Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk / Purificação parcial da enzima isomerase do ácido linoleico da bactéria Lactobacillus paracasei isolada do leite

Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 µg.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

O Ácido Linoleico Conjugado (CLA) tem chamado a atenção de diversos pesquisadores, principalmente aquele de origem microbiana, devido à sua importância biológica para o consumidor. O presente estudo visa extrair a enzima LA Isomerase da bactéria Lactobacillus paracasei do leite e usar essa enzima na produção de CLA. Meios seletivos, incluindo MRS e MRS-Dagatose, foram usados no isolamento de cepas locais. Os isolados bacterianos selecionados foram testados quanto à sua capacidade de produzir a enzima LA-Isomerase. Foi selecionado o isolado com alta atividade enzimática. Após a extração e purificação parcial da enzima, as condições ideais para a produção de ácido graxo conjugado foram estudadas e os produtos da reação foram identificados usando a tecnologia GC-MS. Verificou-se que 11 isolados possuem capacidade de produzir CLA em diferentes concentrações. O isolado H1 apresentou a maior produção de ácido graxo conjugado, na concentração de 120,45 g.ml-1, e este isolado foi selecionado como fonte para extração enzimática. A atividade enzimática do extrato bruto e parcialmente purificado com sulfato de amônio foi estimada por métodos de coloração em comprimento de onda de 233 nm. O efeito das condições ótimas (pH, temperatura, concentração de ácido linoleico e concentração de enzima) no produto CLA foi estudado usando a enzima LA Isomerase parcialmente purificada e as condições ótimas para produção foram 6,5, 45 °C, 100 µg.ml-1 e 0,7 mL, respectivamente. A técnica de GC-MS mostrou a presença de uma série de produtos de reação que são isômeros do ácido linoleico conjugado (C9T11, T9T12, T10C12) com diferentes concentrações.

Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk

Abstract Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 g.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

Resumo O Ácido Linoleico Conjugado (CLA) tem chamado a atenção de diversos pesquisadores, principalmente aquele de origem microbiana, devido à sua importância biológica para o consumidor. O presente estudo visa extrair a enzima LA Isomerase da bactéria Lactobacillus paracasei do leite e usar essa enzima na produção de CLA. Meios seletivos, incluindo MRS e MRS-Dagatose, foram usados no isolamento de cepas locais. Os isolados bacterianos selecionados foram testados quanto à sua capacidade de produzir a enzima LA-Isomerase. Foi selecionado o isolado com alta atividade enzimática. Após a extração e purificação parcial da enzima, as condições ideais para a produção de ácido graxo conjugado foram estudadas e os produtos da reação foram identificados usando a tecnologia GC-MS. Verificou-se que 11 isolados possuem capacidade de produzir CLA em diferentes concentrações. O isolado H1 apresentou a maior produção de ácido graxo conjugado, na concentração de 120,45 g.ml-1, e este isolado foi selecionado como fonte para extração enzimática. A atividade enzimática do extrato bruto e parcialmente purificado com sulfato de amônio foi estimada por métodos de coloração em comprimento de onda de 233 nm. O efeito das condições ótimas (pH, temperatura, concentração de ácido linoleico e concentração de enzima) no produto CLA foi estudado usando a enzima LA Isomerase parcialmente purificada e as condições ótimas para produção foram 6,5, 45 °C, 100 g.ml-1 e 0,7 mL, respectivamente. A técnica de GC-MS mostrou a presença de uma série de produtos de reação que são isômeros do ácido linoleico conjugado (C9T11, T9T12, T10C12) com diferentes concentrações.

Factores asociados al uso, disposición y eliminación de elementos de protección personal y otros insumos de la clí­nica veterinaria de grandes animales / Factors associated with the usage, disposal and elimination of personal protective equipment and other veterinary inputs in large animal practice

Se efectuó un estudio transversal en 330 Médicos Veterinarios de grandes animales en la Provincia de Santa Fe, Argentina para estimar: a) frecuencia de uso y disposición final de elementos de protección personal e insumos para la práctica clínica, b) asociaciones con características sociodemográficas de los entrevistados. El análisis de los datos incluyó: χ2, t de Student, test exacto de Fisher y correlación de Pearson. El 30,8% había sido diagnosticado con zoonosis (brucelosis 27,2%). El descarte de los guantes a campo fue la forma de disposición más frecuente. Uno de cada cinco profesionales los eliminaba junto a la basura domiciliaria y cuatro de cada 10 hacía lo propio con las agujas descartables. El género y la edad de los profesionales estuvieron significativamente asociados con la utilización, disposición y eliminación de los insumos veterinarios luego de diversas prácticas profesionales.

A cross-sectional study was carried out in 330 veterinarians working on large animal practice in the Province of Santa Fe, Argentina to estimate: a) frequency of usage and final disposal of personal protective equipment and supplies for clinical practice, b) associations with socio-demographic characteristics of the interviewees. Data analysis included: χ2, Student's t test, Fisher's exact test and Pearson's correlation coefficient. Thirty percent had been diagnosed with zoonosis (brucellosis: 27.2%). Discarding gloves on the farm was the most frequent means for disposal. One out of five practitioners eliminated the gloves along with household wastes, while four out of 10 had a similar behavior on regards of disposable needles. Practitioners´ gender and time after graduation were significantly associated with usage, disposal and elimination of veterinary inputs following various professional practices.

Structure of the Conjugated Linoleic Acid-Paclitaxel Conjugate Self-Assembled Nanoparticles / 中国药学杂志

OBJECTIVE: To prepare, the conjugated linoleic acid-paclitaxel conjugate self-assembled nanoparticles (CLA-PTX NPs) by nanoprecipitation. METHODS: The Dynamic light scattering, nuclear magnetic resonance spectroscopy, raman spectroscopy, fourier transform infrared spectroscopy and nitrogen element distribution of CLA-PTX NPs were studied. RESULTS: The hydroxyl groups (C-4 and C-10 of PTX) and the acetyl groups (C-1 and C-7 of PTX) were on the surface of CLA-PTX NPs, CLA carbon chain, the benzene ring (C-2 and C-3' of PTX) and the amide bond (C-3' of PTX) were inside the CLA-PTX NPs. CONCLUSION: It is speculated that the self-assembly of CLA-PTX is that the non-polar CLA carbon chain spontaneously aggregates inward due to hydrophobic interaction, and the hydrophilic oxygen-containing groups of PTX (hydroxyl group and carbonyl group) are on the surface of the nanoparticle to form nanoparticles.

