be used together with current legislation to make the use of surgical smoke extractors mandatory to protect all personnel in the operating theatre.