Impact of Distinct Freezing Curves on the Quality of Ram Sperm after Thawing.

BACKGROUND: Automated equipment with customized freezing curves can be used to cryopreserve ram sperm, but none is considered a standard. OBJECTIVE: This study compared the post-thawing quality of ram sperm frozen using a conventional freezing curve and two controlled-rate curves. MATERIALS AND METHODS: Six ejaculates were collected from four rams (n = 24). In the conventional curve (110 min), sperm was cooled at -0.3 to -0.5°C min-1 until 5°C, stabilized for 60 min and exposed to liquid nitrogen (LN2) vapor for 10 min (-80°C) before submersion. The slow-customized (SC) curve (126.2 min) used a rate of -0.25°C min-1 until 5°C, stabilization for 60 min and a rate of -20°C min-1 until -120°C before immersion in LN2. Rates for the fast-customized (FC) curve (75 min) were: -0.3°C min-1 until 5°C; -3°C min-1 until -10°C; -5°C min-1 until -35°C; and -4°C min-1 until immersion in LN2 (-43°C). RESULTS: Velocity in a straight line and beat-cross frequency were lower for spermatozoa frozen with the FC than with the conventional curve (P < 0.05). The FC curve resulted in more membrane and acrosome damages than the other curves (P < 0.05). Mitochondrial membrane potential was lower with the SC than with the other curves (P < 0.05). CONCLUSION: The conventional curve was more efficient than both tested automated freezing curves. The FC curve may be an alternative to the SC curve due to the shorter processing time.

Diagnostic accuracy of thromboelastometry and its correlation with the HPLC-MS/MS quantification test.

The aim of the study was to evaluate the diagnostic accuracy of thromboelastometry for assessing rivaroxaban concentrations. The accuracy of thromboelastometry was compared with the high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method, which is the gold standard for drug plasma monitoring (the reference standard). Forty-six clinically stable patients were treated with 10, 15, or 20 mg of rivaroxaban once daily (OD group) or 15 mg twice a day (BID group) (no particular indication for treatment). Patient samples were collected 2 h after the use of the medication (peak) and 2 h before the next dose (trough). The rivaroxaban plasma concentrations were determined via HPLC-MS/MS, and thromboelastometry was performed using a ROTEM® delta analyzer. There were significant prolongations in clotting time (CT) for the 10, 15, and 20 mg of rivaroxaban treatments in the OD groups. In the 15 mg BID group, the responses at the peak and trough times were similar. At the peak times, there was a positive correlation between the plasma concentration of rivaroxaban and CT (Spearman correlation rho=0.788, P<0.001) and clot formation time (rho=0.784, P<0.001), and a negative correlation for alpha angle (rho=-0.771, P<0.001), amplitude after 5 min (rho=-0.763, P<0.001), and amplitude after 10 min (rho=-0.680, P<0.001). The CT presented higher specificity and sensitivity using the cut-off determined by the receiver characteristics curve. ROTEM has potential as screening tool to measure possible bleeding risk associated with rivaroxaban plasma levels.

Diagnostic accuracy of thromboelastometry and its correlation with the HPLC-MS/MS quantification test

The aim of the study was to evaluate the diagnostic accuracy of thromboelastometry for assessing rivaroxaban concentrations. The accuracy of thromboelastometry was compared with the high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method, which is the gold standard for drug plasma monitoring (the reference standard). Forty-six clinically stable patients were treated with 10, 15, or 20 mg of rivaroxaban once daily (OD group) or 15 mg twice a day (BID group) (no particular indication for treatment). Patient samples were collected 2 h after the use of the medication (peak) and 2 h before the next dose (trough). The rivaroxaban plasma concentrations were determined via HPLC-MS/MS, and thromboelastometry was performed using a ROTEM® delta analyzer. There were significant prolongations in clotting time (CT) for the 10, 15, and 20 mg of rivaroxaban treatments in the OD groups. In the 15 mg BID group, the responses at the peak and trough times were similar. At the peak times, there was a positive correlation between the plasma concentration of rivaroxaban and CT (Spearman correlation rho=0.788, P<0.001) and clot formation time (rho=0.784, P<0.001), and a negative correlation for alpha angle (rho=−0.771, P<0.001), amplitude after 5 min (rho=−0.763, P<0.001), and amplitude after 10 min (rho=−0.680, P<0.001). The CT presented higher specificity and sensitivity using the cut-off determined by the receiver characteristics curve. ROTEM has potential as screening tool to measure possible bleeding risk associated with rivaroxaban plasma levels.