Risk Assessment of "Other Substances" – Conjugated Linoleic Acids

The Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) has, at the request of the Norwegian Food Safety Authority (Mattilsynet; NFSA), assessed the risk of "other substances" in food supplements and energy drinks sold in Norway. VKM has assessed the risk of doses given by NFSA. These risk assessments will provide NFSA with the scientific basis for regulating the addition of "other substances" to food supplements and other foods. "Other substances" are described in the food supplement directive 2002/46/EC as substances other than vitamins or minerals that have a nutritional or physiological effect. It is added mainly to food supplements, but also to energy drinks and other foods. VKM has not in this series of risk assessments of "other substances" evaluated any potential beneficial effects from these substances, only possible adverse effects. The present report is a risk assessment of conjugated linoleic acids (CLAs), and is based on previous risk assessments of CLAs and articles retrieved from literature searches. According to information from the NFSA, CLAs are ingredients in food supplements sold in Norway, and NFSA has requested a risk assessment of the following doses of CLAs in food supplements: 3.0, 3.25 and 3.5 g/day. The daily intakes in Norway of CLAs range between 20 and 170 mg (MoBa 2008, version 4). The CLAs are mostly studied in overweight and obese subjects because of their claimed effects to reduce body weight. CLAs constitute a group of isomeric fatty acids mostly produced by bacterial fermentation in the gut. In the human diet, meat (mainly isomers c9,t11 and t10,c12) and dairy products (mainly isomer c9,t11) are main sources of CLAs. The various isomers may have different metabolic effects. In the food supplements evaluated by EFSA (EFSA, 2010 a; EFSA, 2010 b; EFSA, 2012), Clarinol® and Tonalin®, the t10,c12 and the c9,t11 isomers are present in about equal proportions. In research articles not all authors are consistent in reporting what they have studied, so in this report we sometimes do not distinguish between these isomers; hence they are referred collectively to as CLAs if it is not specified. Most of the cited studies have tested supplemental CLAs in doses of about 3.5 g/day, but ranging from 0.7 to 6.0 g/day. In most of the randomised controlled trials (RCTs) there have been no significant differences in adverse effects between the placebo and CLA-groups. Concerns about indications of an unfavourable effect on biomarkers of lipid- and carbohydrate metabolism in obese men with metabolic syndrome as well as unfavourable effect on antioxidant status; increased markers of oxidative stress after consumption of supplemental CLAs have been reported in previous studies. No clear dose-response effects have been found. It is concluded that supplemental CLAs may impair lipid- and carbohydrate metabolism in obese men with the metabolic syndrome. Intake of supplemental CLAs by lactating women may reduce fat content in breastmilk, and intake of supplemental CLAs by pregnant women may reduce birth weight and –length among their offsprings. Only one randomised controlled trial has included children (6-10 years). These children were all overweight or obese, subjects likely to have a different CLA-metabolism/-effect than normal-weight subjects. VKM considers that current data are too incomplete to evaluate any doses of CLAs for children and adolescents. There are few long-term studies, and adverse health effects were not primary outcomes in these studies. Based on available data no conclusions can be drawn for supplemental use of CLAs longer than six months. VKM concludes that: In adults (≥18 years), the specified doses 3.0, 3.25 and 3.5 g/day CLAs in food supplements are considered to be unlikely to cause adverse health effects if used for up to six months. CLA may cause lipid- and carbohydrate disturbances in obese men with metabolic syndrome as well as in overweight/obese subjects with type 2 diabetes. Use of CLA supplements in lactating and pregnant women may cause reduction in milk production and in the content on milk fat and cause decreased birth weight andlength in their off-springs. No conclusion can be made for children and adolescents. Data are insufficient to conclude regarding use of CLAs for more than six months.

Single Incision Laparoscopic Appendectomy for Management of Complicated Appendicitis: Comparison between Single-Incision and Conventional

PURPOSE: Single incision laparoscopic appendectomy (SILA) is a widely used surgical procedure for treatment of appendicitis with better cosmesis. However, many surgeons generally tend to choose conventional multiport laparoscopic appendectomy regarding with complicated appendicitis. The aim of this study is to demonstrate the safety and feasibility of SILA for treatment of complicated appendicitis by comparison with 3-ports conventional laparoscopic appendectomy (CLA). METHODS: Retrospective chart review of patients diagnosed appendicitis at single hospital during January 2015 to May 2017 collected 500 patients. Among 134 patients with complicated appendicitis, we compared outcomes for 29 patients who got SILA and 105 patients who got CLA. RESULTS: 179 and 321 patients were treated by SILA and CLA, respectively. 134 (26.8%) patients were treated for complicated appendicitis, 29 patients by SILA and 105 patients by CLA, respectively. There was no case converted to open or added additional trocar in both groups. There were no differences in demographics with regard to age, sex, body mass index (BMI), and American society of anesthesiologists (ASA) scores. There was no difference in mean operating time (58.97±18.53 (SILA) vs. 57.57±21.48 (CLA), p=0.751). The drain insertion rate (6.9% vs 37.1%, p=0.001) and the length of hospital stay (2.76±1.41 vs. 3.97±2.97, p=0.035) were lower in SILA group with significance. There was no significant difference in the rate of surgical site infection (6.9% vs. 6.7%, p=1.000). CONCLUSION: This study demonstrates that SILA is a feasible and safe procedure for treatment of complicated appendicitis.

