Culturable micro-organisms in human milk were found to be associated with maternal weight, diet and age during early lactation.

AIMS: This study aimed to evaluate the influence of maternal factors on the total plate count, Staphylococci, Lactobacilli and Bifidobacteria populations in the milk of lactating Filipino women for the first 4 months of lactation period. METHODS AND RESULTS: Milk samples (n = 136), 24 h diet recalls and responses from a qualitative food frequency questionnaire (FFQ) were collected from 34 healthy lactating women classified according to their BMI: underweight (n = 7), normal weight (n = 16) and overweight (n = 11). The FFQ was useful in assessing prebiotic and probiotic food items consumed by the participants. Microbial populations were enumerated using culture-plating method, and showed a nonsignificant decreasing trend suggesting their relative stability throughout the first 4 months of lactation. Carbohydrate and fat intakes were associated with TPC, and with both TPC and Staphylococci respectively (P ≤ 0·05); and consumption of root crop is linked with Staphylococci, Lactobacilli and Bifidobacteria (P ≤ 0·05). Interestingly, age was found to be a positive determinant for Bifidobacteria (P = 0·00), whereas being normal- or overweight as negative determinants of Lactobacilli (P = 0·017). Consumption of milk also seems to positively influence both Lactobacilli (P = 0·00) and Bifidobacteria (P = 0·05) counts. CONCLUSIONS: Certain populations of culturable micro-organisms were found to be associated with maternal diet, weight classification and age. SIGNIFICANCE AND IMPACT OF THE STUDY: This study offered new discoveries in the recently growing endeavor on the role of maternal factors in modulating certain microbial populations in human milk. Ultimately, the findings of this study could provide a basis in crafting lactation policies and guidelines that may help enhance the microbial quality of human milk through adjustments in maternal diet or weight during lactation.

Lipopolysaccharide (LPS)-induced septic shock causes profound changes in myocardial energy metabolites in pigs.

INTRODUCTION: Energy deficiency is a cause for myocardial dysfunction during septic shock. In rodents, septic shock decreases the oxidation of long-chain fatty acids and glucose in the myocardium causing energy deficiency. However, the effect of septic shock on myocardial energy metabolites in large animals and human is unknown. OBJECTIVES: Investigate the effects of septic shock on myocardial energy metabolites in domestic pigs. METHODS: Seventeen female pigs divided into control and lipopolysaccharide (LPS)-induced septic shock groups. Myocardial metabolites were analyzed ex vivo by 1H nuclear magnetic resonance spectroscopy and liquid chromatography-tandem mass spectrometry. Gene and protein expression analysis were analyzed by real-time PCR and western blot. RESULTS: Septic shock was associated with an increase in myocardial levels of short- and medium-chain acylcarnitines, lactate, alanine, and pyruvate dehydrogenase kinase 4 gene expression. COX-2 and prostaglandin E4 receptor gene expression also increased in the septic myocardium, although the only elevated eicosanoid in the septic animals was thromboxane B2. Myocardial levels of niacin, taurine, glutamate, glutamine, and glutathione were higher, and hypoxanthine levels lower in septic pigs than controls. CONCLUSIONS: In pigs, septic shock induced by LPS caused myocardial changes directed to decrease the oxidation of medium- and short-chain fatty acid without an effect on long-chain fatty acid oxidation. The increase in myocardial levels of lactate, alanine, and pyruvate dehydrogenase kinase 4 gene expression suggest that septic shock decreases pyruvate dehydrogenase complex activity and glucose oxidation. Homeostasis of niacin, taurine, glutamate, glutamine, glutathione, hypoxanthine and thromboxane B2 is also affected in the septic myocardium.

Fruit flesh volatile and carotenoid profile analysis within the Cucumis melo L. species reveals unexploited variability for future genetic breeding.