Time collection and storage conditions of lipid profile.

The stability of samples is crucial for getting reliable concentrations of many analytes, including lipid profile. Thus, the goal of this study was to analyze lipid profile under different storage and temperature conditions. This was a prospective study with 809 patients of both genders. Total cholesterol, triglycerides, high-density lipoprotein cholesterol, low density lipoprotein cholesterol and non-high-density lipoprotein were measured within 1 h from collection at room temperature, after 2-3 h of refrigeration (8°C) and after 4-5 h at room temperature. The processing time and storage conditions did not affect the analytes measured. These findings are important for multicenter studies, because of the difficulties related to centrifugation and freezing of samples immediately after collection.

Expression of paraoxonase types 1, 2 and 3 in reproductive tissues and activity of paraoxonase type 1 in the serum and seminal plasma of bulls.

The paraoxonases types 1, 2 and 3 (PON1, PON2 and PON3, respectively) are enzymes that degrade lipid peroxides, preventing oxidative damages relevant for male reproductive function. This study determined the expression of those three paraoxonases in reproductive tissues of bulls and evaluated correlations among the activity of PON1 in the serum and seminal plasma with breeding soundness parameters in bulls. The expression of PON1, PON2 and PON3 was characterised by RT-PCR in samples of testicular parenchyma, vesicular glands and epididymis collected from three slaughtered bulls. All three paraoxonases were expressed in the testicular parenchyma, PON2 and PON3 were both expressed in the epididymis head and PON3 was also expressed in the epididymis tail. The PON1 activity was determined in samples of serum and seminal plasma from 110 bulls submitted to breeding soundness evaluation. There was a strong correlation (r = .90) between the activity of the PON1 in both serum and seminal plasma (p < .0001). The PON1 activity in the seminal plasma was positively correlated with ejaculate's colour, sperm mass activity (p = .04), motility, vigour and viability (all p < .01). Thus, PON1 may be a potential marker for sperm motility and viability in bulls.

Spatial patterns of fish standing biomass across Brazilian reefs.

A large fish-count dataset from the Brazilian province was used to describe spatial patterns in standing biomass and test if total biomass, taxonomic and functional trophic structure vary across nested spatial scales. Taxonomic and functional structure varied more among localities and sites than among regions. Total biomass was generally higher at oceanic islands and remote or protected localities along the coast. Lower level carnivores comprised a large part of the biomass at almost all localities (mean of 44%), zooplanktivores never attained more than 14% and omnivores were more representative of subtropical reefs and oceanic islands (up to 66% of total biomass). Small and large herbivores and detritivores varied greatly in their contribution to total biomass, with no clear geographical patterns. Macrocarnivores comprised less than 12% of the biomass anywhere, except for two remote localities. Top predators, such as sharks and very large groupers, were rare and restricted to a few reefs, suggesting that their ecological function might have already been lost in many Brazilian reefs.

Therapeutic salivary monitoring of IV busulfan in patients undergoing hematopoietic stem cell transplantation: a pilot study.

Individual therapeutic monitoring of busulfan (BU) minimizes its toxicity and improves the therapeutic outcomes during hematopoietic stem cell transplantation (HSCT). For individual dose adjustment, several blood collections are performed that are uncomfortable for patients. The aim of this pilot study was to validate a laboratory method for quantification of BU in saliva and to present the results obtained using this protocol in HSCT patients. We performed analyses of selectivity, precision and accuracy of saliva with standard concentrations of BU using ultra-high-performance liquid chromatography with diode array detection. We also determined salivary and plasmatic concentrations of BU in six HSCT patients. Saliva exhibited excellent selectivity, precision and accuracy for quantification of BU. In the patient samples, significant correlations were noted between plasmatic and salivary concentrations of BU (r=0.97, P<0.001 in the test dose; r=0.93, P<0.001 in the adjusted dose). Passing &Bablok regression revealed good agreement between the two methods (R2=0.956 for test dose; R2=0.927 for adjusted dose). In conclusion, the saliva is safe for laboratory BU measurement. The good agreement with plasma encourages further clinical studies using saliva for BU therapeutic monitoring.

