Primitive neuroectodermal adrenal gland tumour.

Ewing's sarcoma, also called primitive neuroectodermal tumour of the adrenal gland, is extremely rare. Only a few cases have been reported in the literature. We report on a woman with adult-onset primitive neuroectodermal tumour of the adrenal gland presenting with progressive flank pain. Computed tomography confirmed an adrenal tumour with invasion of the left diaphragm and kidney. Radical surgery was performed and the pain completely resolved; histology confirmed the presence of primitive neuroectodermal tumour, for which she was given chemotherapy. The clinical presentation of this condition is non-specific, and a definitive diagnosis is based on a combination of histology, as well as immunohistochemical and cytogenic analysis. According to the literature, these tumours demonstrate rapid growth and aggressive behaviour but there are no well-established guidelines or treatment strategies. Nevertheless, surgery remains the mainstay of local disease control; curative surgery can be performed in most patients. Adjuvant chemoirradiation has been advocated yet no consensus is available. The prognosis of patients with primitive neuroectodermal tumours remains poor.

Paediatric thyroidectomy: When and why? A 25-year institutional experience.

OBJECTIVE: Thyroidectomy in paediatric patients is relatively uncommon. In this study, we reviewed our experience of thyroidectomy in children and identified risk factors associated with postoperative complications. METHODS: We performed a retrospective analysis of paediatric patients who had thyroidectomy in our institution between April 1995 and January 2021. Demographic data, preoperative cytological findings, indications of surgery, surgical complications and histological results were analysed. RESULTS: A total of 87 paediatric patients with 92 thyroidectomy were identified. The indications for surgery were Graves' disease refractory to medical treatments (40.2%), benign thyroid nodules or multinodular goitre (26.4%), thyroid carcinoma (23.0%) and multiple endocrine neoplasm type 2A syndrome (10.3%). Patients presented with thyroid nodules or cervical lymph nodes had a 43.9% risk of malignancy. 66 total thyroidectomy were done with median operation time of 134 min(102-170), while 26 hemi-thyroidectomy were performed (Right side 12/92, Left side 14/92) with median operation time of 65 min(49-102). The median postoperative hospital stay was 2 days(1-4). Intraoperative neck dissection (p = 0.003), drain insertion (p = 0.001) and hypocalcaemia requiring medical treatment (p = 0.004) were associated with longer hospital stay. The median follow-up was 11.3 years (3.0-16.8). 32% patients had immediate postoperative hypocalcaemia and 8% patients had permanent hypoparathyroidism. Transient vocal cord palsy was found in 3 patients(3%) and all resolved within 5-month time upon reassessment direct laryngoscopy. The use of intraoperative recurrent laryngeal nerve monitoring was associated with less vocal cord palsy (p = 0.022). The median disease-free survival was 13.7 years(7.4-17.7) for patients operated for well-differentiated thyroid carcinoma(WDTC). amongst the 9 patients who had prophylactic total thyroidectomy for MEN2A syndrome, 44% were found to have medullary thyroid microcarcinomas on pathology. CONCLUSIONS: Surgical management of paediatric thyroid disease can be complex. Postoperative hypocalcaemia and vocal cord palsy were usually transient after total thyroidectomy. The use of intraoperative recurrent laryngeal nerve monitoring had resulted in less vocal cord palsy. Long-term disease-free survival of patients with thyroid cancer had been achieved with multi-disciplinary management in our centre. LEVEL OF EVIDENCE: Retrospective Comparative Study; Level III.

Prognostic value of lymph node ratio in stage III colorectal cancer.

