Gene expression profiling in melanoma: A view from the clinic.

The treatment of Melanoma, one of the most aggressive human malignancies, has been revolutionised by the advent of novel targeted and immuno-therapies. However, methods utilised to detect early presentations, and to stratify risk for patients diagnosed with invasive melanoma in the clinical setting are lagging. The primary prognostic indicator is restricted to Breslow Thickness, or depth the tumour invades into the dermis. Gene Expression Profiling (GEP), the analysis of molecular gene signatures of an individual tumour, has been implemented with great success in other malignancies, such as breast and prostate cancer. In the setting of melanoma, commercial GEP panels are becoming available, offering patients a personalised approach, though yet to enter widespread clinical use. This short perspective seeks to describe how GEP is currently employed in practice, and its current clinical impact. We also suggest the potential roles for GEP in meeting the key clinical challenges faced by clinicians in melanoma treatment, such as decisions around adjuvant therapy, sentinel lymph node biopsy (SLNB) and surgical resection , thus highlighting areas for future potential research.

Referral of patients to plastic surgeons following self-harm: Opportunities for suicide prevention.

Self-harm is a common source of referral to plastic and hand surgery services. Appropriate management of these patients is complex and includes the need for close liaison with mental health services. Self-harm is the single biggest risk factor for completed suicide, thereby increasing the risk by a factor of 66.1 This study aimed to analyse the clinical pathway and demographics of patients referred to plastic surgeons following self-harm. This 6-year retrospective series included patients referred to plastic surgeons following self-harm within the Galway University Hospital group. Patients were identified through the Hospital inpatient enquiry system, cross-referenced with data from the National Suicide Research Foundation. Data collected included demographics, psychiatric history, details of self-harm injury, admission pathway and operative intervention. Forty-nine patients were referred to plastic surgery services during the study period, accounting for 61 individual presentations. The male-to-female ratio was 26 (53%) to 23 (47%). Mean age was 40 years (range 21-95 years). Alcohol or illicit substance use was recorded in 17 of 61 (28%) presentations. Mortality from suicide occurred in 4 patients (8%). Mental health assessment was not carried out in 9 presentations (15%). Documentation of need for close or one-to-one observation was made in 11 cases (20%) and was not referred to in 43 cases (83%) following mental health assessment. This study demonstrates significant diversity in the management of this vulnerable patient group and may inform development of referral pathways to improve the safety of transfer, surgical admission and discharge of patients following self-harm, in consultation with mental health services.

Reliability of functioning free muscle transfer and vascularized ulnar nerve grafting for elbow flexion in complete brachial plexus palsy.

We compared outcomes of primary vascularized ulnar nerve grafts from the C5 root neurotizing biceps and brachialis muscles, and gracilis functioning free muscle transfer neurotized by the distal spinal accessory nerve, as a primary or salvage procedure after complete brachial plexus injury. At 45 months, three of eight primary vascularized ulnar nerve graft patients regained grade 4 elbow flexion, while one regained grade 3. All 13 primary gracilis transfer patients regained grade 4 elbow flexion. Four patients with vascularized ulnar nerve grafts failed and subsequently had salvage functioning free muscle transfer procedures resulting in delayed recovery. Although vascularized ulnar nerve graft-based primary reconstructions can provide useful elbow flexion, this was achieved in less than half the cases. We consider primary gracilis functioning free muscle transfer neurotized by the distal spinal accessory nerve as the most reliable reconstruction for the restoration of elbow flexion in complete brachial plexus injury. LEVEL OF EVIDENCE: IV.

Relationship between CCL5 and transforming growth factor-ß1 (TGFß1) in breast cancer.

