Food, mood and health: a neurobiologic outlook

Hippocrates was the first to suggest the healing power of food; however, it was not until the medieval ages that food was considered a tool to modify temperament and mood, although scientific methods as we know them today were not in use at the time. Modern scientific methods in neuroscience began to emerge much later, leading investigators to examine the role of diet in health, including mental well-being, with greater precision. This review shows how short- and long-term forced dietary interventions bring about changes in brain structure, chemistry, and physiology, leading to altered animal behavior. Examples will be presented to show how diets alter brain chemistry, behavior, and the action of neuroactive drugs. Most humans and most animal species examined in a controlled setting exhibit a fairly reproducible pattern of what and how they eat. Recent data suggest that these patterns may be under the neurochemical and hormonal control of the organisms themselves. Other data show that in many instances food may be used unconsciously to regulate mood by seemingly normal subjects as well as those undergoing drug withdrawal or experiencing seasonal affective disorders and obesity-related social withdrawal. We will discuss specific examples that illustrate that manipulation of dietary preference is actually an attempt to correct neurochemical make-up.

A simple RT-PCR-based strategy for screening connexin identity

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

Neonatal handling induces anovulatory estrous cycles in rats

Since previous work has shown that stimulation early in life decreases sexual receptiveness as measured by the female lordosis quotient, we suggested that neonatal handling could affect the function of the hypothalamus-pituitary-gonadal axis. The effects of neonatal handling on the estrous cycle and ovulation were analyzed in adult rats. Two groups of animals were studied: intact (no manipulation, N = 10) and handled (N = 11). Pups were either handled daily for 1 min during the first 10 days of life or left undisturbed. At the age of 90 days, a vaginal smear was collected daily at 9:00 a.m. and analyzed for 29 days; at 9:00 a.m. on the day of estrus, animals were anesthetized with thiopental (40 mg/kg, ip), the ovaries were removed and the oviduct was dissected and squashed between 2 glass slides. The number of oocytes of both oviductal ampullae was counted under the microscope. The average numbers for each phase of the cycle (diestrus I, diestrus II, proestrus and estrus) during the period analyzed were compared between the two groups. There were no significant differences between intact and handled females during any of the phases. However, the number of handled females that showed anovulatory cycles (8 out of 11) was significantly higher than in the intact group (none out of 10). Neonatal stimulation may affect not only the hypothalamus-pituitary-adrenal axis, as previously demonstrated, but also the hypothalamus-pituitary-gonadal axis in female rats.

Effects of dietary lipids on renal function of aged rats

Normal aging is accompanied by renal functional and morphological deterioration and dietetic manipulation has been used to delay this age-related decline. We examined the effects of chronic administration of diets containing 5% lipid-enriched diet (LD, w/w) on renal function of rats at different ages. Three types of LD were tested: canola oil, fish oil and butter. Mean systemic tail-cuff blood pressure and glycemia remained within the normal range whatever the age and the diet of the animals. Proteinuria began to rise from the 8th month in the groups ingesting LD, while in the control group it increased significantly (above 10 mg/24 h) only after the 10th month. With age, a significant and progressive decline in glomerular filtration rate (GFR) and renal plasma flow was observed in the LD groups but after 6 months of lipid supplementation, the decline in these parameters was more marked in the butter and fish oil groups. By the 18th month, the lowest GFR level was observed in the group ingesting the butter diet (2.93 ± 0.22 vs 5.01 ± 0.21 ml min-1 kg-1 in control, P<0.05). Net acid excretion, evaluated in 9- and 18-month-old rats, was stimulated in the fish oil group when compared both to control and to the other two LD groups. These results suggest that even low levels of LD in a chronic nutritional regimen can modify the age-related changes in renal function and that the impact of different types of lipid-supplemented diets on renal function depends on the kind of lipid present in the diet.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Validation of the Brazilian Children's Test of Pseudoword Repetition in Portuguese speakers aged 4 to 10 years