Efeito da adição do ácido linoleico conjugado no cultivo in vitro de embriões F1 Holandês x Zebu na sobrevivência pós-vitrificação / Effect of conjugated linoleic acid addition in in vitro culture medium in F1 Holstein X Zebu embryo survival post vitrification

Avaliou-se o efeito da adição do ácido linoleico conjugado (CLA) ao meio de cultivo in vitro na viabilidade pós-vitrificação de embriões F1 Holandês x Zebu. Foram utilizados três meios de cultivo: controle (n=340 oócitos): meio SOF e soro fetal bovino (SFB), sem o CLA; SFB+CLA (n=359 oócitos): meio SOF, SFB e CLA; CLA (n=339 oócitos): meio SOF e CLA, sem o SFB. Todos os blastocistos produzidos foram submetidos à vitrificação, pelo método de Open Pulled Straw. Quinze blastocistos de cada tratamento foram fixados para quantificação lipídica por coloração com Sudan Black B. Para avaliar a viabilidade embrionária, foi observada a capacidade de reexpansão e eclosão pós-aquecimento dos embriões (controle=27; SFB+CLA=30; CLA=17). Foram realizadas transferências em um ou dois embriões por receptora para avaliação da sobrevivência in vivo: T1 [receptoras que receberam um blastocisto (n=17 embriões, sendo controle=5, SFB+CLA=6 e CLA=6)]; T2 [receptoras que receberam dois blastocistos, (n= 54 embriões, sendo controle=18, SFB+CLA=14 e CLA=22)]. Não houve diferença nas taxas de clivagem (62,1%; 74,0%; 74,0% para controle; SFB+CLA; CLA, respectivamente), produção de blastocistos em relação aos clivados (59,7%; 47,7%; 38,3% para controle; SFB+CLA; CLA, respectivamente) e produção de blastocistos em relação ao total de oócitos (37,1%; 35,4%; 28,3% para controle; SFB+CLA; CLA, respectivamente) (P>0,05). Houve diminuição de gotículas lipídicas nos embriões cultivados em meio suplementado com CLA em relação aos embriões cultivados na presença do SFB e na ausência do CLA (P<0,05). A taxa de reexpansão foi maior no grupo controle (70,4%) em relação ao CLA (47,1%) e menor no grupo SFB+CLA (43,3%) (P<0,05). O CLA foi eficaz em reduzir a deposição de lipídeos intracitoplasmáticos nas células embrionárias, porém não houve diferença de viabilidade após a desvitrificação dos embriões.(AU)

The effect of adding conjugated linoleic acid (CLA) to the culture media on the viability after cryopreservation of F1 Holstein X Zebu embryos was evaluated. Three different culture media were tested: control (n = 340 oocytes): SOF medium and fetal bovine serum (FBS) without the CLA; FBS + CLA (n = 359 oocytes): SOF, FBS and CLA; CLA (n = 339 oocytes): SOF and CLA without the FBS. The produced blastocysts were subjected to vitrification, by the Open Pulled Straw method. Fifteen blastocysts per treatment were fixed for lipid quantification by staining with Sudan Black B. Embryo re-expansion and hatching capability were used to assess viability (control = 27; FBS + CLA = 30; CLA = 17). Transfers of one or two embryos to recipients were performed to evaluate in vivo survival: T1 [recipients that received one blastocyst (n=17 embryos, Control=5, FBS+CLA=6 and CLA=6)]; T2 [recipients that received two blastocysts (n =54 embryos, Control=18, FBS+CLA=14 and CLA=22)]. There was no difference in cleavage rate (62.1%; 74%; 74% for Control; FBS + CLA, CLA, respectively), blastocyst production in relation to the cleaved structures (59.7%; 47.7%; 38 3% for Control; FBS + CLA, CLA, respectively) and blastocyst production relative to the total oocytes (37.1%, 35.4%, 28.3% for Control; FBS + CLA, CLA, respectively) between treatments (P> 0.05). A reduction of lipid droplets was observed in embryos cultured in medium supplemented with CLA compared to embryos cultured in the FCS in the absence and presence of CLA (P <0.05). The reexpansion rate was higher in the Control group (70.4%) compared to the CLA (47.1%) and lowest for FBS+CLA (43.3%) (P<0.05). The hatching rates were similar among treatments, 42.1%; 23.1%; 25% for control; SFB + CLA; CLA respectively (P>0.05). Only one pregnancy was observed in early and confirmatory diagnosis, as the result of a Control group embryo transfer. Although embryos cultured with CLA have shown smaller intracytoplasmic lipid content, no difference was observed in viability following vitrification between treatments.(AU)

Qingfei Xiaoyan Wan, a traditional Chinese medicine formula, ameliorates Pseudomonas aeruginosa-induced acute lung inflammation by regulation of PI3K/AKT and Ras/MAPK pathways

Gram-negative pathogen-induced nosocomial infections and resistance are a most serious menace to global public health. Qingfei Xiaoyan Wan (QF), a traditional Chinese medicine (TCM) formula, has been used clinically in China for the treatment of upper respiratory tract infections, acute or chronic bronchitis and pulmonary infection. In this study, the effects of QF on Pseudomonas aeruginosa-induced acute pneumonia in mice were evaluated. The mechanisms by which four typical anti-inflammatory ingredients from QF, arctigenin (ATG), cholic acid (CLA), chlorogenic acid (CGA) and sinapic acid (SPA), regulate anti-inflammatory signaling pathways and related targets were investigated using molecular biology and molecular docking techniques. The results showed that pretreatment with QF significantly inhibits the release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and RANTES), reduces leukocytes recruitment into inflamed tissues and ameliorates pulmonary edema and necrosis. In addition, ATG was identified as the primary anti-inflammatory agent with action on the PI3K/AKT and Ras/MAPK pathways. CLA and CGA enhanced the actions of ATG and exhibited synergistic NF-κB inactivation effects possibly via the Ras/MAPK signaling pathway. Moreover, CLA is speculated to target FGFR and MEK firstly. Overall, QF regulated the PI3K/AKT and Ras/MAPK pathways to inhibit pathogenic bacterial infections effectively.

