Double power-law viscoelastic relaxation of living cells encodes motility trends.

Living cells are constantly exchanging momentum with their surroundings. So far, there is no consensus regarding how cells respond to such external stimuli, although it reveals much about their internal structures, motility as well as the emergence of disorders. Here, we report that twelve cell lines, ranging from healthy fibroblasts to cancer cells, hold a ubiquitous double power-law viscoelastic relaxation compatible with the fractional Kelvin-Voigt viscoelastic model. Atomic Force Microscopy measurements in time domain were employed to determine the mechanical parameters, namely, the fast and slow relaxation exponents, the crossover timescale between power law regimes, and the cell stiffness. These cell-dependent quantities show strong correlation with their collective migration and invasiveness properties. Beyond that, the crossover timescale sets the fastest timescale for cells to perform their biological functions.

Major seminal plasma proteome of rabbits and associations with sperm quality.

The present study was conducted to describe the major seminal plasma proteome of rabbits and potential associations between seminal proteins and semen criteria. Semen samples were collected from 18 New Zealand adult rabbits, and seminal plasma proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Sperm motility, vigor, concentration, morphology and membrane sperm viability were evaluated. Rabbits ejaculated 364 ±â€¯70 million sperm/ml, with 81 ±â€¯6.1% motile cells, 3.8 ±â€¯0.2 vigor and 66.7 ±â€¯2.5% sperm with normal morphology. Based on the viability and acrosome integrity assay, there were 65.8 ±â€¯2.5% live sperm with intact acrosome and most spermatozoa had both intact acrosome and functional membrane. On average, 2-D gels of rabbit seminal plasma had 232 ±â€¯69.5 spots, as determined by PDQuest software (Bio Rad, USA). Mass spectrometry allowed the identification of 137 different proteins. The most abundant proteins in rabbit seminal plasma were hemoglobin subunit zeta-like, annexins, lipocalin, FAM115 protein and albumin. The intensity of the spots associated with these five proteins represented 71.5% of the intensity of all spots detected in the master gel. Multiple regression models were estimated using sperm traits as dependent variables and seminal plasma proteins as independent ones. Also, sperm motility had positive association with beta-nerve growth factor and cysteine-rich secretory protein 1-like and a negative one with galectin-1. The percentage of rabbit sperm with intact membrane was related to seminal plasma protein FAM115 complex and tropomyosin. Then, the population of morphologically normal sperm in rabbit semen was positively linked to carcinoembryonic antigen-related cell adhesion molecule 6-like and down regulated by seminal plasma isocitrate dehydrogenase. Based on another regression model, the variation in the percentage of live sperm with intact acrosome was partially explained by the amount of leukocyte elastase inhibitor and the peptidyl-prolyl cis-trans isomerase A in the rabbit seminal fluid. The current study reports the identification of 137 proteins of rabbit seminal plasma. Major proteins of seminal secretion relate primarily to prevention of damages caused by lipid peroxide radicals and oxidative stress, membrane functionality, transport of lipids to the sperm membrane and temperature regulation. Moreover, finding seminal plasma proteins as indicators of semen parameters will improve assisted reproductive technologies.

Gastroprotective and anti-inflammatory activities integrated to chemical composition of Myracrodruon urundeuva Allemão - A conservationist proposal for the species.

ETHNOPHARMACOLOGICAL RELEVANCE: Myracrodruon urundeuva Allemão (Aroeira-do-Sertão), Anacardiaceae, is one of the most used plants in folk medicine in Northeastern Brazil as an anti-inflammatory, healing and antiulcer. This species is threatened with extinction due to anthropogenic exploitation. The importance of this study is to demonstrate the feasibility of a conservationist model of replacement of the M. urundeuva adult tree (inner bark) for its under developing plants (shoots) in order to ensure the preservation of this species, but also to ensure sufficient raw material for pharmaceutical purposes. AIM OF THE STUDY: To characterize chemically and assess the gastroprotective and anti-inflammatory activities of the fluid extracts from M. urundeuva innebark (adult plant) as well as stem and leaves of shoots (young plant). MATERIALS AND METHODS: The fluid extracts were prepared by maceration-percolation with hydroalcoholic solution according to the methodology described in the Brazilian Pharmacopoeia. These extracts were cleaned-up through solid phase extraction (SPE) and chemically characterized by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-ESI-QTOF MS/MS). Gastroprotective and anti-inflammatory activities of the extracts (700 or 1000 mg/kg) were assessed on ethanol-induced gastric lesions and Croton oil-induced ear edema in rats, respectively. The extracts were evaluated for cytotoxicity in vitro. RESULTS: The UPLC-ESI-QTOF-MS/MS analysis evidenced the presence of chalcones, flavonoids and tannins. Gastroprotective and anti-inflammatory activities achieved with fluid extracts from the stems and leaves was similar to inner bark. The fluid extracts were not toxic. CONCLUSION: It is possible to replace the inner bark of the adult tree for the stems and leaves from the shoots as raw material to be used in the preparation of its the phytotherapeutics. Therefore, this finding may help in the implementation of public policies that ensure the conservation of the species along with its sustainable use for pharmaceutical purposes.

