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BACKGROUND Lumbar kyphosis is a complex spinal deformity occurring in approximately 8% to 20% of patients with myelomeningocele. The resulting gibbosity may cause pressure ulcers, difficulty lying down in the supine position and sitting on the ischia without support, decreasing quality of life (QOL). Surgery is generally performed to correct kyphosis and maintain vertebral alignment, but high complication rates have been reported. Despite satisfactory radiological results, the impact of surgery and its complications on health-related QOL (HRQOL) has not yet been established. QUESTIONS/PURPOSES Among children with myelomeningocele undergoing corrective surgery for lumbar kyphosis: (1) What is the risk of complications and reoperation after this procedure? (2) Does this procedure improve HRQOL scores in these patients? METHODS Between 2012 and 2013, five surgeons at three centers treated 32 patients for myelomeningocele-related kyphosis with kyphectomy and posterior instrumentation. During that period, all surgeons used the same indications for the procedure, which were progressive postural decompensation and chronic ulceration at the apex of the deformity. Data were prospectively collected, and all patients who underwent surgery were considered in this retrospective study. The legal guardians of one patient declined to sign the informed consent form, resulting in 31 patients included. A total of 9.7% (3 of 31) were lost to follow-up before the 2-year period, and the remaining 90.3% (28 of 31) were seen at a mean of 3 years (± 9 months) after surgery. The average age was 10 years, 7 months (± 21 months) at the time of surgery. The patients had a mean kyphosis angle of 130° ± 36° before surgery. This technique involved posterior fixation using S-shaped rods inserted through the foramina of S1 and pedicle screws inserted in the thoracic spine. The patients' caregivers answered both the generic and specific (neuromuscular module) Pediatric Quality of Life Inventory questionnaires preoperatively and 2 years postoperatively. The minimum clinically important difference (MCID) considered for the instruments used was 5. RESULTS Reoperation was performed in 68% of patients (19 of 28), mostly to treat deep infection. In all, 18% of patients (five of 28) underwent implant removal to control infection. Eleven percent (three of 28) had a loss of reduction and pseudarthrosis. The HRQOL increased from 71 ± 11 preoperatively to 76 ± 10 postoperatively (p < 0.001), resulting in a 5-point increase (95% CI 3 to 7) in the generic questionnaire score and from 71 ± 13 to 79 ± 11 (p < 0.001), resulting in an 8-point increase (95% CI 5 to 10) in the neuromuscular Paediatric Quality of Life Inventory questionnaire score, mainly in the physical health domain on both questionnaires. CONCLUSIONS Kyphectomy was associated with a high risk of complications and reoperations and did not seem to deliver a substantial clinical benefit for patients who underwent the procedure. Most of our HRQOL score improvements were below the minimum clinically important difference for the Pediatric Quality of Life Inventory questionnaires. Although it seems that surgeons lack a better surgical alternative when facing the challenging health impairments these patients suffer, efforts should be made to improve the technique and reduce surgical complications. Additionally, patients and caregivers should be advised of the high reoperation rate and notified that the procedure may not result in a better QOL and should thus be avoided when possible. Future studies should verify whether decreasing the complication rate could imply improvement in the HRQOL of these patients after surgery. LEVEL OF EVIDENCE Level IV, therapeutic study.

Zinc (Zn) is an element essential to all living organisms and it has an important role as a cofactor of several enzymes. In fish, Zn deficiency has been associated with impaired growth, cataracts, skeletal abnormalities and reduced activity of various Zn metalloenzymes. Fish meal and fish oil traditionally used in salmon feed preparation are being replaced by plant-based ingredients. Zinc additives are supplemented to salmon feed to ensure adequate Zn levels, promoting good health and welfare in Atlantic salmon (Salmo salar). The main objective of the present study was to evaluate Zn species found in an Atlantic salmon feed. This work describes a Zn extraction method that was optimized using a fractional factorial design (FFD), whereby the effect of six factors could be studied by performing only eight experiments. The effects of the type of extraction solution and its molar concentration, pH, presence of sodium dodecyl sulphate, temperature and extraction time on Zn extraction were investigated. Mild extraction conditions were chosen in order to keep the Zn species intact. Total Zn (soluble fractions and non-soluble fractions) was determined by inductively coupled plasma mass spectrometry (ICP-MS). The highest Zn recovery was obtained using 100 mM Tris-HCl, pH 8.5 at a temperature of 4 °C for 24 h where the total Zn in soluble fraction and non-soluble fraction was 9.9 ± 0.2% and 98 ± 6%, respectively. Zinc speciation analysis (on the soluble fractions) was further conducted by size exclusion inductively coupled plasma mass spectroscopy (SEC-ICP-MS). The SEC-ICP-MS method provided qualitative and semi-quantitative information regarding Zn species present in the soluble fractions of the feed. Four Zn-containing peaks were found, each with different molecular weights: Peak 1 (high molecular weight - ≥600 kDa), peak 2 and peak 3 (medium molecular weight – 32 to 17 kDa) were the least abundant (1–6%), while peak 4 (low molecular weight – 17 to 1.36 kDa) was the most abundant (84–95%). publishedVersion