AIM: Recent reports show that a positive metastatic to examined lymph nodes ratio (LNR) has prognostic value in malignancies. This study aimed to evaluate the prognostic value of LNR in patients having resection for stage III colorectal cancer. METHOD: From January 2000 to December 2006, patients who underwent resection for stage III colorectal carcinoma were included. All clinicopathological and follow-up data were prospectively collected. The impact of LNR and other clinicopathological factors on survival were evaluated. RESULTS: The study included 533 (52.3% male) patients with a median age of 70 years. The median number of lymph nodes harvested and the median number of positive lymph nodes examined were 11 and 2, respectively. The median LNR was 0.263 (range, 0.03-1). After a median follow up of 52.65 months, the 5-year overall survival and disease-free survival were 55.9% and 49.4%. The patients were stratified into four groups according to LNR quartiles (1, LNR ≤ 0.125; 2, 0.1250.500). The 5-year overall and disease-free survival were 72.8%, 63.1%, 50.0%, 39.6% (P<0.001) and 68.5%, 54.1%, 47.2%, 29.9% (P<0.001), respectively, with increasing LNR groups. On multivariate analysis, age, T stage and LNR were independent predictors of both overall and disease-free survival. Subgroup analysis revealed that the LNR had a prognostic value for disease-free survival irrespective of number of lymph nodes harvested and location of tumour. CONCLUSION: The LNR is an independent prognostic factor for survival in colorectal cancer and is superior to the pN category in TNM staging.

Changes in breast cancer patients after psychosocial intervention as indicated in drawings.

OBJECTIVE: The present study attempts to understand the experience of breast cancer patients who had participated in an Eastern-based body-mind-spirit (BMS) psychosocial intervention program by observing changes in the images made by the patients. METHODS: Pre- and post-intervention drawings on the theme of 'my cancer' were collected from 67 primary breast cancer patients. Two creative art therapists compared the drawings according to the structural and formal art elements (body), the symbols used (mind), and the emotions and feelings presented in the drawings (spirit). Numbers of pre- and post-intervention drawings, showing the presence of each element in these three dimensions, were also counted and compared. RESULTS: There were several changes noted between pre- and post-intervention drawings. The use of color, space, and multiplicity increased from 12 to 17%. Images of breasts decreased from 13 to 0%. Representations of cancer decreased from 15 to 7%. There was a slight increase in symbolic representations of natural, landscapes, and social support in post-drawings (3-6%). The portrayal of negative emotions was greatly reduced from 52 to 3%, while positive emotions increased from 28 to 93% in post-drawings. CONCLUSIONS: The comparison of pre- and post-intervention drawings revealed changes in subject matter and accompanying emotions. Overall, there was a trend in changes toward a more peaceful and hopeful attitude. Through the use of realistic and symbolic images, participants depicted a range of emotions. Limitations and recommendations for using art-making, as an assessment tool and intervention, are addressed.

Orthopaedic Resident Burnout: A Literature Review on Vulnerability, Risk Factors, Consequences and Management Strategies.

Introduction: Orthopaedic surgery is physically demanding. Surgeons may have to work long unpredictable hours especially during residency training. This arduous task comes with the risk of burnout leading to negative repercussions to the surgeon and the patient. In view of strategising peer support, we intend to review the literature and analyse whether orthopaedic resident burnout is a global issue. We also intend to derive common strategies to tackle burnout at individual and organisational levels. Materials and Methods: A literature search was carried out in the databases including PubMed, Scopus, SciELO, and Google Scholar to shortlist studies dealing with orthopaedic residency and related burnout. Those studies that used the Maslach Burnout Inventory (MBI) for quantifying burnout were collectively interpreted. Other studies were reviewed to analyse the vulnerability, risk factors, consequences and management strategies related to burnout. Results: Among a total of 72 titles shortlisted, eight studies independently reported burnout among orthopaedic surgery residents/trainees and used MBI as a tool for assessing burnout. Based on the three subscales of MBI, 37.2% had high degree of emotional exhaustion (EE), 48% had high degree of depersonalisation (DP) and 33.1% perceived low personal accomplishment. This signifies the high prevalence of burnout among orthopaedic residents/trainees. Conclusion: Burnout among orthopaedic surgery residents seems to be a universal problem. Risk factors could be multifactorial, influenced by clinical competency and work-home environment. This can be tackled at the individual level by being aware of burnout syndrome, involving in adequate physical activity and spending quality social time; and at the organisational level by duty hour limitation, professional appreciation and mentorship programme.

