Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy.

AIMS: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. METHODS AND RESULTS: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. CONCLUSIONS: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.

Disentangling the effects of foliar vs. floral herbivory of leaf-cutting ants on the plant reproductive success of Miconia nervosa (Smith) Triana (Family Melastomataceae).

Flower and leaf herbivory might cause relevant and negative impacts on plant fitness. While flower removal or damage by florivores produces direct negative effects on plant fitness, folivores affect plant fitness by reducing resource allocation to reproduction. In this study, we examine the effects of both flower and leaf herbivory by leaf-cutting ants on the reproductive success of the shrub species Miconia nervosa (Smith) Triana (Family Melastomataceae) in a fragment of Atlantic Forest in Northeast Brazil. We conducted a randomized block-designed field experiment with nine replicates (blocks), in which three plants per block were assigned to one of the three following treatments: undamaged plants (ant exclusion), leaf-damaged plants (ant exclusion from reproductive organs, but not from leaves), and flower + leaf-damaged plants (no exclusion of ants). We then measured flower production, fruit set, and fruit production. Our results showed that flower + leaf-damaged plants reduced flower production nearly twofold in relation to undamaged plants, while flower set in leaf-damaged plants remained constant. The number of flowers that turned into fruits (i.e., fruit set), however, increased by 15% in flower + leaf-damaged plants, while it slightly decreased in leaf-damaged compared to undamaged plants. Contrastingly, fruit production was similar between all treatments. Taken together, our results suggest a prominent role of ant floral herbivory across different stages of the reproductive cycle in M. nervosa, with no consequences on final fruit production. The tolerance of M. nervosa to leaf-cutting ant herbivory might explain its high abundance in human-modified landscapes where leaf-cutting ants are hyper-abundant.

Detecting reproductive system abnormalities of broiler breeder roosters at different ages.

The objective of this study was to detect the reasons of rooster's fertility decrease at 50 weeks of age. Therefore, the reproductive system of broiler breeder roosters was laparoscopic, macroscopic and histopathology evaluated, and a comparison of the anatomical aspect with the sperm analysis and birds' age was realized. Cobb roosters (n = 59) were distributed into two groups (30 and 50 weeks). Evaluations were performed with laparoscopy, macroscopy and histopathology, and seminal quality, blood serum testosterone concentration and weight were also determined. The old roosters presented smaller testicle size, higher intensity epididymal lithiasis and lower testicle sperm production, compared to the young roosters. The use of the endoscope could easily distinguish a normal-sized testicle than an atrophic one. Four old roosters with severe testicular atrophy did not show spermatogenesis, although three still had sperm in the ejaculate. This would falsely indicate a wrong diagnosis of normal fertility before the testicular atrophy took place. In conclusion, in addition to the weight increase with age, the testicular atrophy and impairment of sperm production seemed to be the main reason to the decrease in the rooster's fertility at 50 weeks of age. Therefore, the use of the laparoscopy as a way to detect the roosters with testicular atrophy before 50 weeks of age and their removal from them flock could be useful as a diagnostic tool to prevent the birds' fertility loss.

Can the chlorophyll-a fluorescence be useful in identifying acclimated young plants from two populations of Cecropia pachystachya Trec. (Urticaceae), under elevated CO2 concentrations?

The physiological behavior of PSII measured by chlorophyll a fluorescence explains stress responses; wonders if it can differentiate plants from different populations. For this purpose, acclimated young plants of two C. pachystachya populations were cultivated from seeds. Chlorophyll-a fluorescence was measured after fertilization and [CO(2)](e). In the first 48 h after fertilization there was a reduction in the maximum quantum yield of PSII, while the means obtained under [CO(2)](e) were significantly higher than in other treatments (0.8 and 0.81). The variable PI best expressed the different conditions tested. Compared to their respective controls, the reduction of DIo/CS was 35.89 % in population (P) and 41.89 % in population (I), while the polyphasic fluorescence kinetics differed between treatments, but not necessarily between populations, except for post-fertilization at I-P steps. The analysis of kinetics between Fo and Fj (Wt) showed no K band during the O-J phase. The interferences found in PSII reinforces the idea of reversible damage to PSII. This effect is directly related to the reduced electron transport rate and increased non-photochemical dissipation and may be similar to those observed under field conditions after planting; adjustment time depends, among other factors, on the genetic potential of the species.