The phonological loop is a component of the working memory system specifically involved in the processing and manipulation of limited amounts of information of a sound-based phonological nature. Phonological memory can be assessed by the Children's Test of Nonword Repetition (CNRep) in English speakers but not in Portuguese speakers due to phonotactic differences between the two languages. The objectives of the present study were: 1) to develop the Brazilian Children's Test of Pseudoword Repetition (BCPR), a Portuguese version of the CNRep, and 2) to validate the BCPR by correlation with the Auditory Digit Span Test from the Stanford-Binet Intelligence Scale. The BCPR and Digit Span were assessed in 182 children aged 4-10 years, 84 from Minas Gerais State (42 from a rural region) and 98 from the city of São Paulo. There are subject age and word length effects causing repetition accuracy to decline as a function of the number of syllables of the pseudowords. Correlations between BCPR and Digit Span forward (r = 0.50; P <= 0.01) and backward (r = 0.43; P <= 0.01) were found, and partial correlation indicated that higher BCPR scores were associated with higher Digit Span scores. BCPR appears to depend more on schooling, while Digit Span was more related to development. The results demonstrate that the BCPR is a reliable measure of phonological working memory, similar to the CNRep.

Effect of naloxone on food competition aggression in food-restricted high and low aggression pigeons (Columba livia)

We determined the effect of the opiate receptor antagonist naloxone on aggression, emotion, feeder control, and eating behavior in high and low aggression female pigeons maintained at 80% of their normal weight and exposed to food competition interactions. Pigeons were divided into pairs by previously ranked high aggression (total time spent in offensive aggression exceeding 60 s/5 min; N = 6 pairs) and low aggression females (time spent in offensive aggression less than 10 s/5 min; N = 6 pairs). A pigeon in each pair received an sc dose of naloxone (1 mg kg-1 ml saline-1) and the other animal received the vehicle. Trials (10 min) were performed 30 min after the naloxone/vehicle administration. The naloxone group of high aggression pigeons showed lower scores of total time spent in offensive aggression (control: 98.6 ± 12.0; naloxone: 46.8 ± 6.6 s; P < 0.05) and higher scores of time spent in emotional responses (control: 3.5 ± 0.6; naloxone: 10.8 ± 2.4 s; P < 0.05) than controls. The other behaviors scored, feeder control and eating behavior, were not affected in this group. The naloxone group of low aggression pigeons, however, showed higher scores of offensive aggression than their controls (5.3 ± 1.3; naloxone: 28.7 ± 8.0 s; P < 0.05). The present results suggest that opiate receptor mechanisms are implicated in offensive aggression responses in high and low aggression pigeons. However, as reported for brain 5-hydroxytryptamine manipulation and GABA-A-benzodiazepine receptor manipulation, the effect of the opiate receptor antagonist on food competition aggression in pigeons was related to their pretreatment level of aggression.

Looking for the GAP effect in manual responses and the role of contextual influences in reaction time experiments

When the offset of a visual stimulus (GAP condition) precedes the onset of a target, saccadic reaction times are reduced in relation to the condition with no offset (overlap condition) - the GAP effect. However, the existence of the GAP effect for manual responses is still controversial. In two experiments using both simple (Experiment 1, N = 18) and choice key-press procedures (Experiment 2, N = 12), we looked for the GAP effect in manual responses and investigated possible contextual influences on it. Participants were asked to respond to the imperative stimulus that would occur under different experimental contexts, created by varying the array of warning-stimulus intervals (0, 300 and 1000 ms) and conditions (GAP and overlap): i) intervals and conditions were randomized throughout the experiment; ii) conditions were run in different blocks and intervals were randomized; iii) intervals were run in different blocks and conditions were randomized. Our data showed that no GAP effect was obtained for any manipulation. The predictability of stimulus occurrence produced the strongest influence on response latencies. In Experiment 1, simple manual responses were shorter when the intervals were blocked (247 ms, P < 0.001) in relation to the other two contexts (274 and 279 ms). Despite the use of choice key-press procedures, Experiment 2 produced a similar pattern of results. A discussion addressing the critical conditions to obtain the GAP effect for distinct motor responses is presented. In short, our data stress the relevance of the temporal allocation of attention for behavioral performance.