Objective: To evaluate awareness of prostate cancer in the population of the city of São Paulo. Methods: A total of 392 adults were randomly interviewed on public spaces in the city of São Paulo, and answered a questionnaire that addressed demographic questions and specific knowledge about the prostate cancer. A score was used to assess awareness of cancer in general, and of prostate cancer, considering satisfactory knowledge a score of 6 points. Results: The mean age was 36.9 years (standard deviation of ±12.6) and 58.2% of participants were male. No previous contact with information related to prostate cancer was reported by 45.5% of participants. For these cases, a greater proportion was observed among men aged over 50 years. As to the score, the mean was 3.7 (standard deviation of ±1.3), with a positive correlation among higher scores, higher income and education level. Less than 5% of participants believed they should only search for prostate cancer screening when symptomatic. Finally, among the less frequent responses to risk factors for prostate cancer, is “ethnic origin” (2.8%). Conclusion: Even though most participants did not have a satisfactory score, the level of awareness demonstrated in this study seems superior to that of other populational series. Hence it suggested the assessed population understood some essential concepts in prostate cancer, such as the importance of screening and the follow-up. The efforts made by the Sociedade Brasileira de Urologia on educational campaigns partially explain this. However, working in some concepts, like identifying risk factors for prostate cancer, might optimize screening outcomes. RESUMO Objetivo: Avaliar o conhecimento da população da cidade de São Paulo em relação ao câncer de próstata. Métodos: Foram entrevistados randomicamente 392 adultos em espaços públicos da cidade de São Paulo, os quais responderam a um questionário que abordava questões demográficas e de conhecimentos específicos sobre o câncer de próstata. Um escore foi utilizado para avaliar o conhecimento de câncer em geral e do câncer de próstata, considerando um conhecimento satisfatório com escore de 6 pontos. Resultados: A média de idade foi de 36,9 anos (desvio-padrão de ±12,6), e 58,2% dos participantes eram do sexo masculino. Ausência de contato anterior com informações relacionadas ao câncer de próstata foi relatada por 45,5% dos participantes. Nesses casos, maior proporção foi observada entre os homens com mais de 50 anos. Quanto ao escore, a média foi 3,7 (desvio-padrão de ±1,3), com correlação positiva entre maiores escores e maiores renda e escolaridade. Menos de 5% dos participantes acreditavam que só deveriam procurar o rastreamento do câncer de próstata quando sintomáticos. Por fim, entre as respostas menos frequentes aos fatores de risco para câncer de próstata, encontrou-se “etnia” (2,8%). Conclusão: Embora a maioria dos participantes não tenha apresentado escore satisfatório, o nível de conhecimento revelado neste estudo parece superior ao de outros estudos populacionais. Assim, sugere-se que a população avaliada tenha compreendido alguns conceitos essenciais do câncer de próstata, como a importância do rastreamento e do acompanhamento. Os esforços da Sociedade Brasileira de Urologia nas campanhas educacionais explicam parcialmente isso. No entanto, trabalhar em alguns conceitos, como identificar fatores de risco para câncer de próstata, pode otimizar os resultados do rastreamento.