Spatial distribution of nominally herbivorous fishes across environmental gradients on Brazilian rocky reefs.

Assemblages of roving herbivores were consistently different between eastern, warmer, sheltered sites and western, colder, more wave-exposed sites. At eastern sites, detritivorous-herbivorous species dominated while omnivores had the highest biomass and were dominant at western sites. Macroalgivores did not show any trends related to location. These distributional patterns, at relatively small spatial scales of a few kilometres, mirror large-scale latitudinal patterns observed for the studied species along the entire Brazilian coast, where cold water associated species are abundant on south-eastern rocky reefs (analogous to the western sites of this study), and tropical species are dominant on north-eastern coral reefs (analogous to the eastern sites). Species-level analyses demonstrated that depth was an important factor correlated with biomasses of Diplodus argenteus, Sparisoma axillare and Sparisoma tuiupiranga, probably due to resource availability and interspecific competition. Herbivorous fish assemblages in the study area have been historically affected by fishing, and combined with the variation in assemblage structure, this is likely to have important, spatially variable effects on the dynamics of benthic communities.

Hazard Analysis and Critical Control Points System for a Bull Semen Production Centre.

Bull semen production centres (SPC) generally present satisfactory quality control for sperm processing, but non-standardized hygiene procedures. This study describes a Hazard Analysis and Critical Control Points (HACCP) system developed for bull SPC and subsequently implemented in a commercial SPC. After the identification of hazards at each step of semen processing and the determination of their risk and severity, monitoring and corrective procedures were designed to assess the system's efficiency. The HACCP system identified six microbiological hazards, 10 physical hazards, four chemical hazards and three critical control points. After the establishment of Good Processing Practices, Standard Operating Procedures and Standard Sanitizing Operating Procedures, the system was validated through an audit, to identify eventual failures and to define measures to correct them.

Between-Habitat Variation of Benthic Cover, Reef Fish Assemblage and Feeding Pressure on the Benthos at the Only Atoll in South Atlantic: Rocas Atoll, NE Brazil.

The Southwestern Atlantic harbors unique and relatively understudied reef systems, including the only atoll in South Atlantic: Rocas atoll. Located 230 km off the NE Brazilian coast, Rocas is formed by coralline red algae and vermetid mollusks, and is potentially one of the most "pristine" areas in Southwestern Atlantic. We provide the first comprehensive and integrative description of the fish and benthic communities inhabiting different shallow reef habitats of Rocas. We studied two contrasting tide pool habitats: open pools, which communicate with the open ocean even during low tides, thus more exposed to wave action; and closed pools, which remain isolated during low tide and are comparatively less exposed. Reef fish assemblages, benthic cover, algal turfs and fish feeding pressure on the benthos remarkably varied between open and closed pools. The planktivore Thalassoma noronhanum was the most abundant fish species in both habitats. In terms of biomass, the lemon shark Negaprion brevirostris and the omnivore Melichtys niger were dominant in open pools, while herbivorous fishes (mainly Acanthurus spp.) prevailed in closed pools. Overall benthic cover was dominated by algal turfs, composed of articulated calcareous algae in open pools and non-calcified algae in closed pools. Feeding pressure was dominated by acanthurids and was 10-fold lower in open pools than in closed pools. Besides different wave exposure conditions, such pattern could also be related to the presence of sharks in open pools, prompting herbivorous fish to feed more in closed pools. This might indirectly affect the structure of reef fish assemblages and benthic communities. The macroalgae Digenea simplex, which is uncommon in closed pools and abundant in the reef flat, was highly preferred in herbivory assays, indicating that herbivory by fishes might be shaping this distribution pattern. The variations in benthic and reef fish communities, and feeding pressure on the benthos between open and closed pools suggest that the dynamics in open pools is mostly driven by physical factors and the tolerance of organisms to harsh conditions, while in closed pools direct and indirect effects of species interactions also play an important role. Understanding the mechanisms shaping biological communities and how they scale-up to ecosystem functioning is particularly important on isolated near-pristine systems where natural processes can still be studied under limited human impact.