Proteomic analysis of seminal plasma from locally-adapted "Curraleiro Pé-Duro bulls" (Bos taurus): identifying biomarkers involved in sperm physiology in endangered animals for conservation of biodiversity.

The present study was aimed at evaluating the seminal plasma proteins and sperm parameters of Curraleiro Pé-Duro bulls. Semen was collected from 10 bulls by electroejaculation, and sperm parameters were evaluated in fresh and frozen-thawed semen. Seminal plasma proteins were analyzed by 2-D SDS-PAGE and mass spectrophotometry. Tools in computational biology were used to generate bioinformatic knowledge and evaluate gene ontology, protein-protein interactions, phylogenetic trees and multiple sequence alignments. Sperm motility in fresh and frozen-thawed semen was 78.8±1.8% and 21.2±1.6%, respectively. Pearson's correlations were evaluated (p<0.05). Sperm motility and vigor in fresh semen were correlated with clusterin, TIMP2 and cathepsin S (r=0.64-0.71) and sperm defects were related to inhibitor of carbonic anhydrase and BSP 5 (r=0.78-0.80). Clusterin, BSP 5, alpha-enolase, creatine kinase M-type, glyceraldehyde-3-phosphate dehydrogenase, BSP 3, albumin, and 5'-nucleotidase and legumain were correlated with acrosome intact live sperm (r=0.80-0.64). Associations were detected between sperm vigor and spermadhesin 1 (r=-0.89), and between sperm defects in fresh semen and spermadhesin 1 and clusterin (r=-0.81). Sperm motility in frozen-thawed semen was associated with BSP 1, spermadhesin 1, clusterin and spermadhesin Z13 (r=0.64-0.85). The percent of motile sperm after freeze-thawing was negatively correlated (r=-0.64) with the amount of spermadhesin 1 in the seminal plasma. Based on in silico analysis, TIMP2 interacted with BSP1, BSP3, BSP5 and metalloproteinases. Molecular functions of proteins associated with sperm parameters were binding, catalytic activity and enzymatic regulation. Amino acid sequences of spermadhesin 1 and BSP 1 from Bos taurus, and other domestic species were similar. Phylogenetic tree analysis demonstrated that clusterin from Bos taurus was related to Ovis aries and domains of clusterin, spermadhesin 1, BSP 1 and inhibitor of carbonic anhydrase were conserved as well. In summary, specific seminal proteins are associated with sperm parameters of locally-adapted bulls. Use of the endangered mammalian as a model may assist in understanding aspects of evolutionary adaptations and could improve assisted reproductive biotechnologies.

Meat quality assessment from young goats fed for long periods with castor de-oiled cake.

Diet can influence both the qualitative and quantitative traits of ruminant meat. This study evaluated the effects of castor de-oiled cake on the meat of mixed-breed male goat kids. After 165days of diet treatment, no alterations (p>0.05) were observed in the in vivo performance, anatomic components, dissection and proximate composition of the Longissimus dorsi muscle, as well as in the color and pH of the carcasses. However, diet had an effect (p<0.05) on energy metabolites, fatty acid profile, and expression of certain proteins of the Longissimus dorsi muscle. To conclude, this study showed that the establishment of castor de-oiled cake diet for a long period to goats led to alterations in meat quality, without compromising its consumption qualities.

Growth, testis size, spermatogenesis, semen parameters and seminal plasma and sperm membrane protein profile during the reproductive development of male goats supplemented with de-oiled castor cake.

The present study was conducted to evaluate the effect of de-oiled castor cake on reproductive traits of crossbreed goats. Fourteen males were grouped into two lots (n = 7/group), as described: group without de-oiled castor cake (WCC) and group fed with de-oiled castor cake (CC). Goats received two diets containing a mixture of Bermudagrass hay and concentrates with the same energy (73% total digestive nutrients) and protein content (15% crude protein) during 150 days, corresponding to ages from 40 (puberty) to 60 weeks. Blood plasma concentrations of urea, albumin, lactate dehydrogenase, creatinine, alanine aminotransferase and testosterone were determined. We also evaluated scrotal circumference, sperm parameters, quantitative aspects of spermatogenesis and daily sperm production (DSP), as well as the proteome of seminal plasma and sperm membrane. Seminal fluid and sperm proteins were analyzed by 2D SDS-PAGE and mass spectrometry. After 150 days of castor cake feeding, animals had no changes in the biochemical composition of blood plasma, suggesting the absence of intoxication by ingestion of ricin. There were no alterations in dry mater intake, weight gain, testis size, peripheral concentrations of testosterone, sperm concentration, motility and morphology. Sertoli and germ cell populations in the testis and DSP were not affected either. However, there were significant variations in the expression of five seminal plasma proteins and four sperm membrane proteins. In conclusion, the replacement of soybean meal by castor cake (with ricin concentrations of 50mg/kg) did not interfere with the growth and core reproductive development of male goats. However, the diet with ricin altered the expression of certain seminal plasma and sperm membrane proteins, which play roles in sperm function and fertilization. Lower expression of these proteins may impair the ricin-fed animals to perform as high-fertility sires.