Background It studies on the factors that affect the stability of fatty acid profiles from human blood specimens are generally performed by evaluating the effect of a single factor on an individual fatty acid and excluding a considerable amount of data from the total fatty acid profiles. Methods The stability of fatty acids from plasma, serum and red blood cells (RBC) was evaluated in terms of time, temperature, antioxidant and thawing. The fatty acids were methylated and analyzed by gas chromatography. The large volume of data is evaluated simultaneously and automatically by observing an Excel-based colour scale that indicates whether the fatty acid profiles have changed significantly as a result of the storage time (0–52 weeks), temperature (− 20 °C/− 80 °C), butylated hydroxytoluene (BHT) antioxidant (presence/absence) or thawing (single/multiple). Results Fatty acids from plasma were stable at both temperatures (− 20 °C/− 80 °C) regardless of BHT. Fatty acids from serum without BHT degrades faster at − 80 °C than − 20 °C and fatty acids from RBC without BHT degrades faster at − 20 °C than − 80 °C. Addition of BHT inhibits this effect in serum and RBC. Multiple thawing of RBC without BHT demonstrated that polyunsaturated fatty acids were generally more susceptible for changes at − 80 °C than at − 20 °C while BHT prevents partially this effect. Conclusions This study draws attention to the importance of pre-analytical considerations when storing blood samples in biobanks and the need of careful judgments when analyzing fatty acids profiles.

The aim was to study the effect of resveratrol on the interplay of inflammatory signals using three different cell models; a metabolic organ (liver), an endocrine organ (visceral adipose tissue, VAT) and an immune organ (head kidney leukocytes, HKL) following lipopolysaccharide challenge (LPS). Atlantic salmon HKL, liver cells and VAT were isolated from the same fish (n = 5). Each cell type was cultured either as mono-cultures, as co-cultures between HKL-liver cells, liver cells-VAT and HKL-VAT. Triple -cultures included all three tissues. In all cultures of HKL, LPS induced transcription of IL-1β, cox2, tnfα, IL-12, ccattβ and Ahr were significantly inhibited by resveratrol (100, 200 μM). Likewise, in all cultures of liver cells, the LPS induced expression of IL-1β was inhibited by resveratrol (100 and 200 μM). HKL, both mono-cultures and triple-cultures and VAT cocultured with liver cells, showed LPS induced cox2 transcription that was inhibited by resveratrol (100 and 200 μM). In contrast, VAT cultured as triple cultures, resveratrol 200 μM particularly, in the presence of LPS, seemed to increase the expression of IL-1β and ccattβ. Resveratrol did not significantly affect lox5 expression in any culture. HKL and VAT are the main producers of PGE2 in response to inflammatory stimuli. VAT showed high endogenous production of eicosanoids, particularly LTB4 and LTB5. Resveratrol inhibited bot LPS induced and endogenous eicosanoid production. Possible targets of resveratrol, Sirt1 and pAMPK were affected differently in the different cells and tissue studied.

The aim of this study was to compare how different dietary vegetable oil n-6/n-3 ratios affect gene responses involved in inflammation, signaling pathways, fatty acid synthesis and oxidation, oxidation and apoptosis as well as eicosanoid production in salmon head kidney tissues and isolated head kidney leukocytes. Salmon smolts (200 g) were fed four different diets where the main lipid components were palm oil (n-6/n-3 ratio = 0.7), rapeseed oil (n-6/n-3 ratio = 0.9), and soybean oil (n-6/n-3 ratio = 2.4) and a high soybean oil diet with an n-6/n-3 ratio = 4. Both head kidney tissue and leukocytes isolated from head kidneys were sampled from the four diets, but from different fish. Leukocytes isolated from the head kidneys were seeded into culture wells and added lipopolysaccharide (LPS) to induce inflammatory responses. Controls without LPS were included. Head kidney leukocytes and the tissues should have the same phenotype reflecting the different diets. Interleukin 1β (IL-1β) transcription was elevated in head kidney tissue and especially in LPS treated leukocytes isolated from soybean oil (n-6/n-3 = 2.4) fed salmon, which confirmed the suitability of the in vitro model in this experiment. Leukocytes, treated with LPS, and isolated from salmon fed the soybean oil diet (n-6/n-3 = 2.4) also upregulated tumor necrosis factor alpha (tnf-α), cyclooxygenase (cox2), prostaglandin D and E synthase (ptgds, ptges), fatty acyl synthase (fas), 5 and 6 desaturases (5des, 6 des) and a fatty acid translocase protein (cd36) when compared to the other diets. The results suggest that diets with a specific n-6/n-3 ratio influence the transcription of pro-inflammatory genes and may be cross-linked to transcription of selected fatty acid metabolism genes. Salmon fed the palm oil diet (n-6/n-3 = 0.7) showed a lower expression of inflammatory genes. Instead, peroxisome proliferator activated receptor β1 (pparβ1), acyl coenzyme A (aco), apoptosis regulator (bax) and superoxide dismutase (sod) were upregulated in leukocytes in vitro, while head kidney tissue transcription of a dendritic marker (cd83) was lower than measured in tissues from fish fed the other diets. The concentration of LTB4 (10-20 ng/mL) were relatively constant in leukocyte supernatants, all diets. Head kidney leukocytes from soybean oil (n-6/n-3 = 2.4) fed fish produced LPS induced PGE2 (mean 0.5 ng/mL) while leukocytes isolated from palm oil diet (n-6/n-3 = 0.7) secreted very high amounts of LTB5 (50-70 ng/mL). In addition, equal amounts of LPS induced PGE2 and PGE3 (mean 0, 5 ng/mL) were produced, indicating that the n-6/n-3 ratio of this saturated fatty acid may have a specific impact on eicosanoid production in the head kidney of salmon.