Contribution of boars to reproductive performance and paternity after homospermic and heterospermic artificial insemination.

Heterospermic AI is commonly used in swine despite preventing precise evaluation of individual boar fertility. The present study compared the contribution of four boars (A, B, C and D) for reproductive performance and for paternity using homospermic and heterospermic (AB, AC, AD, BC, BD and CD) AI (n=204 for homospermic AI; n=307 for heterospermic AI). Blood samples from the four boars, from all sows inseminated with heterospermic doses and from the umbilical cords of their piglets, as well as tissue smears from mummified fetuses, were genotyped using single nucleotide polymorphisms (SNPs). Differences among boars were detected for the in vitro oocyte penetration rate and for the number of spermatozoa per oocyte (P<0.05), but not for sperm motility, mitochondrial functionality and integrity of the membrane, acrosome and DNA (P>0.05). Homospermic and heterospermic AI resulted in similar (P>0.05) farrowing rates (90.5% and 89.9%, respectively) and total litter size (12.4±0.4 and 12.7±0.7, respectively). Farrowing rate was lower for Boar B than for Boar C (P<0.05), but no other differences in reproductive performance among boars were observed with homospermic AI. The SNPs determined the paternity of 94.2% of the piglets sired by heterospermic AI. In the AC pool, paternity contribution per boar was similar (P>0.05), but differences between boars occurred in all other pools (P<0.05). Boar D achieved the greatest paternity contribution in all pools and parity categories (nearly 60%), whereas Boar B sired the fewest piglets (at most 40%). Reproductive performance was similar with homospermic and heterospermic AI, but differences in performance among boars undetected with homospermic AI were only evident after genotyping the piglets sired through heterospermic AI.

Reef fish structure and distribution in a south-western Atlantic Ocean tropical island.

The community structure of the reef fish fauna of Trindade Island, a volcanic oceanic island located 1160 km off the coast of Brazil, is described based on intensive visual censuses. Seventy-six species were encountered in 252 censuses, with mean ± S.E. of 99 ± 3 individuals and 15.7 ± 0.3 species 40 m(-2) transect. The average fish biomass, calculated from length-class estimation, was 22.1 kg 40 m(-2) transect. The species contributing most to biomass were, in decreasing order, Melichthys niger, Cephalopholis fulva, Kyphosus spp., Holocentrus adscensionis, Sparisoma amplum, Sparisoma axillare, Acanthurus bahianus and Epinephelus adscensionis. Carnivorous fishes were the largest trophic group in terms of biomass, followed by omnivores and roving herbivores. The two predominant types of reef habitat, fringing reefs built by coralline algae and rocky reefs made of volcanic boulders, showed significant differences in the biomass and the abundance of the trophic guilds. Within each habitat type, significant differences in species richness, density and biomass were detected among crest, slope and interface zones. Although similar in overall species composition to coastal reefs in Brazil, the fish fauna of Trindade Island shares certain characteristics, such as a high abundance of planktivores, with other Brazilian oceanic islands. Despite comparatively high fish biomass, including the macro-carnivorous species habitually targeted by fisheries, signs of overfishing were evident. These findings highlight the urgency for a conservation initiative for this isolated, unique and vulnerable reef system.