BACKGROUND: Aroma profile and carotenoids content of melon flesh are two important aspects influencing the quality of this fruit that have been characterized using only selected genotypes. However, the extant variability of the whole species remains unknown. RESULTS: A complete view of the volatile/carotenoid profiles of melon flesh was obtained analyzing 71 accessions, representing the whole diversity of the species. Gas chromatography-mass spectrometry and high-performance liquid chromatography were used to analyze 200 volatile compounds and five carotenoids. Genotypes were classified into two main clusters (high/low aroma), but with a large diversity of differential profiles within each cluster, consistent with the ripening behavior, flesh color and proposed evolutionary and breeding history of the different horticultural groups. CONCLUSION: Our results highlight the huge amount of untapped aroma diversity of melon germplasm, especially of non-commercial types. Also, landraces with high nutritional value with regard to carotenoids have been identified. All this knowledge will encourage melon breeding, facilitating the selection of the genetic resources more appropriate to develop cultivars with new aromatic profiles or to minimize the impact of breeding on melon quality. The newly characterized sources provide the basis for further investigations into specific genes/alleles contributing to melon flesh quality. © 2018 Society of Chemical Industry.

Serum levels of IGF1 are a useful predictor of retinopathy of prematurity.

OBJECTIVE: To ascertain whether insulin-like growth factor 1 (IGF1) is associated with retinopathy of prematurity (ROP) and is a useful predictor of the disease. Although its aetiopathogenesis is multifactorial, development of the disease appears to be related to a deficiency in IGF1, a hormone that acts together with vascular endothelial growth factor in the normal angiogenesis in the retina. DESIGN: Prospective study for a 30-month period. PARTICIPANTS: A total of 74 premature newborn babies, of less than 1500 g and/or 32 weeks' gestational age or less. TESTING: To determine the development and severity of ROP. MAIN OUTCOME MEASURES: Serum levels of IGF1 were measured once a week from birth until 40 weeks corrected gestational age in each subject. RESULTS: Of our subjects, 32.4% developed some form of ROP, and all those ROP patients had the following characteristics at birth (median +/- standard deviation scores): low weight (1098 +/- 188 vs. 1393 +/- 285 g), short length (36.74 +/- 1.77 vs. 38.89 +/- 3.08 cm), small cranial perimeter (26.03 +/- 1.74 vs. 27.93 +/- 1.81 cm) and young gestational age (29.7 +/- 1.78 vs. 31.3 +/- 1.79 weeks) (p < 0.05). Other factors previously associated with ROP that were also observed with statistically significant frequency in our ROP patients were bronchopulmonary dysplasia, intracranial haemorrhage, the need for erythrocyte transfusion or treatment with erythropoietin and sepsis (all p < 0.05). Levels of IGF1 at the 3rd week post-partum, independent of gestational age at birth, were clearly lower in the group who developed ROP (29.13 vs. 43.16 ng/mL, p < 0.05). A value of 30 ng/mL of IGF1 in the third week post-partum was found to have a 90% sensitivity in the diagnosis of ROP. A rapid rise in IGF1 levels between the 3rd and 5th weeks appeared to be related to the development of a higher stage of ROP. CONCLUSION: Determination of IGF1 serum levels in the 3rd week post-partum, independent of gestational age at birth, provides a sufficient and reliable prognostic tool and allows the identification of a group of patients at high risk of developing the disease.

[Pediatric nephrology in developing countries]. / Néphrologie pédiatrique dans les pays en développement.

Pediatric nephrology is not a priority medicine in developing countries, but it should improve along with the development of both preventive and curative simple measures since the mortality rate is still important. This can be applied to the management of urinary tract infection, acute nephritis and some other cases of acute renal failure. The identification and management of chronic kidney diseases is mainly based on blood pressure measurement, proteinuria screening and plasma creatinine assessment. However renal replacement therapy (i.e., dialysis and transplantation) is highly expensive and its access is therefore limited to selected growing countries. The improvement in the care of renal children therefore requires better medical knowledge, nurse training and population information.

Sospecha clínica y estudios diagnósticos iniciales en enfermedades metabólicas hereditarias / Clinical suspicion and initial diagnostic studies in inherited metabolic diseases