Residual entropy and waterlike anomalies in the repulsive one dimensional lattice gas.

The thermodynamics and kinetics of the one dimensional lattice gas with repulsive interaction are investigated using transfer matrix technique and Monte Carlo simulations. This simple model is shown to exhibit waterlike anomalies in density, thermal expansion coefficient, and self-diffusion. An unified description for the thermodynamic anomalies in this model is achieved based on the ground state residual entropy which appears in the model due to mixing entropy in a ground state phase transition.

Neonatal innate immunity response in invasive candidiasis.

Infections caused by Candida spp. are frequent in critically hospitalized patients, especially among premature neonates, representing one of the most common healthcare-related infections. Although there is considerable production of current knowledge about the mechanisms of immune response, aspects involved in the newborn's innate defense are not fully understood. The aim of this study was to describe the innate immune mechanisms involved in the defense of neonates against invasive candidiasis. This is an integrative literature review from the Scopus, Scifinder, Medline, Web of Science databases and the electronic libraries ScienceDirect and Scielo, in the period between 2002 and 2020, with rescue based on primary descriptor Immunity Innate plus secondary descriptors Candidiasis Invasive AND Infant Newborn. We have observed the involvement of various mechanisms in the neonatal response against invasive candidiasis, including the recognition, signaling, recruitment, and initiation of an effective immune response. These mechanisms encompass the presence of antimicrobial peptides, phagocytosis, synthesis of reactive oxygen species, inflammatory mediators, and complex cell signaling systems mediated by Pattern Recognition Receptors (PRRs). With this study, it is expected to contribute to the expansion of knowledge about the immunological mechanisms involved in the innate immune response of the newborn against disseminated infections caused by Candida species, and in the same sense, highlight the importance of this knowledge as a reflex in the decrease in mortality in the neonatal period.

Effect of a Toothpaste for Sensitive Teeth on the Sensitivity and Effectiveness of In-office Dental Bleaching: A Randomized Clinical Trial.

OBJECTIVES: The present study aimed to evaluate the desensitizing effect of toothpaste for sensitive teeth on patient tooth sensitivity and on bleaching efficacy of the 38% hydrogen peroxide bleaching agent used for in-office bleaching compared to a regular toothpaste in a randomized clinical trial. METHODS AND MATERIALS: Forty-eight patients having maxillary right central incisors with darkness greater than A1 were selected for the present double-blind randomized clinical trial. Patients were randomly allocated into two groups: the placebo group, which used regular toothpaste, and the experimental group, which used sensitivity toothpaste. The intervention consisted of applying toothpaste with the aid of an individual tray for a period of 4 minutes daily, starting one week before the first bleaching session and interrupting use immediately after the second session. After allocation to one of the groups, individuals received in-office dental bleaching with a 40-minute application of 38% hydrogen peroxide for two sessions with an interval of one week. The incidence and intensity of sensitivity were assessed using a visual analogue scale and a numeric analogue scale. Sensitivity was measured immediately before each session, 1 hour, 24 hours, and 48 hours after each bleaching session and four weeks after the second bleaching session. Tooth shade was evaluated using a spectrophotometer and by comparison with the VITA Classical Shade Guide (Vita Zahnfabrik, Bad Säckingen, Germany). Tooth shade was evaluated before the first bleaching session, one week after the first bleaching session, one week after the second bleaching session and four weeks after the second bleaching session. Participants and professionals who performed the bleaching, shade, and sensitivity assessments were blinded to the group of patients they were treating or assessing. For the incidence of hypersensitivity, the results were evaluated by comparing the groups at different evaluation times with the Mann-Whitney test for comparison between groups, the Friedman test for repeated measures, and the Tukey test for comparison of times. Shade change on the guide was analyzed using the Mann-Whitney test for comparison between groups and the Wilcoxon test for comparison between times. Shade change by the spectrophotometer was analyzed using the t-test for comparison between groups and the paired t-test for comparison between times. All analyses were performed with a significance level of 5%. RESULTS: There was no difference in the pattern of dental hypersensitivity between groups. For all shade measures, there was no difference between the bleaching results, and no statistically significant difference was observed between the study groups. CONCLUSION: The use of arginine-based desensitizing toothpaste did not interfere with the bleaching ability of hydrogen peroxide and was not effective in reducing the sensitivity caused by in-office tooth bleaching.