Atividade anti-HSV-1 in vitro de galactomanana quimicamente sulfatada de sementes de Leucaena leucocephala / In vitro anti-HSV-1 activity of a chemically sulfated galactomannan from Leucaena leucocephala seeds

Obteve-se uma galactomanana quimicamente sulfatada (LLS-2) a partir de polissacarídeo extraído de sementes de Leucaena leucocephala, a qual apresentou 15.2% de sulfato e grau de derivatização de 0,60, e, seu efeito antiviral sobre a replicação do vírus Herpes simplex tipo 1 (HSV-1) em células Vero foi avaliado pela metodologia de redução do número de unidades formadoras de placas. LLS-2 apresentou 93.7% de inibição da replicação viral à concentração de 2,5 ?g/ml, quando adicionado durante as etapas iniciais de replicação, com um índice de seletividade maior que 1.000, sugerindo que LLS-2 inibe a ligação de HSV-1 às células hospedeiras.

A galactomannan extracted from the seeds of Leucaena leucocephala was sulfated chemically, yielding a polymer (LLS-2) with 15.2% sulfate by weight (degree of sulfation 0.60), and its effect on the replication of Herpes simplex virus 1 (HSV-1) in Vero cells was investigated. When added during infection and early replication, LLS-2 showed 93.7% inhibition of HSV-1 replication at a concentration of 2.5 ?g/mL, according to the reduction in the number of viral plaques, and a selectivity index higher than 1,000, suggesting that it inhibits HSV-1 binding to the host cell.

Parallelisation of a multi-neighbourhood local search heuristic for a phylogeny problem.

In this work we study a phylogeny problem. That is, given a collection of organisms, we want to reconstruct the evolutionary history of the organisms. We are interested in inferring relationships between the organisms. For a number of reasonable biological hypotheses the problem becomes NP-hard. Besides that, the problem data is large enough to inhibit anyone using exact algorithms to solve, in practical computational time, real instances of the problem. In this work, we propose an innovative technique based on local search procedures that use multiple starts and diversified neighbourhoods.

Diet and trophic plasticity of an herbivorous blenny Scartella cristata of subtropical rocky shores.

The goals of the present study were to investigate the feeding rates of Scartella cristata, accounting for different water temperature regimes, and to analyse spatial and seasonal variations in its diet. The feeding rate of S. cristata was higher in warmer waters and was not correlated with season. A typical herbivorous diel feeding pattern was observed, with a steady increase in bite rate throughout the morning until a plateau in the early afternoon, followed by an abrupt decrease until the end of the daylight period. The gut contents of S. cristata included at least 41 different items. Detritus and algae (mainly filamentous) comprised most of the bulk of S. cristata's diet. Scartella cristata also exhibited significant seasonal and spatial variability in its diet. This study highlights the importance of detritus within the epilithic algal matrix in the diet of S. cristata and pinpoints that, as in coral reefs, the detrital pathway exerts an important role in the trophodynamics of subtropical rocky shores where these fish are abundant.

Identification of COL6A1 as a differentially expressed gene in human astrocytomas.

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.

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.

Time-varying modeling of gene expression regulatory networks using the wavelet dynamic vector autoregressive method.

MOTIVATION: A variety of biological cellular processes are achieved through a variety of extracellular regulators, signal transduction, protein-protein interactions and differential gene expression. Understanding of the mechanisms underlying these processes requires detailed molecular description of the protein and gene networks involved. To better understand these molecular networks, we propose a statistical method to estimate time-varying gene regulatory networks from time series microarray data. One well known problem when inferring connectivity in gene regulatory networks is the fact that the relationships found constitute correlations that do not allow inferring causation, for which, a priori biological knowledge is required. Moreover, it is also necessary to know the time period at which this causation occurs. Here, we present the Dynamic Vector Autoregressive model as a solution to these problems. RESULTS: We have applied the Dynamic Vector Autoregressive model to estimate time-varying gene regulatory networks based on gene expression profiles obtained from microarray experiments. The network is determined entirely based on gene expression profiles data, without any prior biological knowledge. Through construction of three gene regulatory networks (of p53, NF-kappaB and c-myc) for HeLa cells, we were able to predict the connectivity, Granger-causality and dynamics of the information flow in these networks. SUPPLEMENTARY INFORMATION: Additional figures may be found at http://mariwork.iq.usp.br/dvar/.