O Território do Cerrado brasileiro é constantemente vítima da exploração desenfreada, sem planejamento político e enraizada em uma história de um modelo sócio-econômico ultrapassado e desenvolvimentista, repleto de conflitos, interesses e de atendimento à demanda externa por meio de exportação. O presente trabalho teve como objetivo principal colocar em pauta discussões e reflexões a respeito da sua (in)sustentabilidade sociopolítica, econômica e ambiental, devido às atividades de produção agro-industrial. Baseou-se em revisão bibliográfica especializada e reflexões críticas, contextualizando diversos clássicos da crítica literária e de autores referendados no mundo em se tratando de pesquisas e publicações na área social, política e ambiental. O resultado obtido consiste em concepções críticas e embasadas em uma nova perspectiva de conceber a exploração dos recursos naturais no bioma cerrado brasileiro. Tornam-se relevantes estudos desta magnitude, visto que não só podem oferecer maiores subsídios para trabalhos futuros, mas também contribuir para o desenvolvimento social e econômico local sustentável.

A simple method coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated for the extraction of prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) from human plasma. The extraction protocol consisted of adding formic acid (10 μL) and acetonitrile (140 μL) to human plasma (50 μL) and further injection of the supernatant (25 μL) into the LC–MS/MS. The method was selective for PGE 2 and LTB 4 and the regression models, based on deuterated internal standards (30 ng mL −1 PGE 2 - d 4 and 40 ng mL −1 LTB 4 - d 4 ), were linear over the concentration range 1.0–50.0 ng mL −1 with limits of detection (3 × σ blank ) and quantification (6 × σ blank ) of 0.5 ng mL −1 and 1 ng mL −1 for both eicosanoids. The recovery ranges were 95.1–104.7% for PGE 2 and 86.4–103.2% for LTB 4 . The developed method was successfully implemented on plasma samples from patients before and after exposure to certain anti-inflammatory treatment.

This study assess which pathways and molecular processes are affected by exposing salmon head kidney cells or liver cells to arginine supplementation above the established requirements for growth support. In addition to the conventional mono cultures of liver and head kidney cells, co cultures of the two cell types were included in the experimental set up. Responses due to elevated levels of arginine were measured during inflammatory (lipopolysaccharide/LPS) and non -inflammatory conditions. LPS up regulated the genes involved in polyamine turnover; ODC (ornithine decarboxylase), SSAT (spermidine/spermine-N1-acetyltransferase) and SAMdc (S-adenosyl methionine decarboxylase) in head kidney cells when co cultured with liver cells. Regardless of treatment, liver cells in co culture up regulated ODC and down regulated SSAT when compared to liver mono cultures. This suggests that polyamines have anti-inflammatory properties and that both salmon liver cells and immune cells seem to be involved in this process. The transcription of C/EBP β/CCAAT, increased during inflammation in all cultures except for liver mono cultures. The observed up regulation of this gene may be linked to glucose transport due to the highly variable glucose concentrations found in the cell media. PPARα transcription was also increased in liver cells when receiving signals from head kidney cells. Gene transcription of Interleukin 1β (IL-1β), Interleukin-8 (IL-8), cyclooxygenase 2 (COX2) and CD83 were elevated during LPS treatment in all the head kidney cell cultures while arginine supplementation reduced IL-1β and IL-8 transcription in liver cells co cultured with head kidney cells. This is probably connected to p38MAPK signaling as arginine seem to affect p38MAPK signaling contrary to the LPS induced p38MAPK signaling, suggesting anti-inflammatory effects of arginine/arginine metabolites. This paper shows that co culturing these two cell types reveals the connection between metabolism and inflammation, suggesting different pathways and candidate biomarkers to be further explored.