Las enfermedades metabólicas hereditarias (EMH) constituyen un grupo diverso y complejo de trastornos genéticos que, aunque individualmente son poco frecuentes, en su conjunto representan una importante causa de morbilidad y mortalidad. Se lleva a cabo una revisión exhaustiva de la literatura con el objetivo de proporcionar información que oriente la sospecha clínica y el enfoque en los estudios diagnósticos iniciales, cuya clave reside en una anamnesis completa que abarque los antecedentes familiares y obstétricos, junto con una consideración cuidadosa de las manifestaciones clínicas del paciente. Es crucial tener en cuenta la naturaleza multisistémica de estas enfermedades que pueden manifestarse desde el periodo neonatal, generalmente como intoxicaciones agudas, hasta una presentación más insidiosa en adultos jóvenes. Si la evaluación clínica sugiere la posibilidad de una EMH, es fundamental llevar a cabo medidas de apoyo general y realizar investigaciones de laboratorio de manera simultánea. En neonatos, donde la presentación de una EMH puede representar una emergencia médica que requiere una respuesta inmediata, esta acción es especialmente crítica. El diagnóstico de las EMH representa un desafío debido a su variabilidad clínica y sintomatología heterogénea. Sin embargo, la identificación temprana de estas enfermedades es fundamental para iniciar un tratamiento oportuno y mejorar el pronóstico de los pacientes.(AU)

Inherited metabolic diseases (IMDs) constitute a diverse and complex group of genetic disorders that, although individually rare, collectively represent a significant cause of morbidity and mortality. A comprehensive literature review is carried out with the aim of providing information to guide clinical suspicion and the approach to initial diagnostic studies, the key of which lies in a complete medical history encompassing family and obstetric backgrounds, along with careful consideration of the patient’s clinical manifestations. It is crucial to consider the multisystemic nature of these diseases, which can manifest from the neonatal period, usually as acute intoxications, to a more insidious presentation in young adults. If clinical evaluation suggests the possibility of an IMD, it is essential to implement general supportive measures and simultaneously perform laboratory investigations. In neonates, where the presentation of an IMD can represent a medical emergency requiring an immediate response, this action is especially critical. The diagnosis of IMDs poses a challenge due to their clinical variability and heterogeneous symptomatology. However, early identification of these diseases is crucial for initiating timely treatment and improving patient prognosis.(AU)

[Importance of early diagnosis and treatment in the prognosis of type I Glutaric Acidaemia]. / Importancia del diagnóstico precoz y el tratamiento temprano en el pronóstico de la aciduria glutárica tipo I.

INTRODUCTION: Glutaric Acidaemia type I (GA-I) is an autosomal recessive progressive neurodegenerative inborn error of metabolism caused by deficient activity of the enzyme glutaryl-CoA dehydrogenase (GCDH). In most cases, the diagnosis is established biochemically by the detection of glutaric acid and 3-hydroxy glutaric acid in urine and glutarylcarnitine in plasma. Patients excreting small amounts of glutaric acid may be overlooked. OBJECTIVE: To investigate the value of expanded newborn screening by adding the measurement of urine glutarylcarnitine to conventional chromatography-mass spectrometry (GC-MS) in the diagnosis of GA-1. MATERIAL AND METHODS: We report clinical and biochemical data in 5 GA-I patients diagnosed in our Hospital. Details regarding biochemical diagnosis are emphasised and the absence or presence of symptoms was correlated with neuroimaging findings, age at diagnosis and treatment. RESULTS: Two patients showed high glutarylcarnitine levels in plasma and were identified by routine newborn GC-MS screening. Following early appropriate treatment they are asymptomatic 6 years later. Two patients with delayed diagnosis displayed neurological sequels in spite of treatment. The remaining patient, who presented with encephalopathic episode at age 8 months showed normal glutarylcarnitine levels in routine plasma GC-MS but high urine glutarylcarnitine levels in a retrospectively screened urine sample from the newborn period. CONCLUSIONS: Early treatment seems to positively influence the clinical evolution of GA-I patients. Thus, improving the identification of GA-I represents an important diagnostic challenge. The urinary excretion of glutarylcarnitine is a specific biochemical marker of GA-I and allows the identification of patients without glutaric aciduria and with normal plasma acylcarnitine profiles.

[Advances in the diagnosis and treatment of maple syrup urine disease: experience in Galicia (Spain)]. / Avances en el diagnóstico y tratamiento de la enfermedad de jarabe de arce, experiencia en Galicia.