A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children

We report a fast (less than 3 h) and cost-effective melting temperature assay method for the detection of single-nucleotide polymorphisms in the MBL2 gene. The protocol, which is based on the Corbett Rotor Gene real time PCR platform and SYBR Green I chemistry, yielded, in the cohorts studied, sensitive (100%) and specific (100%) PCR amplification without the use of costly fluorophore-labeled probes or post-PCR manipulation. At the end of the PCR, the dissociation protocol included a slow heating from 60º to 95ºC in 0.2ºC steps, with an 8-s interval between steps. Melting curve profiles were obtained using the dissociation software of the Rotor Gene-3000 apparatus. Samples were analyzed in duplicate and in different PCR runs to test the reproducibility of this technique. No supplementary data handling is required to determine the MBL2 genotype. MBL2 genotyping performed on a cohort of 164 HIV-1-positive Brazilian children and 150 healthy controls, matched for age and sex and ethnic origin, yielded reproducible results confirmed by direct sequencing of the amplicon performed in blind. The three MBL2 variants (Arg52Cys, Gly54Asp, Gly57Glu) were grouped together and called allele 0, while the combination of three wild-type alleles was called allele A. The frequency of the A/A homozygotes was significantly higher among healthy controls (0.68) than in HIV-infected children (0.55; P = 0.0234) and the frequency of MBL2 0/0 homozygotes was higher among HIV-1-infected children than healthy controls (P = 0.0296). The 0 allele was significantly more frequent among the 164 HIV-1-infected children (0.29) than among the 150 healthy controls (0.18; P = 0.0032). Our data confirm the association between the presence of the mutated MBL2 allele (allele 0) and HIV-1 infection in perinatally exposed children. Our results are in agreement with the literature data which indicate that the presence of the allele 0 confers a relative risk of 1.37 for HIV-1 infection through vertical transmission.

Bioeffects of albumin-encapsulated microbubbles and real-time myocardial contrast echocardiography in an experimental canine model

Myocardial contrast echocardiography has been used for assessing myocardial perfusion. Some concerns regarding its safety still remain, mainly regarding the induction of microvascular alterations. We sought to determine the bioeffects of microbubbles and real-time myocardial contrast echocardiography (RTMCE) in a closed-chest canine model. Eighteen mongrel dogs were randomly assigned to two groups. Nine were submitted to continuous intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) plus continuous imaging using power pulse inversion RTMCE for 180 min, associated with manually deflagrated high-mechanical index impulses. The control group consisted of 3 dogs submitted to continuous imaging using RTMCE without PESDA, 3 dogs received PESDA alone, and 3 dogs were sham-operated. Hemodynamics and cardiac rhythm were monitored continuously. Histological analysis was performed on cardiac and pulmonary tissues. No hemodynamic changes or cardiac arrhythmias were observed in any group. Normal left ventricular ejection fraction and myocardial perfusion were maintained throughout the protocol. Frequency of mild and focal microhemorrhage areas in myocardial and pulmonary tissue was similar in PESDA plus RTMCE and control groups. The percentages of positive microscopical fields in the myocardium were 0.4 and 0.7% (P = NS) in the PESDA plus RTMCE and control groups, respectively, and in the lungs they were 2.1 and 1.1%, respectively (P = NS). In this canine model, myocardial perfusion imaging obtained with PESDA and RTMCE was safe, with no alteration in cardiac rhythm or left ventricular function. Mild and focal myocardial and pulmonary microhemorrhages were observed in both groups, and may be attributed to surgical tissue manipulation.