A Plackett-Burman design was used for screening 12 variables that could affect the mass spectrometry signals of phosphatidylcholines (PtdCho) and proposing optimal fragmentation conditions to study the fragmentation mechanisms of PtdCho standards. Under optimal conditions, three well-established methods for extracting phospholipids from biological samples, specifically liquid-liquid extraction (LLE), normal phase liquid chromatography (NPLC) and high performance thin layer chromatography (HPTLC), were compared in terms of the number of regiospecifically characterized PtdCho by means of liquid chromatography three-stage mass spectrometry (LCMS 3 ). The same number of PtdCho species (35) were characterized by LCMS3 in the LLE and NPLC fractions, whereas only 15 in the HPTLC fraction.

Understanding pathogen recognition and mechanisms in Atlantic cod are of significant importance for both basic research on wild populations and health management in aquaculture. A microarray approach was utilized to search for effects of viral (polyinosinic acid:polycytidylic acid), bacterial (lipopolysaccharide) and polyclonal activator (phytohaemoagglutinin) stress in Atlantic cod head kidney cells. LPS cell activation increased mRNA expression of interleukin 8; interleukin-1β; cyclooxygenase 2; leukocyte derived chemotaxin 2; carboxyl-esterase 2 and environmental biomarker cytochrome P450 1A. Mitogen activated protein kinase p38 and cathepsin F were down regulated by LPS. The antiviral responses induced by double stranded RNA clearly increased transcription of Toll like receptor 3 and interferon stimulating gene 15. The phytohaemoagglutinin response seemed to be more non-specific. Special for the phytohaemoagglutinin induction was the increase in major histocompatibility complex class I. CC chemokine type 2 mRNA expression was increased by phytohaemoagglutinin, lipopolysaccharide and polyinosinic acid:polycytidylic acid, while mitogen activated protein kinase p38 and leukocyte derived chemotaxin 2 were down regulated by phytohaemoagglutinin. Oxidative stress related genes like catalase and glutaredoxin and the anti-apoptotic gene Bcl-2 showed no transcriptional changes compared to control in any of the treatments. Eicosanoids like prostaglandin 2, leukotriene B4 and B5 were constitutively produced by cod head kidney cells in vitro. The most remarkable feature of eicosanoid secretion is the higher production of leukotrienes against prostaglandins, indicating that the lipooxygenase pathway is preferred over the cyclooxygenase pathway. Although there were no significant differences in eicosanoid secretion between the groups, polyinosinic acid:polycytidylic acid showed a clear tendency to increase the levels of leukotriene B4 and B5. This study reveals distinct signatures of bacteria and virus transcriptional responses in cod head kidney cells. In addition, the novel finding that cytochrome P450 1A was upregulated during the antibacterial response indicates a connection between immunity and aryl hydrocarbon receptor activation in Atlantic cod.

Hydrolyzed fish proteins (H-pro) contain high concentrations of free amino acids and low molecular peptides that potentially may benefit fish health. The following study aimed to test whether the water-soluble phase of H-pro could attenuate lipopolysaccharide (LPS) provoked inflammation in liver cells and head kidney cells isolated from Atlantic salmon. Cells were grown as mono cultures or co cultures to assess possible crosstalk between immune cells and metabolic cells during treatments. Cells were added media with or without H-pro for 2 days before LPS exposure and harvested 24 h post LPS exposure. Respective cells without H-pro and LPS were used as controls. H-pro alone could affect expression of proteins directly as H-pro increased catalase protein expression in head kidney- and liver cells, regardless of culturing methods and LPS treatment. Leukotriene B4 (LTB4) production was also increased by H-pro in head kidney cells co cultured with liver cells. H-pro increased LPS induced interleukin 1β (IL-1β) transcription in liver cells co cultured with head kidney cells. All cultures of head kidney cells showed a significant increase in IL-1β transcription when treated with H-pro + LPS. H-pro decreased caspase-3 transcription in liver cells cultured co cultured with head kidney cells. Peroxisome proliferator activated receptor α (PPAR α) was upregulated, regardless of treatment, in liver cells co cultured with head kidney cells clearly showing that culturing method alone affected gene transcription. H-pro alone and together with LPS as an inflammation inducer, affect both antioxidant and inflammatory responses.