Associação entre o óleo de soja e o óleo de peixe na dieta de cabras em lactação como estratégia para melhorar o perfil de ácidos graxos do leite / Association between soybean oil and fish oil in the diet of lactating goats as a strategy to improve milk fatty acids profile

Os objetivos neste experimento foram avaliar os efeitos da associação entre o óleo de soja e o óleo de peixe na dieta de cabras em lactação sobre o consumo de matéria seca e de nutrientes, a variação do peso corporal, a produção e composição do leite, assim como o perfil de ácidos graxos. As cabras foram alocadas em baias individuais, onde receberam dieta composta por 50% de feno de "coastcross" e 50% de concentrado. Foram utilizadas nove cabras mestiças Boer x Saanen multíparas, distribuídas em três quadrados latinos 3 X 3. O experimento teve duração de 51 dias, divididos em três períodos de 17 dias, sendo os 13 primeiros dias para adaptação dos animais às dietas e os 4 dias subsequentes para colheita de amostras e de dados. Os tratamentos experimentais foram: a) dieta controle (CT), sem adição de óleo; b) dieta contendo 3% de óleo de soja (OS); e c) dieta contendo 2,5% de óleo de soja + 0,5% de óleo de peixe (OS+P). A inclusão dos óleos reduziu (P<0,05) o consumo de matéria seca, no entanto aumentou (P<0,05) a eficiência alimentar dos animais, sem afetar (P>0,05) a produção de leite. Houve efeito (P<0,05) da dieta no perfil de ácidos graxos do leite, sendo que ambos os tratamentos com adição de óleo elevaram as concentrações de ácidos graxos de cadeia média e longa, reduzindo os de cadeia curta. O tratamento com a combinação do óleo de soja com o óleo de peixe foi o que promoveu os maiores aumentos na concentração de ácido vacênico (398%), rumênico (352%) e de CLA total (341%) no leite. Os resultados permitem concluir que a suplementação lipídica elevou a eficiência alimentar dos animais e que o fornecimento de óleo de soja em combinação ao óleo de peixe aumentou a concentração no leite dos ácidos graxos benéficos à saúde humana.

The aims in this experiment were to determine the effects of the association between soybean and fish oils on dry matter intake (DMI) and nutrient intake, body weight change, milk production and composition and milk fatty acid profile of dairy goats. The animals were housed in tie stalls and fed a 50% of coastcross hay and 50% concentrate diet. Nine multiparous crossbred Boer x Saanen goats were assigned in tree 3 X 3 Latin Squares. The experimental period lasted 51 days; divided into three periods of 17 days, being the first nine days used to adapt goats to diets and the 4 other days for data collection. Experimental diets were: a) control diet (CT) without oil; b) control diet supplemented with 3% of soybean oil (SO); and c) control diet supplemented with 2.5% of soybean oil plus 0.5% of fish oil (SO+F). DMI was negatively influenced by oil addition. However, feed efficiency was higher in diets with oils, maintaining similar milk production (P>0.05 for all comparisons). The supply of oils changed milk fatty acids profile (P<0.05), increasing the concentrations of medium and long-chain fatty acids and reducing short-chain. Milk from goats fed the SO+F diet had higher concentration of vaccenic (398%), rumenic acid (352%) and total CLA (341%). The results indicate that the oil supply increased the feed efficiency of goats, and that the combination of soybean and fish oils caused a higher elevation of fatty acids considered to have health benefits.

Perfil de ácidos graxos do leite de vacas holandês x Gir em pastagem de capim-marandu suplementado com quantidades crescentes de grão de soja tostado / Milk fatty acid profile of holstein x Gyr cows grazing on marandugrass supplemented with increasing levels of roasted soybeans

O experimento foi realizado na Embrapa Gado de Leite, em delineamento quadrado latino (QL) 4x4, com o objetivo de avaliar o perfil de ácidos graxos (AG) e os índices de qualidade nutricional da gordura do leite de vacas Holandês x Gir (n = 16) sob pastejo em Brachiaria brizantha cv. Marandu suplementada com concentrado (6kg/vaca/dia) contendo 0; 1,3; 2,6 e 3,9kg (base matéria natural) de grão de soja tostado (GST). Foram observados decréscimos lineares (P<0,0001) nas concentrações e nas secreções dos AG láurico, mirístico e palmítico e dos AG de cadeia ímpar linear e ramificada, bem como incrementos lineares (P<0,0001) nas concentrações e secreções dos AG α-linolênico, linoleico, oleico e esteárico na gordura do leite, com a inclusão do GST no concentrado. As concentrações e as secreções dos AG vacênico e rumênico apresentaram comportamento quadrático (P<0,001) em resposta à inclusão de quantidades crescentes de GST na dieta. As alterações observadas no perfil de AG do leite com o aumento da inclusão de GST no concentrado resultaram em reduções lineares (P<0,0001) nos índices de aterogenicidade e de trombogenicidade e em incrementos lineares (P<0,0001) nas relações entre AG hipo:hipercolesterolêmicos e entre AG ω-6:ω-3 da gordura do leite. Os resultados deste estudo mostraram que a inclusão de quantidades crescentes de GST na dieta de vacas Holandês x Gir pastejando capim-marandu apresenta potencial para a secreção de leite com gordura enriquecida com ácidos graxos benéficos à saúde humana.

The experiment was carried out at Embrapa Dairy Cattle in order to evaluate the milk fatty acid (FA) composition and indices of nutritional quality of milk fat from Holstein x Gyr cows (n = 16) grazing on Brachiaria brizanta cv. Marandu supplemented with concentrate (6 kg/cow/d) containing 0, 1.3, 2.6 and 3.9 kg (as-fed basis) of roasted soybeans (RS). A linear decrease (P<0.0001) in the content and secretion of lauric, myristic and palmitic FA, and odd- and branched-chain FA was observed, whereas the content and secretion of α-linolenic, linoleic, oleic, and stearic FA in milk fat increased linearly (P<0.0001) as the amount of RS increased in the diet. The milk fat content and secretion of vaccenic and rumenic FA increased quadratically (P<0.001) in response to dietary RS level. As a consequence of the above-mentioned changes in milk FA composition, both atherogeniticy and trombogenicity indices of milk fat were reduced linearly (P<0.0001), whereas hypo:hypercholesterolemic and ω-6:ω-3 FA ratios increased linearly (P<0.0001) in milk fat from cows fed increasing levels of RS. The results showed that dietary supplementation with increasing amounts of RS have the potential to secrete fat milk enriched fatty acids beneficial to human health.