Immune factors and immunoregulation in tuberculosis

Pathogens causing tuberculosis and other chronic infectious diseases of major public health importance commonly have complex mechanisms involved in their persistence in the host despite specific and sometimes strong immune responses. These diseases are also associated with the lack of efficient vaccines, difficult therapeutics and a high mortality rate among susceptible individuals. Here, we will review features of the host immune response that contribute to the occurrence of disease. In addition, we propose that the immune responses observed in tuberculosis cannot be interpreted solely on the basis of a Th1-Th2 counter-regulatory paradigm since there is growing evidence that natural regulatory T cells may play an important role in the regulation of host immune responses against Mycobacterium tuberculosis. Thus, the development of more effective vaccines against this bacterial disease should take into account the role of natural regulatory T cells in the progression to severe disease and persistence of infection. Finally, new treatments based on manipulation of regulatory T cells should be investigated.

Green welding in practice

Efficient production and consumption of energy has become the top priority of national and international policies around the world. Manufacturing industries have to address the requirements of the government in relation to energy saving and ecologically sustainable products. These industries are also concerned with energy and material usage due to their rising costs. Therefore industries have to find solutions that can support environmental preservation yet maintain competitiveness in the market. Welding, a major manufacturing process, consumes a great deal of material and energy. It is a crucial process in improving a product’s life-cycle cost, strength, quality and reliability. Factors which lead to weld related inefficiencies have to be effectively managed, if industries are to meet their quality requirements and fulfil a high-volume production demand. Therefore it is important to consider some practical strategies in welding process for optimization of energy and material consumption. The main objective of this thesis is to explore the methods of minimizing the ecological footprint of the welding process and methods to effectively manage its material and energy usage in the welding process. The author has performed a critical review of the factors including improved weld power source efficiency, efficient weld techniques, newly developed weld materials, intelligent welding systems, weld safety measures and personnel training. The study lends strong support to the fact that the use of eco-friendly welding units and the quality weld joints obtained with minimum possible consumption of energy and materials should be the main directions of improvement in welding systems. The study concludes that, gradually implementing the practical strategies mentioned in this thesis would help the manufacturing industries to achieve on the following - reduced power consumption, enhanced power control and manipulation, increased deposition rate, reduced cycle time, reduced joint preparation time, reduced heat affected zones, reduced repair rates, improved joint properties, reduced post-weld operations, improved automation, improved sensing and control, avoiding hazardous conditions and reduced exposure of welder to potential hazards. These improvement can help in promotion of welding as a green manufacturing process.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). β-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Cell therapy in dilated cardiomyopathy: from animal models to clinical trials

Dilated cardiomyopathy can be the end-stage form and common denominator of several cardiac disorders of known cause, such as hypertensive, ischemic, diabetic and Chagasic diseases. However, some individuals have clinical findings, such as an increase in ventricular chamber size and impaired contractility (classical manifestations of dilated cardiomyopathy) even in the absence of a diagnosed primary disease. In these patients, dilated cardiomyopathy is classified as idiopathic since its etiology is obscure. Nevertheless, regardless of all of the advances in medical, pharmacological and surgical procedures, the fate of patients with dilated cardiomyopathy (of idiopathic or of any other known cause) is linked to arrhythmic episodes, severe congestive heart failure and an increased risk of sudden cardiac death. In this review, we will summarize present data on the use of cell therapies in animal models of dilated cardiomyopathies and will discuss the few clinical trials that have been published so far involving patients affected by this disease. The animal models discussed here include those in which the cardiomyopathy is produced by genetic manipulation and those in which disease is induced by chemical or infectious agents. The specific model used clearly creates restrictions to translation of the proposed cell therapy to clinical practice, insofar as most of the clinical trials performed to date with cell therapy have used autologous cells. Thus, translation of genetic models of dilated cardiomyopathy may have to wait until the use of allogeneic cells becomes more widespread in clinical trials of cell therapies for cardiac diseases.