INTRODUCTION: Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder caused by an inherited deficiency of branched chain alpha-ketoacid dehydrogenase activity. Accumulation of the amino acids leucine, isoleucine, valine and alloisoleucine and their metabolic products in cells and biological fluids results in severe brain dysfunction. PATIENTS AND METHODS: We present three cases of MSUD diagnosed in Galicia since 2000, the year in which the Extended Newborn Screening Program by tandem mass spectrometry was started in this region. One of the patients was diagnosed on the basis of early clinical presentation and the others by neonatal screening. Enzymatic and molecular studies confirmed two classic cases of MSUD and an intermediate variant. We describe the clinical and biochemical details at confirmation of diagnosis and the long-term outcome of the three patients. Throughout follow-up, all the patients maintained adequate leucine levels, which were near the normal range (mean levels: 220, 177 and 252 micromol/L, respectively). Several moderate metabolic decompensations were observed but leucine levels only occasionally exceeded 1000 micromol/L (one day in two patients). IQ tests were performed in all patients and scores were within the normal range. In view of our results, we believe the following measures are essential to improve the prognosis of MSUD: inclusion of this disease in Expanded Neonatal Screening Programs with early samples (at 2-3 days of life); aggressive treatment in the initial phase and during acute decompensations; strict metabolic control to prevent crises, monitoring of branched-chain amino acids (dried blood spot sample), and maintenance of long term plasma leucine levels below 300 micromol/L.

Study of chimerism in long-term survivors after bone marrow transplantation for severe acquired aplastic anemia.

Chimerism studies employing PCR and Southern techniques targeting VNTR loci were performed in 17 severe acquired aplastic anemia patients who were long-term survivors after BMT. They were studied a median of 4 years after BMT (1-12). All patients had normal blood counts. All patients conditioned with radiation-based schemes showed a full donor pattern of hemopoiesis. Conversely, out of five patients who received only cyclophosphamide as conditioning therapy, two of them had a late graft failure (2.4 and 3 years after BMT). One of these relapsing patients had a durable mixed chimerism, which was first detected 1 month after BMT. Our results seem to suggest that durable mixed chimerism can antecede graft failure in some patients conditioned only with cyclophosphamide, and that a more stringent monitoring can be clinically rewarding in this group of patients.

Gas exchange and pulmonary haemodynamic responses to fat emulsions in acute respiratory distress syndrome.

OBJECTIVE: To investigate the gas exchange and pulmonary haemodynamic responses to two different intravenous fat emulsions in patients with acute respiratory distress syndrome (ARDS). DESIGN: Prospective, randomized, double-blind, placebo-controlled study. SETTING: Intensive care unit in a university-affiliated hospital. PATIENTS: 21 patients with ARDS [mean age, 57 +/- 3 (SEM) years; Acute Physiology and Chronic Health Evaluation II, 20 +/- 3; Murray's score, 2.85 +/- 0.12] consecutively admitted. INTERVENTIONS: Patients were assigned to three groups (n = 7 each): group A (LCT) received long-chain triglycerides (20% LCT), group B (MCT/LCT), medium-chain triglycerides/long-chain triglycerides (20% MCT/LCT: 50/50) and group C placebo (0.9% sodium chloride, NaCl). The infusion was always given at the rate of 2 mg/kg min over a total period of 12 h, with a volume infusion of 500 ml in each group. MEASUREMENTS: Data were collected before, immediately after and 12 h after infusion ceased. Pulmonary and systemic haemodynamic and gas exchange variables were measured at each time point. Serum triglyceride cholesterol, and non-esterified fatty acids levels were measured. RESULTS: During LCT infusion, cardiac output, oxygen consumption and oxygen delivery increased (all p < 0.05), whereas pulmonary haemodynamics, arterial oxygen tension, mixed venous partial pressure of oxygen and venous admixture ratio remained essentially unaltered. No changes were observed following MCT/LCT infusion. CONCLUSIONS: The administration of LCT emulsion given at a slow rate did not alter arterial oxygenation because of the beneficial effect of a high cardiac output, hence offsetting the detrimental effect of increased O2 consumption.

Fatty acid composition of platelet membrane lipids after administration of two different fat emulsions in critically ill patients.