Comparison of MAST-CLA System Results and Allergen Detection Rates between Acute and Chronic Urticaria Patients in Gangwon, Yeongseo Province, Korea / 대한피부과학회지

BACKGROUND: The multiple allergosorbent test chemiluminescent assay (MAST-CLA) is a simple system with high sensitivity and specificity for measuring total and allergen-specific IgE antibodies in patients with allergic skin diseases such as urticaria. OBJECTIVE: The purpose of this study was to evaluate total IgE class, allergen frequencies, and their correlations in MAST-CLA in acute and chronic urticaria. METHODS: We performed a retrospective study using medical documents of 2,028 urticaria patients (average age= 34.85 years) who visited Yonsei Wonju Severance Christian Hospital, Yeongseo Province, Korea, between 2003 and 2012. Positive rates of specific IgE and total serum IgE levels in acute and chronic urticaria patients were analyzed. RESULTS: Among the total 2,028 patients, 1,263 (62.3%) had acute urticaria, and 765 (37.7%) had chronic urticaria. The number of patients with higher than class 2 level of serum total IgE was 1,496 (73.8%): 964 (76.3%) of the acute urticaria group and 532 (69.5%) of the chronic urticaria group. More than half of the patients (1,048; 51.7%) showed a positive reaction to at least one allergen-specific IgE. The positive rate of acute urticaria was 53.0% and chronic urticaria 49.5%. In both acute and chronic urticaria, the most commonly detected allergen was Dermatophagoides farinae, followed by D. pterony and house dust. Most patients showed lower positive reactions to food allergens. Peach was the most common food allergen in both groups. Of the patients who showed positive total IgE, 40.4% did not react to any allergen-specific IgE. Also, 26.3% of the patients who were negative for total IgE showed a positive reaction to more than one allergen-specific IgE. The level of total IgE and the number of positive allergen-specific IgE responses were positively correlated in both acute and chronic urticaria patients. CONCLUSION: Our results showed that the positive rate of total IgE was higher in acute urticaria patients than in chronic urticaria patients. Both acute and chronic urticaria patient groups showed the highest positive rates for D. farinae. Positive rates for response to food allergens, such as soybean, chicken, yeast, tuna, and salmon, were lower in the chronic urticaria group. For inhalant allergens, the positive rates of response to white oak and Aspergillus were higher in the acute urticaria group. The level of total IgE and the number of positive allergen-specific IgE responses showed a positive correlation in both groups. Thus, MAST-CLA appears to be a simple and convenient method for determining causative allergens in the occurrence and exacerbation of urticaria, but other studies are necessary to confirm these preliminary findings.

Transferencia del ácido linoleico conjugado (CLA) de la leche cruda a leche en polvo y condensada / Conjugated linoleic acid transfer (CLA) from raw milk to powdered milk and condensed milk

The objective of this study was to assess the effect of processing fresh milk into dairy products on conjugated linoleic acid (CLA) content of total and individual CLA isomers (cis-9, trans-11; trans-10, cis-12; cis-10. cis-12) of dairy cow products, raw milk powdered and condensed milk. Additionally, samples of raw milk from two regions of southern Chile (Los Angeles and Los Lagos), obtained by a dairy plant were collected and analyzed. The processing of fresh milk dairy products affected the CLA and its isomers, finding a significant interaction between product type and sampling month, with the total average values of 1.35 g CLA / 100g of fatty acids (AG) in fresh milk and 1.45 and 0.93 g/ 100 g AG transferred to the milk powder and condensed respectively. As isomers, the cis-9, trans-11 had higher levels in the powdered milk that fresh milk in 7 months of the 10 months studied with values from 0.50 to 0.84 g/ 100 g AG. The isomer trans-10, cis-1 + cis-10, cis-12 CLA was found in a range of 0, 24 to 0.64 g/ 100 g AG always low in fresh milk. It is concluded that further research is necessary to elucidate the precise effect of processing and storage of milk and milk products on the content of CLA needed.

El objetivo de este estudio fue evaluar la transferencia del ácido linoleico conjugado (CLA) y de sus isómeros (cis-9, trans-1; trans-10, cis-12; cis-10 cis-12) de leche cruda a productos lácteos como la leche en polvo y la leche condensada. Se recogieron y analizaron muestras de leche cruda de dos regiones del sur de Chile (Los Ángeles y Los Lagos), recepcionada por una planta de lácteos. El procesamiento de la leche fresca a productos lácteos afectó al CLA y a sus isómeros, encontrándose una interacción significativa entre el tipo de producto y el mes de muestreo, con valores promedios de CLA total de 1,35 g/100 g AG en la leche fresca y de 1,45 y 0,93 g/100 g AG traspasados a la leche en polvo y condensada respectivamente. En cuanto a los isómeros, el cis-9, trans-11 presentó niveles más altos en leche la en polvo que el leche fresca en 7 meses de los 10 estudiados con valores que fueron de 0,50 a 0,84 g/100 g AG. El CLA trans-10, cis-1+ CLA cis-10, cis-12 se encontró en un rango de 0, 24 a 0,64 a g/100 g AG siempre bajo de la leche fresca en el caso de la leche. Se concluye que es necesario una mayor investigación para dilucidar el efecto exacto del procesamiento y almacenamiento de la leche y los productos lácteos sobre el contenido de CLA.