Infection with Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in solid organ transplantation.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae is spreading globally and represents a challenge in infection control and treatment. Solid organ transplant (SOT) recipients are especially at risk for infection by multidrug-resistant bacteria, and little is known about infection with KPC-producing organisms in this setting. The aim of this study was to describe the clinical and microbiologic aspects of KPC-producing K. pneumoniae infections in SOT recipients. A KPC-2-producing K. pneumoniae outbreak was identified in a public teaching tertiary care hospital in São Paulo, Brazil, in June 2009. During the outbreak, cases of KPC-2-producing K. pneumoniae infection in SOT recipients occurred between July 2009 and February 2010; these cases were retrospectively reviewed. Overall, 12 episodes of infection with KPC-producing K. pneumoniae occurred in 2 heart, 4 liver, and 6 kidney transplant recipients with incidence rates of 16.7%, 12.9%, and 26.3% in heart, liver, and kidney transplantation, respectively. Infection occurred at a median time of 20 days after transplantation. Primary infection sites were as follows: 4 urinary tract infections, 4 bloodstream infections, 2 pneumonias, and 2 surgical site infections. All patients except one had received antibiotics in the last 30 days, mostly piperacillin-tazobactam or glycopeptides. All strains exhibited susceptibility to amikacin and gentamicin. Patients were treated with tigecycline plus polymyxin B (3 cases), polymyxin B plus carbapenem (3 cases), polymyxin B alone (3 cases), or tigecycline plus imipenem (1 case). In 2 cases, patients received only carbapenem, and death occurred before the final culture result. The overall 30-day mortality rate was 42%. In this series of KPC-producing K. pneumoniae infection in SOT recipients, the infection occurrence was high during an institutional outbreak and was potentially life threatening.

Nutritional value and sensory properties of common carp (Cyprinus carpio L.) fillets enriched with sustainable and natural feed ingredients.

Declines across global fishery stocks forced aquaculture feed manufacturers to search for new and sustainable components. Therefore, the aim of study was assessing nutritional value and sensory properties of meat of common carp (Cyprinus carpio L.) fed for 116 days with two blends. The control feed contained 5% of fishmeal and vegetable oils (rapeseed and soybean) as sole fat sources. While in the experimental diet half of the fishmeal was replaced with a blend of microalgae (Spirulina sp., Chlorella sp.), macroalgae (Laminaria digitata) and vegetable oil was replaced with salmon oil. Proximate composition, energy value, fatty acid profile of meat, nutritional characteristics of fat and protein as well as culinary properties of fillets were assessed. Fillets of carp fed experimental diet had a higher level of protein, lower level of fat and energy value. Intramuscular fat of fish fed with the experimental diet had a better parameters of quality. Protein in the meat of fish from both groups was characterized by a high quality comparing to the protein standard. Our study showed that meat of carp fed with experimental feed enriched with sustainable and natural feed ingredients can be a sensorily attractive source of nutritious ingredients in the human diet.

Plasma biochemistry, gene expression and liver histomorphology in common carp (Cyprinus carpio) fed with different dietary fat sources.

Demand for omega-3 long chain polyunsaturated fatty acids has become global challenge for aquaculture and different components have been used to increase nutritional value of fillets. The aim of this study was to evaluate influences of feeds on zootechnical parameters, biochemical plasma parameters, expression of lipid-dependent genes, hepatocyte histomorphologies, and fatty acid profiles in common carp fillets. We compared a control diet (CTRL), mimicking a commercial feed formulation for common carp, with three diets containing blends of vegetable oils and a DHA-rich alga (Schizochytrium sp.) included at 3.125% (CB1) or 1.563% (CB2), and 2.1% salmon oil (CB3). The study revealed no differences in final body weight of fish fed CB1-3 diets in comparison with significantly lower CTRL. Concentrations of all biochemical parameters in plasma increased gradually in fish fed CB1-3 diets when compared to CTRL diet, with exception of triacylglycerol levels. Expression of hepatic fas, elovl-5a and pparα genes increased significantly in fish fed CB1 and CB2. Additionally, eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) accumulation in muscle tissue was directly proportional to the amounts supplied in the diets. Our study revealed that carp fillet profiles can be manipulated for DHA and EPA-contents using enriched diets, depending on the source of fat.