Sulphate induces very fast cell rounding and detachment.

Within a few minutes of incubation with SO4(2-), cultured monolayer cells retract into round shapes with drastically reduced surface area. Concomitant elevation of phosphoinositide second messenger levels, viz, 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), is observed. A causal relationship with sulphation seems to be suggested by finding (a) sulphation of an added acceptor, 4-methylumbelliferone, (b) sulphation of endogenous glycosaminoglycan (GAG) polymers, (c) inhibition by phenol sulphotransferase inhibitor, DCNP (2,6-dichloro-4-nitrophenol). DCNP also inhibits the second messenger production and cell rounding. Reduced surface area appears to be caused by massive plasma membrane internalization in a distinctive endocytosis which is also seen in cell rounding from directly imposed ionic gradients. Reducing the surface area would reduce the adhesive or attachment sites. Besides demonstrating a highly efficient cell detachment potential, huge macromolecules appear to be readily internalized. The association of sulphation, signal transduction and cell detachment is novel.

Mechanism of clofibrate hepatotoxicity: mitochondrial damage and oxidative stress in hepatocytes.

Peroxisome proliferators have been found to induce hepatocarcinogenesis in rodents, and may cause mitochondrial damage. Consistent with this, clofibrate increased hepatic mitochondrial oxidative DNA and protein damage in mice. The present investigation aimed to study the mechanism by which this might occur by examining the effect of clofibrate on freshly isolated mouse liver mitochondria and a cultured hepatocyte cell line, AML-12. Mitochondrial membrane potential (Delta Psi(m)) was determined by using the fluorescent dye 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM). Application of clofibrate at concentrations greater than 0.3 mM rapidly collapsed the Delta Psi(m) both in liver cells and in isolated mitochondria. The loss of Delta Psi(m) occurred prior to cell death and appeared to involve the mitochondrial permeability transition (MPT), as revealed by calcein fluorescence studies and the protective effect of cyclosporin A (CsA) on the decrease in Delta Psi(m). Levels of reactive oxygen species (ROS) were measured with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (DCFDA) and dihydrorhodamine 123 (DHR123). Treatment of the hepatocytes with clofibrate caused a significant increase in intracellular and mitochondrial ROS. Antioxidants such as vitamin C, deferoxamine, and catalase were able to protect the cells against the clofibrate-induced loss of viability, as was CsA, but to a lesser extent. These results suggest that one action of clofibrate might be to impair mitochondrial function, so stimulating formation of ROS, which eventually contribute to cell death.

Mitochondrial damage by the "pro-oxidant" peroxisomal proliferator clofibrate.

Clofibrate is a peroxisome proliferator that can cause hepatic cancer in rodents. It has been suggested that oxidative damage is involved in this hepatocarcinogenesis, although the data are conflicting. We confirmed that clofibrate causes oxidative damage in nuclei from the livers of mice treated with this substance, measured both as protein carbonyls and levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA. In addition, clofibrate also affects mitochondria, causing elevated levels of carbonyls and 8-OHdG, increased state 4 respiration and decreased adenosine triphosphatase (ATPase) activity. No evidence for clofibrate-induced lipid peroxidation in mitochondria was obtained. We propose that mitochondria may be a major target of injury and a source of oxidative stress in clofibrate-treated animals.

Active transport of glutathione S-conjugate in human colon adenocarcinoma cells.

The formation of the glutathione S-conjugate of monochlorobimane (GSH-bimane) in human colon adenocarcinoma cells was identified by HPLC-fluorimetry and its transport from the cells was found to be temperature-sensitive, saturable and ATP-dependent. The apparent K(m) and Vmax values were 2.4 +/- 0.5 nmol GSH-bimane/10(6) cells and 0.5 +/- 0.1 nmol GSH-bimane/min per 10(6) cells, respectively. This active transport of GSH-bimane was inhibited by low micromolar concentrations of classical uncouplers of oxidative phosphorylation, namely carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), carbonylcyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP). The efflux of GSH-bimane was competitively inhibited by chlorambucil (CMB) and 1-chloro-2,4-dinitrobenzene (CDNB), two other substrates of GST. This study demonstrates the presence and kinetic measurements of the glutathione S-conjugate export (GS-X) pump in human colon cancer cells, an export pump whose function has been implicated in the phenomenon of multidrug resistance.