Semen variables and sperm membrane protein profile of Saanen bucks (Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S).

The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks (n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant (p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher (p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758).

This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=-0.87; P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.

Membrane-associated proteins of ejaculated sperm from Morada Nova rams.

The objective was to describe the profile of membrane proteins from sperm of tropically adapted Morada Nova rams (N = 5). Samples from protein-enriched fractions of ejaculated sperm (containing 400 µg of protein) were separated by two-dimensional electrophoresis and respective maps analyzed using PDQuest software (version 7.3.0; Bio-Rad). Proteins were identified using tandem mass spectrometry. Also, membrane proteins were incubated with antibodies against binder of sperm protein (BSP) 1 and bodhesin 2 (Bdh-2), components of vesicular gland secretion. For membrane proteins of ejaculated sperm, an average of 133 ± 4.6 spots were detected per gel, of which, 107 spots were consistently present on all gels. Sixty-eight spots and 37 proteins were identified using mass spectrometry, corresponding to 71.6% of the intensity of all spots detected. Three major spots identified as ram seminal vesicle protein (RSVP) 14 represented approximately 30% of the intensity of all spots. Two of the most intense spots in the gel reacted against anti-BSP1, at 14 kDa. In addition, four low molecular weight spots reacted with anti-Bdh-2 antibodies. Proteins RSVP and Bdh-2 belong to the BSP and spermadhesin families, respectively, and were previously reported as major components of ram seminal proteins. Additional proteins identified in the sperm membrane two-dimensional maps included alpha-2-heparan sulfate-glycoprotein, plasma glutamate carboxypeptidase, arylsulfatase A, cathelicidin, heat shock protein 70 kDa, angiotensin-converting enzyme, leucine aminopeptidase, and clusterin. Some proteins were present as multiple isoforms, such as tubulin (12), alpha-2-heparan sulfate-glycoprotein (5), ATP synthase (5), Bdh-2 (4) and RSVP14 (3). Based on gene ontology analysis, the most common biological processes associated with the membrane proteins were cellular processes (34%), response to stimulus (14%), and metabolic processes (11%). Binding (37%) and catalytic activity (32%) corresponded to the most frequent molecular functions for those proteins. In conclusion, we identified a diverse cohort of components of membrane proteins in ram sperm. Major proteins previously reported in seminal plasma, such as RSVP14 and Bdh-2, were also extracted from sperm membranes. Knowledge of sperm proteins is crucial for elucidating mechanisms underlying their association with sperm function.

New insights into the complex mixture of latex cysteine peptidases in Calotropis procera.

The latex of Calotropis procera is a rich source of proteolytic activity. This latex is known to contain two distinct cysteine peptidases: procerain and procerain B. In this study, new cysteine peptidases were purified from C. procera latex. The enzymes were purified by two sequential ion-exchange chromatography steps (CM-Sepharose plus Resource S(®)) at pH 5.0 and 6.0. The purified enzymes had molecular mass spectra corresponding to CpCP-1=26,213, CpCP-2=26,133 and CpCP-3=25,086 Da. These enzymes exhibited discrete differences in terms of enzymatic activity at a broad range of pH and temperature conditions and contained identical N-terminal amino acid sequences. In these respects, these three new proteins are distinct from those previously studied (procerain and procerain B). Circular dichroism analysis revealed that the new peptidases contain extensive secondary structures, α(15-20%) and ß(26-30%), that were stabilized by disulfide bonds. The purified enzymes exhibited plasma-clotting activity mediated by a thrombin-like mechanism. The set of results suggest the three isolated polypeptides correspond to different post-translationally processed forms of the same protein.

A novel xyloglucan film-based biosensor for toxicity assessment of ricin in castor seed meal.

Oil from the seed of the castor plant (Ricinus communis L.) is an important commodity for a number of industries, ranging from pharmaceuticals to renewable energy resources. However, the seed and subsequent seed meal contain ricin (RCA60), a potent cytotoxin, making it an unusable product for animal feed. In order to investigate the efficiency of reducing the toxicity of the seed meal, a biosensor is proposed by exploring the lectin-carbohydrate binding. A gold electrode was assembled with a film of Xyloglucan (XG) extracted from Hymenaea courbaril L. The analytical response to RCA60 was obtained using a polyclonal antibody against RCA60 conjugated to peroxidase. The current responses were generated by reaction with H2O2 and amplified with hydroquinone as chemical mediator. Voltammetric studies showed that the XG film was tightly bound to the gold electrode. This biosensor allows discriminate lectins in native and denatured forms. The limit of detection of native RCA60 was 2.1 µg mL(-1). This proposed biosensor showed to be a potential and accurate method for toxicity assessment of the ricin in castor seed meal by simple polysaccharide film-electrode strategy.