Quality improvement of common carp (Cyprinus carpio L.) meat fortified with n-3 PUFA.

The effect of carp feeding with n-3 PUFA-enriched feed (Schizochytrium sp. meal or salmon oil) on nutritional quality indicators (proximal composition, fatty acid profile of fat) and culinary quality (color parameters, texture, sensory properties) was evaluated. Highly significant effects of carp nutrition on chemical composition and fat characteristics, L* and a* color parameters, muscle fiber size, endomysium thickness, moisture and taste of fillets were determined. Fillets obtained from carps fed with the experimental feed contained less protein and more crude fat and had larger muscle fibers, but scored more highly in the sensory evaluation of moisture and fishy taste. In the fat of carp fed the enriched feed, a greater share of total PUFA, n-3 PUFA, total EPA and DHA, n-3/n-6 ratio, and a smaller share of total MUFA were observed compared with control fish. However, no effect of nutrition on the texture of carp fillets, assessed either instrumentally or using sensory methods, was found. The use of Schizochytrium sp. meal as a source of EPA and DHA gave much better results than salmon oil, as it allowed a higher content of these valuable fatty acids to be achieved, without compromising quality.

The effect of the Rochagan on radiolabeling with (99m)Tc.

Radionuclides are used in nuclear medicine by variety of diagnostic procedures. The labeling of red blood cells (RBC) with (99m)Tc is a current method applied in clinical nuclear medicine. Drugs can alter this labeling and modify the disposition of the radiopharmaceuticals. The influence of Rochagan on the labeling of blood constituents with (99m)Tc was reported. Samples of blood were incubated with different concentrations of Rochagan (0%; 6.25%; 12.5%; 25%; 50%; 100%). Stannous chloride and (99m)Tc (3.7MBq/mL) were added. Plasma (P) and (RBC) were isolated and precipitated with thricloroacetic acid 5%. The insoluble (IF) and soluble fractions (SF) were separated. The %ATI in RBC, IF-P and IF-RBC were calculated. The %ATI on RBC decreased significantly (p<0.05) from control to all concentrations of Rochagan, respectively: 90.15 + or - 0.14(control) to 70.80 + or - 4.21; to 64.36 + or - 0.33; to 57.30 + or - 1.56; to 50.28 + or - 2.71; to 42.41 + or - 2.24; on IF-RBC, respectively: 84.70 + or - 0.87(control) to 67.16 + or - 4.38; to 63.63 + or - 2.92; to 59.02 + or - 3.17; to 43.75 + or - 1.00; to 24.15 + or - 0.94 and also on IF-P, respectively: 83.46 + or - 1.09(control) to 50.90 + or - 3.36; to 35.46 + or - 4.13; to 35.78 + or - 2.31; to 28.74 + or - 3.09; to 19.66 + or - 1.34. The analyses were performed by T-Student and Mann Whitney tests, p<0.05. This effect was probably due to products present in Rochagan that may complex with ions or have a direct/indirect effect on intracellular stannous ion concentration.

Cryoprotective effects of antifreeze proteins delivered into zebrafish embryos.

Fish embryo cryopreservation, which is useful in aquaculture or biodiversity conservation, is still far from being achieved. Structural barriers reduce the entrance of cryoprotectants into embryo compartments. Previous studies demonstrated a better ability for freezing in Arctic species which naturally express antifreeze proteins (AFPs). In this study, AFPs were delivered in early zebrafish embryos by incubation in media containing protein. Their cryoprotective effects were then analyzed. Chilling sensitivity was evaluated at 4 degrees C and -10 degrees C. Survival rates significantly increased in embryos incorporating AFPI and kept at -10 degrees C. To analyze their effects on cryopreservation, 5-somite embryos were vitrified. Incorporation of AFPI reduced the percentage of embryos that collapsed at thawing (14.2% of AFPI-treated embryos and 48.9% of controls). Cellular damage caused by vitrification was assessed after thawing by cell dissociation and further analysis of cell survival in culture (SYBR-14/IP labeling). The percentage of viable cells at thawing ranged from 25 to 50%, considered incompatible with embryo development. Cells recovered from frozen-control embryos did not survive in culture. However, the incorporation of AFPs allowed survival similar to that of cells recovered from non-frozen embryos. Blastomere cryopreservation trials incorporating AFPI in the extender also demonstrated a significant increase in viability after freezing. Our findings demonstrated that delivery of AFPs into zebrafish embryos by incubation in media containing protein at early stages is a simple and harmless method that increases cryoprotection of the cellular compartment. This beneficial effect is also noticed in blastomeres, encouraging their use in further protocols for embryo cryopreservation.