PURPOSE: Investigate circulating CCL5 in breast cancer patients and healthy controls, along with gene expression levels in corresponding tumour tissue and isolated primary stromal cells. Hormonal control of CCL5, and a potential relationship with TGFß1, was also investigated. METHODS: Circulating levels of CCL5 and TGFß1 were measured in 102 breast cancer patients and 66 controls using ELISA. Gene expression levels (CCL5, CCR5, TGFß1, TGFßRII) were quantified in corresponding tumour tissue (n = 43), normal tissue (n = 16), and isolated tumour (n = 22) and normal (n = 3) stromal cells using RQ-PCR. CCL5 and circulating menstrual hormones (LH, FSH, Oestradiol, Progesterone) were analysed in serum samples from healthy, premenopausal volunteers (n = 60). RESULTS: TGFß1 was significantly higher in breast cancer patients (Mean(SEM) 27.4(0.9)ng/ml) compared to controls (14.9(0.9)ng/ml). CCL5 levels decreased in the transition from node negative (59.6(3.7)ng/ml) to node positive disease (40.5(6.3)ng/ml) and increased again as the number of positive lymph nodes increased (⩾3 positive 50.95(9.8)ng/ml). A significant positive correlation between circulating CCL5 and TGFß1 (r = 0.423, p<0.0001) was observed, and mirrored at the gene expression level in tumour tissue from the same patients (r = 0.44, p<0.001). CCL5, CCR5 and TGFß1 expression was significantly higher in tumour compared to normal breast tissue (p < 0.001). A significant negative correlation was observed between circulating CCL5, Oestradiol and Progesterone (r = -0.50, r = -0.39, respectively, p < 0.05). CONCLUSION: CCL5 expression is elevated in the tumour microenvironment. The data support a role for hormonal control of circulating CCL5 and also highlight a potentially important relationship between CCL5 and TGFß1 in breast cancer.

An assessment of skin preparation in upper limb surgery.

Postoperative wound infections remain a major source of upper limb morbidity. The effectiveness of peri-operative human upper limb preparation was determined using a clear fluid antiseptic and an iodine-based solution over 60 and 90 seconds. Less area was missed using iodine over both times and increasing clear solution preparation time from 60 to 90 seconds improved coverage. Surgical experience had little outcome relevance and a 90-second preparation time with either solution was insufficient, with fingers being the sites most commonly missed.

Effects of thyrotropin-releasing hormone on human myometrium and umbilical vasculature in vitro.

OBJECTIVE: The purpose of this study was to investigate the direct effects of thyrotropin-releasing hormone on isolated human myometrium that was obtained during pregnancy and on human umbilical vasculature in vitro. STUDY DESIGN: Isolated human myometrial strips were dissected from biopsy specimens that were obtained at elective cesarean delivery and suspended for isometric recording under physiologic conditions. The effects of cumulative additions of thyrotropin-releasing hormone (10(-9)-10(-4) mol/L) on oxytocin-induced myometrial contractility were evaluated. The effects of thyrotropin-releasing hormone (10(-9)-10(-4) mol/L) on umbilical vessel (artery and vein) resistance in vitro were investigated with the use of isolated ring preparations. RESULTS: Thyrotropin-releasing hormone exerted a significant concentration-dependent relaxant effect on pregnant human myometrial tissue, which ranged from 3.54% (10(-9) mol/L, P=.935) to a net cumulative total of 21.06% (10(-4) mol/L, P<.001). Thyrotropin-releasing hormone also exerted a concentration-dependent relaxant effect on human umbilical vasculature that ranged from 12.51% (10(-9) mol/L, P=.994) to a net cumulative total of 23.27%+/-4.87% (SEM, 10(-4) mol/L, P<.01) in umbilical artery. For umbilical vein, the relaxant effect ranged from 1.80% (10(-9) mol/L, P=.998) to a net cumulative total of 14.64% (10(-4) mol/L, P<.009). CONCLUSION: Thyrotropin-releasing hormone exerts a significant relaxant effect in human myometrium and in human umbilical vasculature and highlights a potential physiologic role for this neuropeptide in these tissues. These findings have clinical implications for the therapeutic use of thyrotropin-releasing hormone antenatally.

Adverse effects of fetal cocaine exposure on neonatal auditory information processing.