Acidification-and-recovery induces nuclear accumulation of neutral red and DNA into human KB oral carcinoma cells.

In cultures of oral cancers, gene transfer has been achieved by delivery systems which introduce DNA into cells but not specifically into the nucleus. We observed that acidification and recovery converted the nuclear-insulated interphase KB carcinoma cells into intense nuclear-accumulating states. Nuclear sequestration of the vital dye neutral red and T7 oligonucleotide was demonstrated qualitatively, and quantitatively by image analysis of single cells. pGem beta gal plasmids of 6.8 kb were introduced into KB cells by similar acidification and recovery pulses. Successful integration into the host KB genome was shown by expression of the lacZ gene of the plasmids. Enhanced nucleo-cytoplasmic transport demonstrated here in KB oral epidermoid carcinoma cells could potentially facilitate gene therapy in oral cancers.

Glutathione-related factors are not correlated with sensitivity of human tumour cells to actinomycin D.

Glutathione (GSH) contents and activities of glutathione S-transferases (GST), glutathione reductase (GSH-RD), glutathione peroxidase (GSHpx) and glutathione conjugate export pump (GS-X pump) were determined in eight human tumour cell lines with different sensitivities to melphalan, a substrate of glutathione conjugation, and actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms. Chang liver cells with highest GSH content and highest activities of GST, GSH-RD, GSHpx and GS-X pump were found to be most resistant to melphalan. Statistical analysis showed significant correlations between sensitivities of the human tumour cells to melphalan and the glutathione-related factors (r = 0.72-0.79; except for GST, r = 0.65, P = 0.08), while there were no significant correlations observed between sensitivities of the human tumour cells to actinomycin D and all the glutathione-related factors tested (r = -0.25-0.14). Significant correlations of the glutathione-related factors to resistance of human tumour cells to melphalan, a substrate of glutathione conjugation, but not to resistance of the human tumour cells to actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms suggested that glutathione-related mechanisms contribute to drug resistance by increased detoxification of the drugs involved.

A simple sensitive fluorimetric assay of APS-kinase activity.

Adenosine phosphosulphokinase (APS-kinase or ATP:adenylylsulphate 3'-phosphotransferase; EC 2.7.1.25) catalyses the formation of 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Its activity in various tissues was measured by transferring the sulphate from PAPS, a product of APS-kinase reaction, to 4-methylumbelliferone (4-MU) to form 4-MU-sulphate (4-MUS) using phenolsulphotransferase (PST) extracted from rat liver. Desalting with Sephadex G-25, together with the addition of EDTA effectively removed the Mg2+ ions from the rat liver extract and thereby inhibited the APS-kinase activity therein in the subsequent PST reaction. 4-MUS formed was measured indirectly by a decrease in the fluorescence of 4-MU by a continuous fluorimetric assay. Kinetic data showed that the substrate, APS, at concentrations at and above 132 microM inhibited the APS kinase reaction. Pyrophosphate (PP) also inhibited the reaction. The apparent Km for APS was 14 microM. Two apparent Km values of 0.12 mM and 1.06 mM were obtained for ATP, while that for Mg2+ was 0.09 mM.

Sulphate conjugation of isoprenaline by lung, small intestine and other organs.

Isoprenaline, a sympathomimetic drug used in the treatment of asthma, was found to be sulphated by the bronchial tissues of the monkey and dog. Enzyme preparations of the liver, small intestine and kidney of various animals are also able to catalyze this sulphate conjugation reaction from ATP and inorganic sulphate and from a commercial preparation of adenosine 3'-phosphate 5'-sulphatophosphate (PAP35S) or PAP35S generated from Na235SO4 in vitro. The Km values for isoprenaline for the sulphotransferase of mouse liver and monkey lung, are respectively, 51,3 microM and 138 microM . The significance of this detoxication reaction is discussed in relation to (a) the importance of lung as a potential biotransformation site of isoprenaline, (b) asthma deaths supposed to be associated with the use of isoprenaline in the form of pressurised aerosols and (c) the ability of the different tissues to synthesize PAPS in vitro.