It is suggested that power analysis should be formally incorporated into quantification experiment reports in order to substantiate the conclusions derived from experimental data more effectively. The article addressed the issues of power analysis calculation, sample size estimation and appropriate data reporting in quantitative analytical comparisons. Illustrative examples from the literature are used to show how the described power analysis theory could be applied in practice.

Future feed for farmed fish are based on untraditional feed ingredients, which will change nutrient profiles compared to traditional feed based on marine ingredients. To understand the impact of oils from different sources on fish health, n-6 and n-3 polyunsaturated fatty acids (PUFAs) were added to salmon head kidney cells, in a fully crossed design, to monitor their individual and combined effects on gene expression. Exposing salmon head kidney cells to single fatty acids, arachidonic acid (AA) or decosahexaenoic acid (DHA), resulted in down-regulation of cell signaling pathway genes and specific fatty acid metabolism genes as well as reduced prostaglandin E2 (PGE2) secretion. Eicosapentaenoic acid (EPA) had no impact on gene transcription in this study, but reduced the cell secretion of PGE2. The combined effect of AA + EPA resulted in up-regulation of eicosanoid pathway genes and the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), Bclx (an inducer of apoptosis) and fatty acid translocase (CD36) as well as increased cell secretion of PGE2 into the media. Adding single fatty acids to salmon head kidney cells decreased inflammation markers in this model. The combination AA + EPA acted differently than the rest of the fatty acid combinations by increasing the inflammation markers in these cells. The concentration of fatty acid used in this experiment did not induce any lipid peroxidation responses.

An overview on experimental design in quantification experiments using the internal standard technique is described. Particular emphasis is given to the modelling of the relationship analyte/internal standard and the estimation of the optimal amount of internal standard to be used throughout the analytical concentration range by using a uniform shell design with a minimum number of seven experiments. The described approach is demonstrated in the analysis of cholesterol in various reference materials by gas chromatography (GC), using alpha-5 cholestan as internal standard. The outlined approaches can be applied in the determination of any kind of analyte.

The production of prostaglandins (PGE2, PGE3) and leukotrienes (LTB4, LTB5) in salmon head kidney cell cultures, exposed to different combinations of 20:4ω-6, 20:5ω-3 and 22:6ω-3 polyunsaturated fatty acids (PUFAs), was evaluated by means of a two level factorial design and LC-MS/MS. The method was selective for the pro- and anti-inflammatory analytes and their corresponding stable-isotope labelled internal standards. The regression models were linear over the concentration range 0.5-150 ng/ml with limits of detection of 0.25 ng/ml and quantification of 0.40 ng/ml for the analysed metabolites. The recovery ranged from 78 to 107% for prostaglandins and 73 to 115% for leukotrienes. The analysis of the samples exposed to different combinations of PUFAs revealed that the presence of single ω-3 PUFAs brought an enhancement of the metabolites from the lipooxygenase pathway, specially LTB4, and a reduction of the metabolites from the cyclooxygenase pathway (PGE2 and PGE3), while the two-term interactions generated the opposite effect (high concentration of prostaglandins and low concentrations of leukotrienes). To our knowledge, this is the first implementation of a fully crossed design for investigating the impact of ω-6 and ω-3 PUFAs on the production of eicosanoids not only through their individual but also through their combined effects on Atlantic salmon head kidney cells.

Primary head kidney leukocytes from Atlantic cod were isolated to evaluate the use of arachidonic acid and eicosapentaenoic acid by cyclooxygenases and the production of prostaglandins E 2 and E 3 . The expression of cyclooxygenase genes and selected interleukin genes like Interleukin 1β, Interleukin 6, interleukin 8 and interleukin 10 were monitored. Increasing concentrations of eicosapentaenoic acid and arachidonic acid in equal amounts increased cyclooxygenase2 transcription as well as cell secretion of prostaglandin E 2 . Even though the ratio of the two fatty acids was 1:1, the ratio between prostaglandin E 2 and E 3 was 50:1. The addition of arachidonic acid alone increased prostaglandin E 2 secretion but did not induce cyclooxygenase2 transcription. However, when the concentration of eicosapentaenoic acid was increased, maintaining arachidonic acid constant, both prostaglandin E 3 and prostaglandin E 2 production was induced and the prostaglandin E 2 production was higher than in cell cultures only added arachidonic acid. An up-regulation of cyclooxygenase2 transcription was also observed. The addition of the two fatty acids also affected the immune response by alteration of leukocytic cytokines gene expression. According to our results the Cyclooxygenase in cod seem to prefer arachidonic acid as substrate. Therefore, we suggest that the shift from marine oils (rich in n-3 fatty acids) to plant oils (higher in n-6 fatty acids) in the diet of commercially reared Atlantic cod could have negative effects on the whole organism through the increase in the production of prostaglandins belonging to those derived from n-6 fatty acids.