BACKGROUND: Studies with animals have shown that in utero exposure to cocaine interferes with fetal brain development by disrupting the processes of neuronal proliferation, differentiation, and migration, often leading to subsequent neurobehavioral deficits. However, studies with humans have produced inconsistent findings. Although neurobehavioral abnormalities have been observed among cocaine-exposed infants in several studies and in some cases dose-response effects have been found, the specific neurobehaviors affected vary from one study to the next. Researchers studying the effects of fetal cocaine-exposure are faced with many difficult challenges. For example, women who use cocaine typically use other substances in addition to cocaine, many of the methods available for identifying cocaine-exposed neonates are not reliable, and the available methods for assessing cocaine-exposed newborns may not be sufficiently sensitive to detect the subtle effects of cocaine on the developing central nervous system. Despite these difficulties, there is a growing body of research that suggests that fetal cocaine exposure is associated with subsequent language deficits among children exposed in utero. However, it is virtually impossible to disentangle the effects of the impoverished environments in which these children are often raised from the effect, if any, of fetal cocaine exposure. To determine the effects of fetal cocaine exposure independent of postnatal environmental effects, cocaine-exposed neonates would ideally be tested within the first few weeks of birth, and to identify early risks for subsequent language delay, well-researched auditory information processing measures could be used. OBJECTIVE: The purpose of the present study was to assess the effects of fetal cocaine exposure on neonatal auditory information processing ability. To overcome limitations of some previous studies on the neuroteratogenic effects of cocaine, such as unreliable subject identification techniques, inadequate control over confounding variables, and questionable measures of central nervous system integrity, a valid measure of auditory information processing was used in a rigorous, case-control design. METHOD: Newborn information processing was assessed using habituation and recovery of head-turning toward an auditory stimulus across the 3 phases of the procedure: familiarization, novelty, and dishabituation. During the familiarization phase, the infant orients and habituates to a repeated word; during the novelty phase, the infant recovers head-turning to a novel word and subsequently habituates to this word; and during the dishabituation phase the infant displays renewed head-turning to the return of the original stimulus. Testing takes approximately 20 minutes. This procedure has been shown previously to discriminate among infants at high-, moderate-, and low-risk for subsequent developmental delay. Twenty-five cocaine-exposed and 25 nonexposed control neonates, identified by meconium analysis, urine analysis, and/or maternal self-report, were tested on the auditory information processing procedure. The majority of infants were tested within the first few days of birth. Cocaine-exposed and control neonates were matched on birth weight, gestational age, Apgar scores, age at testing, and socioeconomic status as reflected by household income. Mothers were matched on age, weight gain, cigarette smoking, and alcohol consumption. RESULTS: Fetal cocaine exposure was associated with impaired auditory information processing. Both cocaine-exposed and nonexposed control neonates oriented to the familiarization stimulus, but cocaine-exposed neonates displayed impaired habituation. Moreover, cocaine-exposed neonates did not recover or habituate to the novel stimulus or dishabituate to the return of the familiarization stimulus. (ABSTRACT TRUNCATED)

In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV.

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.

Influence of sulfur-amino acid content variation in plant vs animal protein on serum and tissue lipids in rats.

The objective of the study was to determine the effects of methionine and cysteine supplementation of soy protein isolate and casein on serum and tissue lipid levels in rats. Sixty male, weanling, Wistar-Kyoto rats were fed two sources of protein (casein or soy protein isolate) and three variations of sulfur-amino acid supplementation (none, methionine, or cysteine). At this level of protein intake (10% by weight), rats fed soy-based diets had similar serum lipid concentrations than rats fed casein-based diets. Choline was not added to the diet in order to be able to assess independent influences of methionine and cysteine on lipid metabolism. Overall, serum lipid values were greater in rats fed proteins supplemented with methionine while the addition of cysteine produced lower lipid levels. Liver lipid concentrations were increased tremendously upon cysteine supplementation of soy protein isolate. Protein quality, as determined by protein efficiency ratio, was improved by supplementation of either sulfur-amino acid; however, methionine had the greatest effect. Results indicate that the sulfur-amino acids influence lipid metabolism in the absence of dietary choline. The mechanism by which this occurs is not known.