2,6-Dichloro-4-nitrophenol (DCNP), an alternate-substrate inhibitor of phenolsulfotransferase.

2,6-Dichloro-4-nitrophenol (DCNP)-35sulfate was identified and quantified by an HPLC-radiometric assay following its biosynthesis in vitro from 35S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAP35S) by phenolsulfotransferase (PST) of rat liver cytosol. Acid hydrolysis of DCNP-35sulfate produced almost stoichiometric release of inorganic 35sulfate and DCNP. In two-substrate experiments of sulfation of p-nitrophenol (p-NP) or dopamine (prototype substrates for P and M human PST forms), 10 microM DCNP inhibited the reactions by about 15 and 78%, respectively. This contrasts with its action on PST of human origin where the P-PST was more sensitive to DCNP inhibition. In all mixed bi-substrate experiments, a reciprocal relationship between the two sulfated products was observed. Kinetic data showed that p-NP inhibited the sulfation of DCNP competitively. Likewise the sulfation of p-NP and dopamine was competitively inhibited by DCNP. However, non-competitive inhibition was observed in the sulfation of p-NP by DCNP, measured at varying concentrations of PAP35S. The above kinetic data suggest that DCNP is an alternate-substrate inhibitor of rat liver PST.

Biosynthesis of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in rat skin.

A rapid and sensitive radiometric assay was developed for the measurement of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis in rat skin extract. The formation of PAP35S from sodium 35sulfate and ATP was quantified by the transfer of the 35sulfate to minoxidil by rat liver minoxidil sulfotransferase (MST). The assay is sensitive enough for the detection of as little as 2 pmol of PAP35S. The PAPS-generating system showed a pH optimum of 8.6, with an apparent Km value of 1 mM for the ATP-Mg2+ complex and 68 microM for sodium 35sulfate. ATP and Mg2+, present individually or together in equimolar concentrations, were inhibitory above 8 mM. Excess (or free) ATP was a competitive inhibitor with respect to the ATP-Mg2+ complex; the apparent Ki measured was 0.32 mM. The specific activity of the PAPS-generating system, measured in rat skin cytosol was 0.15 nmol PAPS/min/mg protein. The importance of PAPS generation in detoxification and bioactivation of xenobiotics in skin is discussed.

Direct measurement and regulation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) generation in vitro.

3'-Phosphoadenosine 5'-phospho[35S]sulfate (PAPS) biosynthesized from inorganic [35S]sulfate and ATP was separated from its radiolabeled precursor by reversed-phase paired-ion HPLC and quantified by on-line radiometric detection. This single-step procedure circumvents several problems inherent in conventional sulfotransferase-coupled assays employed in the measurement of PAPS formation. A good correlation was observed between the rate of PAPS generation assayed in several mammalian tissues measured by direct HPLC-radiometry and by coupling to the sulfation of minoxidil or 4-methylumbelliferone. Both AMP and ADP inhibited the rat liver sulfate-activating enzymes competitively with respect to MgATP2-, and the rate of PAPS production was decreased with decreasing ratios of [ATP]:[ADP] and [ATP]:[AMP]. It is possible that these adenine nucleotides regulate sulfate activation by kinetic control and by negative feedback modulation.

Inhibition of the efflux of glutathione S-conjugates by plant polyphenols.