A protocol for the analysis of multiple prostaglandins and leukotrienes in cell culture media by using multiple internal standards was validated. A two-factor Doehlert design was used to determine the behaviour of the relationship analyte/internal standard (namely: PGE 2 /PGE 2 - d 4 , PGE 3 /PGE 2 - d 4 , LTB 4 /LTB 4 - d 4 and LTB 5 /LTB 4 - d 4 ) and to select the optimal amounts of deuterated internal standards for quantifying simultaneously the prostaglandins and leukotrienes in cell culture media by LC–MS/MS. The selection of optimal amounts of internal standards was based on mathematical models that allow visualizing concentration regions where the response factors remain constant over a wide range of analytical concentrations. The linearity of the calibration curves for each analyte at the optimal levels suggested by the mathematical models was statistically confirmed by means of the ratio lack-of-fit to pure error. The validated protocol was successfully applied in the simultaneous quantification of pro- and anti-inflammatory eicosanoids in stimulated cod head kidney cell culture media. The two-factor Doehlert design has permitted to estimate the experimental response as a function of six variables (PGE 2 , PGE 3 , LTB 4 , LTB 5 , PGE 2 - d 4 and LTB 4 - d 4 ) which represents a substantial reduction of resources, time and experiments of approximately 84% (7 × 3 experiments) when compared with the full six-factor Doehlert design (43 × 3 experiments).

OBJETIVO: A haste intramedular (HIM) é o padrão ouro no tratamento das fraturas diafisárias da tíbia. Uma das complicações frequentes é a dor no joelho após o procedimento. MÉTODOS: Vias alternativas, como a suprapatelar para a introdução da haste como opção para evitar a dor no joelho no período pós-operatório tardio, têm sido estudadas. A questão é se nesta nova via ocorre alguma lesão às estruturas intra-articulares do joelho. RESULTADOS: Este estudo analisa a via suprapatelar e o risco para as estruturas adjacentes através da sua reprodução em 10 joelhos de cinco cadáveres. CONCLUSÃO: Foi visto facilidade para a localização do ponto de entrada por esta via, lesando apenas a gordura de Hoffa. Em três dos nossos casos houve lesão da superfície condral, sendo um obstáculo de difícil transposição e indicando necessidade de fabricação de material específico para minimizar a lesão das estruturas intra-articulares por essa via. OBJECTIVE: Intramedullary nails are the gold standard for treating tibial shaft fractures. Knee pain is a frequent complication after the procedure. Alternative routes such as the suprapatellar approach for nail insertion are seen as an option for avoiding late postoperative knee pain. The question is whether this approach might give rise to any injury to intra-articular structures of the knee. METHODS: This study analyzed the suprapatellar approach and the risk to adjacent structures by reproducing it in 10 knees of five cadavers. RESULTS: This approach was seen to make it easy to locate the entry point, with lesions only occurring in the Hoffa fat. In three of our cases, there were lesions of the chondral surface, which is an obstacle that is difficult to overcome. CONCLUSION: There is a need to develop specific material to minimize injury to intra-articular structures when using this route.

The principles of the Doehlert uniform shell designs (aka Doehlert designs) and their importance in the context of chromatography are discussed. The confidence of different models generated by Doehlert designs is studied by means of the uncertainty of the experimental points. The article provides an overview of analytical applications in chromatography with focus on single and coupled techniques and also discusses some reported blunders regarding Doehlert designs.