Comparison of MAST-CLA System Results between Children and Adults with Atopic Dermatitis (2005~2012, Gangwon, Yeongseo Province) / 대한피부과학회지

BACKGROUND: The multiple allergosorbent test chemiluminescent assay (MAST-CLA) system is a simple method with high sensitivity and specificity for measuring total and allergen-specific IgE antibodies in patients with allergic skin diseases such as atopic dermatitis and urticaria. OBJECTIVE: The purpose of this study was to evaluate total IgE class, allergen frequencies, and their correlations in MAST-CLA in children and adults with atopic dermatitis. METHODS: We performed a retrospective study using medical documents of 2,154 atopic dermatitis patients who visited Yonsei Wonju Severance Christian Hospital between 2005 and 2012. Positive rates of specific IgE and total serum IgE levels in children and adults were analyzed. RESULTS: Among 2,154 total patients, 1,189 (55.2%) were children and 965 (44.8%) were adults. The mean age was 20.2 years. The number of patients with greater than class 2 total serum IgE levels was 1,612 (74.8%), 814 children (68.5%) and 798 adults (82.7%). In total, 1,213 patients (56.3%) showed positive reactions to at least one allergen-specific IgE. The positive rates were 55.5% and 57.3% in children and adults, respectively. The most commonly detected allergen was Dermatophagoides farinae, followed by D. pterony and house dust in both children and adults. Most patients showed lower positive reactions to food allergens. The most common food allergen was white egg in children and shrimp in adults; 35.3% of the patients who showed positive total IgE did not react to any allergen-specific IgE. In addition, 31.4% of patients with negative total IgE showed positive reactions to more than one allergen-specific IgE. The total IgE level and number of positive allergen-specific IgE results showed good correlation in both children and adults. CONCLUSION: According to our results, positive rates of both total IgE and allergen-specific IgE were higher in adults with atopic dermatitis than in children. Both adults and children showed higher positive rates for inhalant allergens. Positive rates for food allergens such as egg whites, milk, and soybean were higher in children. The total IgE level and number of positive allergen-specific IgE results showed good correlation in both groups. MAST-CLA would be a simple and convenient method to determine the causative allergen in atopic dermatitis occurrence and exacerbation, but other studies would be necessary for confirmation.

Oferta de pasto kikuyo (Pennisetum clandestinum), tercio de lactancia y perfil de ácidos grasos lácteos / Kikuyo grass (Pennisetum clandestinum) allowance, state of lactation and milk fatty acid profile

Objetivo. Evaluar el efecto de la oferta de kikuyo y el tercio de lactancia sobre el perfil de ácidos grasos en la grasa láctea de bovinos en pastoreo. Materiales y métodos. Se utilizaron 18 vacas de la raza Holstein con un peso promedio de 585 ±10 kg entre dos y cuatro partos, en primer y segundo tercio de lactancia. Las vacas se dividieron en tres grupos y fueron asignados al azar a uno de los tres tratamientos (ofertas de 2.6; 3.3 ó 4.0 kg MS/100 kg PV). Se determinó el perfil de ácidos grasos en la leche los días 14 y 21 del periodo experimental. Resultados. Los ácidos grasos C6:0, C16:0, C18:1c9 y C18:3 permanecieron constantes entre las diferentes ofertas de forraje y tercios de lactancia. En las mayores ofertas se presentó un aumento entre el día 14 y 21 de los ácidos C10:0 y C12:0 mientras que disminuyeron su concentración en estos mismos días los ácidos C18:1t11, C18:2c9c12, C18:2c9t11 y poliinsaturados. Se presentó una mayor concentración del ALC en animales de segundo tercio respecto a los de primero. Conclusiones. Debido a las variaciones entre los días de muestreo en el perfil de ácidos grasos de la grasa láctea fue imposible determinar si el aumento en la oferta mejora las concentraciones de ácidos grasos que han presentado efecto benéfico en la salud humana. La movilización de reservas de grasa podría explicar el comportamiento diferente de la concentración de ácidos grasos entre los días de recolección de muestras.

Objective. To evaluate the effect of kikuyu offer and stage of lactation on fatty acids profile on milk fat of grazing dairy bovines. Materials and methods. Eighteen Holstein cows with average weight of 585±10 Kg in two to four calvings on first and second lactation stages. Cows were divided in three groups and assigned randomly to one of three treatments (pasture allowances of 2.6, 3.3 or 4.0 KgDM/100 Kg LW). Milk fatty acid profiles were determined on days 14 and 21 of the experimental period. Results. Fatty acids C6:0, C16:0, C18:1cis9, C18:3 remained constant between different forage allowances and lactation stage were used. On the treatments of highest allowance, there was an increase between days 14 and 21 of C10:0 andC12:0 acids while C18:1t11,C18:2c9c12, C18:2c9,t11, and polyunsaturated fatty acids decreased their concentration. The lowest allowance had a different behavior. A higher concentration of ALC was observed in animals of second stage compared to first stage of lactation. Conclusions. It was not possible to conclude on the effects of forage allowance on the fatty acid profile of milk, due to variations between sampling dates. It is possible that mobilization of fat storage can explain the different behavior of fatty acid concentrations between sampling days.

Qualidade nutricional e estabilidade oxidativa de manteigas produzidas do leite de vacas alimentadas com cana-de-açúcar suplementada com óleo de girassol / Nutritional quality and oxidative stability of butter obtained from cows fed sugar-cane supplemented with sunflower oil