The formation of dinitrophenylglutathione (DNP-SG) in human colon adenocarcinoma cells was identified and quantified by an HPLC-UV method, following exposure to 1-chloro-2,4-dinitrobenzene (CDNB) at 10 degrees for 40 min. The rate of efflux of DNP-SG at 37 degrees likewise, was measured by monitoring the DNP-SG content in the extracellular medium. Among the polyphenols examined for their action on DNP-SG export, butein was the most potent inhibitor with an IC50 value of 15 microM. The others, in order of decreasing potencies, were quercetin, tannic acid, 2'-hydroxychalcone, 2-hydroxychalcone anIIC50 values in the micromolar range. These polyphenols did not affect the ATP or the glutathione content of the cells. Mg(2+)-ATPase extracted from the plasma membrane of the cells was activated by DNP-SG in a concentration-dependent manner, and the reaction showed saturation kinetics with K(m) and Vmax values of 110 microM and 12.3 nmol/min/mg protein, respectively. However, the six polyphenols mentioned above had negligible effects on the Mg(2+)-ATPase activity, suggesting that this was probably not the target of their inhibitory action. Probenecid, p-trifluoromethoxy-phenylhydrazone (FCCP) and chlorambucil also showed varying degrees of inhibition of the export of DNP-SG.

Importance of extrahepatic sulphate conjugation.

A similar trend in the sulphate conjugation of isoprenaline and harmol was observed in the hepatic and three extrahepatic tissues, namely the kidney, small intestine and lung of some experimental animals. All the hepatic and some extrahepatic tissues exhibit this sulphating capability. In fact, some extrahepatic tissues, e.g. monkey lung, kidney and small intestine, mouse kidney and guinea-pig small intestine surpass their respective livers in this sulphate-conjugating reaction. When no or low activity was observed, a consistent pattern was found for both substrates. In general, isoprenaline is a better acceptor than harmol; the greatest difference was obtained with the mouse kidney preparation where an 18-fold difference was attributed partly to the higher sulphotransferase activity for isoprenaline than for harmol. The importance of extrahepatic sulphate conjugation is discussed.

Kinetics of sulphate conjugation in extracts of human foetal liver cells in culture.

Sulphate conjugation was investigated using extracts of human foetal liver cells in culture. The three reactions which are involved in sulphate conjugation were measured singly or in combination: they are (i) the PAPS generation catalyzed by ATP-sulphurylase and adenosine 5'-phosphosulphate (APS)-kinase, (ii) the phenolsulphotransferase (PST) reaction, and (iii) the overall sulphate conjugation which comprises the above three reactions. All were radiometric assays employing PAP35S or sodium 35sulphate. N-acetyldopamine (NADA) was the substrate of choice although the reactions were also demonstrated with dopamine and 1-naphthol. Kinetic studies with NADA showed two pH optima of 6.7 and 8.6 for the overall sulphate conjugation and the PST reaction while the PAPS generation occurred maximally at pH 8.0. The apparent Km value for NADA measured by both the PST and the overall sulphate conjugation reactions was the same, being 38 microM, while that for inorganic sulphate, of 107 microM and 240 microM (measured by the overall sulphate conjugation reactions and by PAPS generation, respectively) was two orders of magnitude higher than that of PAPS, which was 2.57 microM. It was possible to maintain a relatively constant level of the three activities of sulphate conjugation in confluent, quiescent cultures. The importance of sulphate conjugation for detoxification in foetal cells is discussed.

Sulphate conjugation in human liver. Direct measurement of N-acetyldopamine-sulphate.

The overall three-step sulphate conjugating activity of the human liver was determined by the direct measurement of N-acetyldopamine (NADA)-sulphate from ATP and inorganic sulphate. NADA-sulphate was separated from NADA and quantified by HPLC-ECD (high-pressure liquid chromatography-electrochemical detection). The overall sulphate conjugation of NADA showed at pH optimum of 8.0 with apparent Km values for sodium sulphate and NADA of 103 microM and 4.8 microM. respectively. The optimum concentration of ATP was 5 mM and that for Mg2+ ions was 7 mM. The specific activities of 17 samples of human liver, measured by this HPLC-ECD procedure ranged from 940 to 23,128 pmol NADA-sulphate/hr/mg protein. A comparison of these values with those determined by the radiometric method developed previously showed a direct correlation coefficient, r = 0.89. The rates of overall sulphate conjugation of NADA and dopamine measured in these samples by the radiometric assay procedure were also significantly correlated with r = 0.82.