Identifying differences in protein expression levels by spectral counting and feature selection.

Spectral counting is a strategy to quantify relative protein concentrations in pre-digested protein mixtures analyzed by liquid chromatography online with tandem mass spectrometry. In the present study, we used combinations of normalization and statistical (feature selection) methods on spectral counting data to verify whether we could pinpoint which and how many proteins were differentially expressed when comparing complex protein mixtures. These combinations were evaluated on real, but controlled, experiments (yeast lysates were spiked with protein markers at different concentrations to simulate differences), which were therefore verifiable. The following normalization methods were applied: total signal, Z-normalization, hybrid normalization, and log preprocessing. The feature selection methods were: the Golub index, the Student t-test, a strategy based on the weighting used in a forward-support vector machine (SVM-F) model, and SVM recursive feature elimination. The results showed that Z-normalization combined with SVM-F correctly identified which and how many protein markers were added to the yeast lysates for all different concentrations. The software we used is available at http://pcarvalho.com/patternlab.

[Effect of an intensive nutritional treatment in nutritional status of head and neck cancer patients in stages III and IV]. / Evaluación del impacto de un tratamiento nutricional intensivo sobre el estado nutricional de pacientes con cáncer de cabeza y cuello en estadio III y IV.

OBJECTIVE: Malnutrition and accelerated weight loss are frequent secondary diagnosis in patients with cancer. Head and neck cancer (H & N Cancer) affects nutritional status because of the tumor type and localization. The aim of the study was to assess the effect of an intensive nutritional treatment (INT) on nutritional status of H & N cancer patients, stages III and IV and to compare that with a historical control whose nutritional treatment was conventional (CT). METHODS: All patients were nutritionally assessed before oncological treatment started (anthropometrical, biochemical, and dietetically). A nutritional feeding plan according to their nutritional personal need was calculated. In case it was impossible to cover all the nutritional requirements orally, a feeding tube was placed. Nutritional follow up was performed each 21 days, during their oncological treatment in four occasions. The results obtained were compared with those obtained from the patients who received the CT that consisted only in nutritional counseling by the attending physician; the statistical test used was Mann Whitney U test. RESULTS: The INT was associated with maintenance of the nutritional status along their oncological treatment. The statistical significant parameters were: weight 55 to 47 kg in the CT group vs 59 a 58 kg in the INT group (p < 0.0001), and hemoglobin: from 13 to 11 g/dl in the CT group vs 14 to 13 g/dl in the INT group (p < 0.002) as the most important ones. In the rest of the data we can observe a clear tendency of increasing the nutrition parameters in patients the INT group, while in the CT group, they showed a persistent decrease. CONCLUSION: We concluded that patients who received the INT as part of their oncological treatment deteriorated less their nutritional status than those who received a CT.

Better Selection Criteria With Prognostic Factors for Liver Transplantation.