O objetivo deste estudo foi avaliar o perfil de ácidos graxos (AG), os índices de qualidade nutricional e a estabilidade oxidativa (EO) de manteigas produzidas do leite de vacas alimentadas com dietas à base de cana-de-açúcar contendo níveis crescentes de óleo de girassol (OG): 0 (controle); 1,5; 3,0 e 4,5% da matéria seca (MS). O perfil de AG das manteigas foi analisado por cromatografia gasosa, e a EO foi determinada utilizando-se o equipamento Rancimat®, modelo 743, operado a 120ºC e fluxo de ar de 20L/h. As concentrações dos AG rumênico (CLA cis-9, trans-11), vacênico (C18:1 trans-11) e oleico (C18:1 cis-9) na gordura das manteigas foram aumentadas em 867, 687 e 148%, respectivamente, à medida que se aumentou de 0 para 4,5% o nível de OG na dieta. Por outro lado, as concentrações dos AG saturados de cadeia média foram linearmente reduzidas (P<0,0001) na gordura das manteigas, em razão do incremento de OG nas dietas. Quanto aos índices de qualidade nutricional, houve redução linear (P<0,0001) no índice de aterogenicidade e no de trombogenicidade e aumento da relação entre AG hipo e hipercolesterolêmicos, em resposta ao aumento do nível de OG na dieta. Consistente com o incremento (P<0,0001) nas concentrações totais dos AG mono e poli-insaturados, a EO da gordura das manteigas foi linearmente reduzida (P<0,0001) em razão do incremento de OG nas dietas. Concluiu-se que a suplementação com OG melhorou a qualidade nutricional das manteigas produzidas do leite de vacas Holandês x Gir devido a mudanças positivas no perfil de AG da gordura. Entretanto, tais mudanças na composição dos AG da gordura foram acompanhadas de redução da EO das manteigas, associada à menor vida de prateleira.

The objective of this study was to evaluate the fatty acid (FA) profile, nutritional quality and oxidative stability (OE) indexes of butter obtained from milk of cows fed sugar cane-based diets containing increasing levels of sunflower oil (SO): 0 (Control); 1.5; 3.0 and 4.5% of diet DM. The butter FA profile was analyzed by gas chromatography and OE was determined using the Rancimat® equipment (model 743) operated at 120ºC and air flow of 20 L/h. The concentrations of rumenic acid (cis-9 trans-11 CLA), vaccenic acid (trans-11 C18:1) and oleic acid (cis-9 C18:1) in butter fat were increased by 867, 687 and 148%, respectively, as the dietary SO level increased from 0 to 4.5%. In contrast, the concentrations of medium chain saturated FA were linearly reduced (P<0.05) in butter fat from cows fed increasing levels of SO. Regarding the butter nutritional quality, a linear decrease (P<0.0001) in atherogenicity and thrombogenicity indexes and a linear increase (P<0.0001) in the hypocholesterolemic/ hypercholesterolemic ratio were observed compared to control, 1.5, 3.0 and 4.5% SO, respectively. Consistent with the increased (P<0.0001) concentration of mono and polyunsaturated FA, the OE of butter fat was linearly reduced (P<0.0001) as the dietary SO level increased. It was concluded that diet supplementation with SO improved the nutritional quality of butter fat of Holstein x Gir dairy cows as a result of positive changes in milk FA profile. However, these changes in milk FA composition were accompanied by a reduction in the SO of butter, which in turn is associated with a shorter shelf life.

Effect of isomalto-oligosaccharide and gentio-oligosaccharide on the growth and fatty acid profile of Lactobacillus plantarum

Background: Lactobacillus sp. are probiotic microorganisms, and some of them are able to produce conjugated linoleic acid (CLA) via the bio-hydrogenation of linoleic acid (LA). Both CLA and LA are polyunsaturated fatty acids commonly used in the prevention and control of cardiovascular disease, high cholesterol, and cancer, among other ailments. The carbon source is one variable that can affect the growth and characteristics of these bacteria. Molecules called prebiotics are known to benefit human health by stimulating the growth and activity of probiotic bacteria present in the intestinal microflora. The aim of this study was to evaluate how different oligosaccharides affect the growth and fatty acid profile of Lactobacillus plantarum (NRRL - B4496). L. plantarum cultivation was performed in Man-Rogosa-Sharpe (MRS) medium, and the original carbon source (glucose) in this medium was partially or totally replaced by an oligosaccharide (isomalto-oligosaccharide (IMO) or gentio-oligosaccharide (GTO)). Then, the biomass concentration and fatty acid profile were determined using spectrophotometry and gas chromatography, respectively. Results: When 50% of the glucose in the MRS medium was replaced with IMO, the maximum growth was 2.6 g/L at 37ºC. Under the same culture conditions, the incorporation of GTO only produced 2 g/L of biomass. At 45ºC, the growth of the bacterial culture was lower than that observed at 37ºC, reaching only 0.4 g/L. When cultivated at 37ºC in a mixture of glucose and GTO (1:1), CLA (34%, c9t11) was obtained from cells of L. plantarum. However, when the cultivation was performed at 45ºC, CLA was not obtained. When IMO was used, differences in CLA content were not observed between L. plantarum cultivated with glucose or with IMO present; however, vaccenic acid was produced. Conclusions: Lactobacillus plantarum grow well when a mixture of IMO and glucose is used as the carbon source. However, this mixture does not improve the CLA content, most likely due to high enzymatic activity that promotes the conversion of CLA to vaccenic acid. Additionally, GTO is likely less readily metabolized by this strain. Thus, the enzymatic activity is likely lower and less CLA is converted to vaccenic acid, resulting in an accumulation of CLA.

Anti-proliferative efficacy comparison of conjugated linoleic acid on human cancer cell lines / 동물의과학연구지

The anti-proliferative efficacy of t,t-conjugated linoleic acid (t,t-CLA), c9,t11-CLA, and t10,c12-CLA was compared in several human cancer cell lines. Gastric NCI-N87, liver Hep3B, pancreas Capan-2, and lung NCI-H522 cancer cells were incubated with 50 microM CLA isomers over a period of 6 days. The t,t-CLA inhibited the growth of all cancer cell lines to different extents, but c9,t11-CLA and t10,c12-CLA inhibited or stimulated the growth of the cancer cell lines. NCI-N87 cells were the most sensitive to growth inhibition and apoptosis from all CLA isomers tested. In NCI-N87 cells, CLA isomers reduced the release of arachidonic acid (AA) via the inhibition of cytosolic phospholipase A2 (cPLA2) activity, consequently reducing the production of PGE2 through the inhibition of cyclooxygenase-2 (COX-2). The efficacies of CLA isomers were in the following order (from most to least effective): t,t-CLA, t10,c12-CLA and c9,t11-CLA. Overall, these results imply that the anti-proliferative efficacy of t,t-CLA on cancer cells, especially NCI-N87 cells, was greater than other CLA isomers due to its induction of apoptosis through the inhibition of cPLA2 and COX-2 activities.