Kine Gregersen1,2, Jørgen Valeur1,3, Kristine Lillestøl1,3, Livar Frøyland2, Pedro Araujo2, Gülen Arslan Lied1,3, Arnold Berstad1,31Institute of Medicine, University of Bergen, 2National Institute of Nutrition and Seafood Research; 3Department of Medicine, Section for Gastroenterology, Haukeland University Hospital, Bergen, NorwayBackground: Food hypersensitivity is commonly suspected, but seldom verified. Patients with subjective food hypersensitivity suffer from both intestinal and extraintestinal health complaints. Abnormalities of the enterochromaffin cells may play a role in the pathogenesis. The aim of this study was to investigate enterochromaffin cell function in patients with subjective food hypersensitivity by measuring serum chromogranin A (CgA) and 5-hydroxytryptamine (5-HT, serotonin) in gut lavage fluid.Methods: Sixty-nine patients with subjective food hypersensitivity were examined. Twenty-three patients with inflammatory bowel disease and 35 healthy volunteers were included as comparison groups. CgA was measured in serum by enzyme-linked immunosorbent assay. Gut lavage fluid was obtained by administering 2 L of polyethylene glycol solution intraduodenally. The first clear fluid passed per rectum was collected and 5-HT was analyzed by liquid chromatography tandem mass spectrometry.Results: Serum levels of CgA were significantly lower in patients with subjective food hypersensitivity than in healthy controls (P = 0.04). No differences were found in 5-HT levels in gut lavage fluid between patients with subjective food hypersensitivity and the control groups. There was no correlation between serum CgA and gut lavage 5-HT.Conclusion: Decreased blood levels of CgA suggest neuroendocrine alterations in patients with subjective food hypersensitivity. However, 5-HT levels in gut lavage fluid were normal.Keywords: food hypersensitivity, chromogranin A, serotonin, gut lavage fluid, liquid chromatography

Objective: Intramedullary nails are the gold standard for treating tibial shaft fractures. Knee pain is a frequent complication after the procedure. Alternative routes such as the suprapatellar approach for nail insertion are seen as an option for avoiding late postoperative knee pain. The question is whether this approach might give rise to any injury to intra-articular structures of the knee. Methods: This study analyzed the suprapatellar approach and the risk to adjacent structures by reproducing it in 10 knees of five cadavers. Results: This approach was seen to make it easy to locate the entry point, with lesions only occurring in the Hoffa fat. In three of our cases, there were lesions of the chondral surface, which is an obstacle that is difficult to overcome. Conclusion: There is a need to develop specific material to minimize injury to intra-articular structures when using this route.

Method validation may be regarded as one of the most well-known areas in analytical chemistry as is reflected in the substantial number of articles submitted and published in peer review journals every year. However, some of the relevant parameters recommended by regulatory bodies are often used interchangeably and incorrectly or are miscalculated, due to few references to evaluate some of the terms as well as wrong application of the mathematical and statistical approaches used in their estimation. These mistakes have led to misinterpretation and ambiguity in the terminology and in some instances to wrong scientific conclusions. In this article, the definitions of various relevant performance indicators such as selectivity, specificity, accuracy, precision, linearity, range, limit of detection, limit of quantitation, ruggedness, and robustness are critically discussed with a view to prevent their erroneous usage and ensure scientific correctness and consistency among publications.

A simplified method for quantitative analysis of fatty acids in various matrices by gas chromatography is proposed as an alternative to the conventional method and the variables of the protocol examined to optimize the processing conditions. The modified method involves direct methylation of fatty acids in homogenized samples with boron trihalide (BF(3) or BCl(3) in methanol) followed by extraction with hexane. The addition of hexane to the reaction mixture after the methylation process can enhance the efficiency of fatty acid methylation and is critical for those samples that contain high levels of triglycerides. A mechanism underlying this effect is proposed.

A new extraction method has been developed for the extraction of prostaglandin E(2) (PGE(2)) from human plasma of patients suffering chronic inflammatory disorders. The extraction solvents were optimised systematically and simultaneously by using a central composite design. The optimised method involves precipitation of the protein fraction, centrifugation, evaporation and dissolution of the supernatant in the mobile phase, screening to confirm the presence of the analyte, and quantification of the positive samples by liquid chromatography tandem ion-trap mass spectrometry. Tandem mass spectrometry in negative mode was performed by isolating and fragmenting the ion [PGE(2)-H](-) signal m/z 351. Identification and quantification was carried out by extracting the ion fragment chromatograms at 333, 315 and 271 m/z. The quantitative determination was linear for the low nanogram (1-50 ng/ml) and upper picogram (400-1000 pg/ml) range studied, using 15 and 0.5 ng/ml of internal standard, respectively. The lower limit of detection was 2.5 pg for an injection volume of 25 microl. The optimised extraction method showed high reproducibility (coefficients of variation