BACKGROUND: Liver transplantation has evolved significantly in recent years, with each advancement part of the effort toward increasing patient and graft survival as well as quality of life. The objective of this study was to evaluate the prognostic factors and selection criteria for liver transplantation. METHODS: Our study was a statistical analysis, logistic regression, and survival evaluation of a total of 80 liver transplants that were performed between June 1, 2016 and September 24, 2016. Recipient factors evaluated included age, retransplantation, hemodialysis, cardiac risk, portal vein thrombosis, hospitalization, fulminant hepatitis, previous surgery, renal failure, and Model for End-stage Liver Disease (MELD) score. Donor factors included age, cardiac arrest, acidosis, days in the intensive care unit, steatosis, and vasoactive drug use. RESULTS: Of the 80 patients transplanted, 65 deceased donor liver transplants (DDLTs) and 15 living donor liver transplants (LDLTs) were performed. LDLT overall 1-year patient survival was 77.5% and graft survival 75%, and DDLT overall patient survival was 89.23% and graft survival was 86.15%. On evaluated score criteria analyzed we observed a significant score on recipient (P = .01) and not significant on donor (P =.45). Isolated factors evaluated included recipient age (relative risk [RR] 3.15, 95% confidence interval [CI] 0.89 to 11.09; P = .074), retransplant (RR 4.22, 95% CI 1.36 to 13.1; P = .013), and hemodialysis (RR 4.23, 95% CI 1.45 to 12.31, P = .008). On donor evaluation, we observed moderate and severe steatosis (RR 3.8, 95% CI 0.86 to 16.62; P = .06). CONCLUSION: In conclusion, we demonstrate a relevant model of criteria selection of liver transplant patients that is able to make a better match between the donor and recipient allocation for a better graft and patient survival.

Bioremediation of trace organic compounds found in precious metals refineries' wastewaters: a review of potential options.

Platinum group metal (PGM) refining processes produce large quantities of wastewater, which is contaminated with the compounds that make up the solvents/extractants mixtures used in the process. These compounds often include solvesso, beta-hydroxyxime, amines, amides and methyl isobutyl ketone. A process to clean up PGM refinery wastewaters so that they could be re-used in the refining process would greatly contribute to continual water storage problems and to cost reduction for the industry. Based on the concept that organic compounds that are produced biologically can be destroyed biologically, the use of biological processes for the treatment of organic compounds in other types of waste stream has been favoured in recent years, owing to their low cost and environmental acceptability. This review examines the available biotechnologies and their effectiveness for treating compounds likely to be contained in precious metal extraction process wastewaters. The processes examined include: biofilters, fluidized bed reactors, trickle-bed bioreactors, bioscrubbers, two-phase partitioning bioreactors, membrane bioreactors and activated sludge. Although all processes examined showed adequate to excellent removal of organic compounds from various gaseous and fewer liquid waste streams, there was a variation in their effectiveness. Variations in performance of laboratory-scale biological processes are probably due to the inherent change in the microbial population composition due to selection pressure, environmental conditions and the time allowed for adaptation to the organic compounds. However, if these factors are disregarded, it can be established that activated sludge and membrane bioreactors are the most promising processes for use in the treatment of PGM refinery wastewaters.

The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures.

To date, all attempts at fish embryo cryopreservation have failed. One of the main reasons for this to occur is the high chilling sensitivity reported in fish embryos thus emphasizing the need for further testing of different methods and alternative cryoprotective agents (CPAs) in order to improve our chances to succeed in this purpose. In this work we have used the antifreeze protein type I (AFP I) as a natural CPA. This protein is naturally expressed in sub-arctic fish species, and inhibits the growth of ice crystals as well as recrystallization during thawing. Embryos from Sparus aurata were microinjected with AFP I at different developmental stages, 2 cells and blastula, into the blastomere-yolk interface and into the yolk sac, respectively. Control, punctured and microinjected embryos were subjected to chilling at two different temperatures, 0 degrees C (1h) and -10 degrees C (15min) when embryos reached 5-somite stage. Embryos were subjected to -10 degrees C chilling in a 3M DMSO extender to avoid ice crystal formation in the external solution. Survival after chilling was established as the percentage of embryos that hatch. To study the AFP I distribution in the microinjected embryos, a confocal microscopy study was done. Results demonstrate that AFP I can significantly improve chilling resistance at 0 degrees C, particularly in 2-cell microinjected embryos, displaying nearly 100% hatching rates. This fact is in agreement with the confocal microscopy observations which confirmed the presence of the AFP protein in embryonic cells. These results support the hypothesis that AFP protect cellular structures by stabilizing cellular membranes.

Septicemia caused by Vibrio cholerae O1 biotype El Tor, in São Paulo, Brazil.

We reported a case of septicemia by Vibrio cholerae O1, in São Paulo, Brazil. A 70-year-old male patient, living in an urban area, entered the emergency service having sepsis, dying 12 hours later. Blood culture was positive for Vibrio cholerae O1. This is the first case of bacteremia by Vibrio cholerae O1 reported in South America.