Influence of fish oil in the concentration of conjugated linoleic acid and omega 6 and 3 in buffalo milk / Influência do óleo de peixe na concentração de ácido linoléico conjugado e Omega 6 e 3 no leite de búfalas

The aim of this research work was to investigate the influence of fish oil supplementation on the concentration of conjugated linoleic acid (CLA) and omega 6 and 3 in samples of buffalo milk. A total of 24 female buffaloes separated at random into three groups were fed for 49 days with: natural pasture (group I), supplemented with 70mL of fish oil (group II) and 140mL of fish oil (group III). In the experiment the concentration of CLA showed differences (P<0.05) among the three groups, with a maximum of 7.14mg/g fat in group II. No significant differences were found in omega-6 among the three groups. The highest value of 3.82mg/g fat corresponded to group I, whicht had not been supplemented with fish oil. Significant differences were observed in omega 3 (P<0.05) in groups II and III with respect to group I. The highest average value of 2.42mg/g fat was obtained in group III. The closest relationship omega 6/3 (1.37:1) was observed in group III. As a result, the diets of groups II and III, which included fish oil, increased significantly the content of CLA and omega 3 with reductions in levels of omega 6.

Estudou-se a influência da suplementação com óleo de peixe sobre a concentração dos ácidos graxos ômega 6 e 3, mostrado no leite de búfala. Foram utilizadas 24 búfalas aleatoriamente selecionadas e distribuídas em três grupos e alimentados com pastagem natural por 49 dias com: nenhum suplemento (grupo I), suplementado com 70mL de óleo de peixe (grupo II) e com 140mL de óleo de peixe (grupo III). A concentração de CLA diferiu (P<0,05) entre os três grupos, com valores máxima de até 7,14mg/g de gordura no grupo II. Não foram observadas diferenças significativas para os ácidos graxos omega 6, sendo o maior valor de 3,82mg/g de gordura no grupo I. Foi observada diferença significativa quanto aos ácidos graxos ômega 3 (P<0,05) nos grupos II e III em relação ao grupo I. O mais elevado valor, 2,42mg/g de gordura foi observado no grupo III. A relação ômega 6/3 mais estreita, 1,37:1, foi observada no grupo III. Os resultados mostram que os animais dos grupos II e III que receberam suplementação de óleo de peixe aumentou significativamente o teor de CLA e de omega 3, com diminuição de omega 6.

Body measurements and serum lipid profile of overweight adult dogs fed diet with containing conjugated linoleic acid / Medidas corporais e perfil lipídico plasmático de cães adultos com sobrepeso alimentados com dieta contendo ácido linoléico conjugado

Studies with human beings and animals have shown that dietary conjugated linoleic acid (CLA) promotes changes in body structure, markedly, body fat reduction. This research aimed to assess the effect of CLA (60 percent of conjugated linoleic acid methyl ester, at the proportion of 1:1 of the 9, 11: 10, 12 isomers) on changes in body weight, subcutaneous tissue thickness, body fat mass and concentration of serum lipids (triacylglycerols, total cholesterol, LDL and HDL) in overweight dogs. Dogs of different breeds weighting 16.85±7.13 assigned in a randomized assay. Two types of diets were offered for 120 days: control (-CLA) and test (+CLA, 0.3 percent). There was no difference between control and test groups considering body weight and serum levels of triacylglycerols, total cholesterol and LDL portion. There was a significant difference between the final and initial results for subcutaneous tissue thickness and body fat mass and for serum concentration of HDL in dogs supplemented with CLA. The addition of 0.3 percent of CLA in the diet of overweight dogs decreased the fat deposition on subcutaneous tissue and the development of body fat mass but increased blood levels of HDL.

Estudos em humanos e animais têm demonstrado que o uso do ácido linoléico conjugado (ALC) promove alterações na composição corporal, principalmente reduzindo a deposição de gordura. O objetivo deste trabalho foi avaliar as alterações sobre o peso corporal, tecido adiposo subcutâneo, gordura corporal e concentração sérica de lípideos (triacilglicerol, colesterol total, LDL e HDL) de cães com sobrepeso alimentados com ALC (60 por cento de ácido linoléico conjugado metil ester, na proporção de 1:1 de 9, 11: 10, 12 isômeros) na dieta. Duas dietas foram fornecidas por quatro meses, para 14 cães com sobrepeso (16,85±7,13) de diferentes raças: dietas controle (-ALC) e teste (+ALC, 0,3 por cento). O experimento seguiu delineamento inteiramente casualizado. Não houve diferença entre os grupos para peso corporal e nível sérico de triacilglicerol, colesterol total e LDL. No entanto, foram observadas diferenças significativas entre os períodos final e inicial para tecido adiposo subcutâneo, gordura corporal e nível sérico de HDL nos cães que receberam ALC. A suplementação de 0,3 por cento de ALC na dieta de cães com sobrepeso diminuiu a deposição de lipídios no tecido subcutâneo e a gordura corporal total, mas aumentou os níveis sanguíneos de HDL.

Winnicott clínico / Winnicott as a clinician

De início, o presente artigo apresenta, de maneira esquemática, a concepção freudiana dos distúrbios que fazem parte do domínio de aplicação da psicanálise. Em seguida, dedica-se, de modo mais detalhado, ao estudo da teoria winnicottiana da etiologia, natureza, classificação, solubilidades e de procedimentos de resolução desse mesmo tipo de problemas clínicos. Um dos resultados obtidos é um conjunto de evidências adicionais a favor da tese de que Winnicott operou uma mudança paradigmática na psicanálise.

This paper begins with an outline of the Freudian view of the disorders that are dealt with in psychoanalysis. It then describes in a more detailed manner the Winnicottian theory of the etiology, nature, classification, solutions, and procedures for resolving those same kinds of clinical problems. One of the results obtained is a set of additional evidence for the thesis that Winnicott performed a paradigm shift in psychoanalysis.