OBJECTIVE: To determine the effects on platelet membrane fatty acid composition following administration of two different fat emulsions. DESIGN: Prospective, randomized, double-blind study. SETTING: Intensive care unit in a university-affiliated hospital. PATIENTS: 12 adult critically ill patients in need of total parenteral nutrition. INTERVENTIONS: Patients were treated with total parenteral nutrition (TPN) for 7 days, receiving for fat intake either a long-chain triglyceride (20% LCT) emulsion (group 1, n=6) or a medium-chain triglyceride-LCT (20% MCT/LCT) emulsion (group 2, n=6). MEASUREMENTS AND RESULTS: High-performance liquid chromatography of membrane fatty acids was carried out before and after 7 days of TPN. In the LCT group, an increase in C18:2n-6 and a decrease in caprylic acid and docosahexaenoic acid, which resulted in a decreased ratio of n-3/n-6 fatty acid content, was observed. In the MCT/LCT group, a reduced percentage of palmitoleic acid and arachidonic acid was shown. CONCLUSIONS: The observed changes in fatty acid composition are in agreement with the lipid composition of the fat emulsions used. Because the C18:2n-6/C18:3n-3 ratio in both emulsions is close (approximately 9.0), the observed changes in the fatty acid composition of platelets may not be relevant for platelet function.

Anticoagulative effect of nitric oxide inhalation in ARDS.

Some studies have suggested that nitric oxide (NO) may cause platelet dysfunction. We present an ARDS patient who need this treatment, with a transient alteration of platelet function and a significant prolongation of bleeding time.

Tuberculosis in Spain: epidemiological pattern and clinical practice.

SETTING: Thirteen Autonomous Regions in Spain. OBJECTIVE: To study the incidence of all forms of tuberculosis (TB) and investigate clinical practice in TB. DESIGN: Cases of all forms of tuberculosis diagnosed in the study setting from May 1986 to April 1997 were identified though active search of different databases. Clinical and epidemiological information on cases was collected from clinical records or by interview with physicians. RESULTS: The overall incidence of all forms of tuberculosis was 38.5/100,000 and the incidence of smear-positive disease was 13.83/100,000. Most cases (97.1%) were Spanish nationals, with rates higher in men than in women (52.7/100,000 vs. 24.87/100,000) and in groups aged 25-34 and 75 years and over (61.35/100,000 and 59.35/100,000, respectively). Disseminated forms were frequent (6.6%), and the most common risk factor was human immunodeficiency virus (HIV) infection (17.7% of cases). Hospitalisation was common (71.6%). Microbiological confirmation of diagnosis was sought for 87.7% of the cases (91.8% of pulmonary vs. 75.5% of extra-pulmonary cases), and 65.2% were culture-positive (73.8% of pulmonary vs. 39.7% of extra-pulmonary cases). HIV-infected patients were treated in almost equal proportions with three or four drugs (49.7% and 48.2%, respectively), while HIV-negative cases or those whose HIV status was unknown were usually treated with three drugs. CONCLUSION: The epidemiological pattern of TB in Spain is different to other industrialised countries in the age distribution of cases and the proportions of foreigners and cases with HIV infection. Microbiological confirmation of diagnosis is more common in pulmonary than in extra-pulmonary disease, and treatment with four drugs more frequent in HIV-positive cases.

Identification and characterization of the peptidic component of the immunomodulatory glycoconjugate Immunoferon.

Inmunoferon is a glycoconjugate of natural origin, formed by the noncovalent association of a protein from Ricinus communis and a polysacharidic moiety, and endowed with immunomodulatory as well as pharmacological activities. This study investigated the nature of polypeptidic component of Inmunoferon. Through biochemical procedures and comparison with protein databases, the isolated protein was identified as the processed form of the seed of Ricinus communis 2S storage polypeptide, which has been termed RicC3. Further analysis of the isolated protein has revealed that it is composed of two different subunits, alpha and beta, which form an heterodimer of high stability and resistance to denaturation, acidic pH and proteolytic cleavage. These findings confirm the excellent properties of the product after oral administration and provide additional support for the pharmacological activities of Inmunoferon.

[The prognostic value of eicosanoids in the acute respiratory distress syndrome]. / Valor pronóstico de los eicosanoides en el síndrome de distrés respiratorio agudo.

BACKGROUND: There is a great number of agents involved in the acute respiratory distress syndrome (ARDS) physiopathology, and some of them may have a prognostic value. The objective of the present study has been to analyse the prognostic value of eicosanoids in this syndrome. MATERIAL AND METHOD: A prospective study with 21 consecutive ARDS patients admitted to the intensive care unit of a therapy hospital in Barcelona, Spain, was carried out. In the first 48 h of the ARDS diagnosis, at baseline, the plasma levels, (in peripheral arterial and pulmonary arterial samples) of thromboxane B2 (TXB2), prostaglandin F1-alpha) (PGF1-alpha) and leukotriene B4 (LTB4) were analysed by RIA. Simultaneously we measured different pulmonary and systemic hemodynamical variables, as well as the pulmonary gas exchange data. We also studied the venous levels of the same eicosanoids in 17 healthy adults, used as reference. RESULTS: Plasma levels of eicosanoids in the ARDS patients were higher than reference subjects (p < 0.05). No differences were observed between systemic arterial and pulmonary arterial values. From all the eicosanoids, only LTB4, (in both systemic arterial and pulmonary blood), was correlated with LIS (r = 0.49, p < 0.05; and r = 0.45, p < 0.05, respectively). Patients who did not survive presented a lower systemic-pulmonary arterial gradient of eicosanoids levels than survivors (-1.27 vs -0.10 ng/ml; p < 0.01). CONCLUSIONS: In our ARDS patients only LTB4 plasma levels correlated with the severity of respiratory failure. Patients who did not survive presented a lower LTB4 gradient than survivors.

[Platelet function study in primary Raynaud's phenomenon and Raynaud's phenomenon associated with scleroderma]. / Estudio del funcionalismo de las plaquetas en el fenómeno de Raynaud primario y fenómeno de Raynaud asociado a esclerodermia.

We evaluated platelet function in 59 patients with Raynaud's phenomenon (RP): 24 had primary RP (PRP) and in 35 RP was associated with diffuse scleroderma (DS). In the group with PRP there were 10 males and 14 females, with a mean age of 43 +/- 12 years and a time of evolution of 5 +/- 5 years. In the group with RP associated with DS there were 31 females and 4 males with a mean age of 53 +/- 12 years and a time of evolution of 9 +/- 7 years. The control group consisted of 20 healthy individuals (14 males and 6 females with a mean age of 40 +/- 12 years). In all patients and controls beta-thromboglobulin (BTG) and platelet factor 4 (PF4) levels were measured in plasma, and platelet aggregation was evaluated in the presence of adenosine diphosphate (ADP), collagen and arachidonic acid. The patients with RP associated with DS had BTG and PF4 higher than those with PRP and controls (p less than 0.02). The BTG/PF4 ratio was also significantly greater in patients with DS (p less than 0.005). The platelets from patients with DS had a greater aggregation with ADP (1 microM and 0.5 microM) than those from PRP (p less than 0.03). We concluded that patients with RP associated with DS had in vivo activation and a greater aggregation of platelets, in contrast with the absence of these findings in the group with PRP and in controls.

[Multicenter study on the efficacy of advice for the prevention of alcoholism in primary health care]. / Estudio multicéntrico sobre la eficacia del consejo para la prevención del alcoholismo en atención primaria (EMPA).

BACKGROUND: The aim of this study was to analyse and to verify the efficacy of systematic advice for alcoholism prevention, assessing the reduction of the number in risk drinkers. PATIENTS AND METHODS: A multicenter randomized controlled clinical trial was designed, to perform in general practitioner setting, on a sample of risk drinkers (alcohol intake > 280 g weekly, without dependence) sent by random in intervention group (systematic brief advice with support material and a five visit program during a year) and control group (once brief advice and a control in 1 year). The procedure to incorporate in both groups included physical exam, a blood test and the MALT questionnaire. A descriptive and analytic study on included variables was realised, assessing the percentage of drinkers who reduced alcohol intake below risk limit at the end of a year follow up, as well as the reduction intake in each group. RESULTS: Of the 139 included males, 75 were in the intervention group and 64 in the control group. The percentage of patients not excluded by MALT > 10, and/or liver disease, that finished the 1 year follow up, was 46%, being the sample average age of 43 +/- 11.8. Patients included in both groups were initially comparable. At the end of a year follow up there were statistically significant differences in: percentage of risk drinkers who decreased alcohol intake below 280 g weekly (82% intervention group; 47% control group); percentage of reduction in GPT, GGT, triglycerides, systolic blood pressure and the MALT questionnaire. CONCLUSIONS: The efficacy of isolated advice of general practitioner was proved to achieve the alcohol intake reduction below the risk limit accepted in male risk drinkers without alcohol dependence. The systematic follow up during a year significantly improves the results achieved with the isolated advice.