Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris)

Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement, indicating that separate reference intervals should be established for serum and plasma. Finally, storage of these enzymes at -70 degrees C for 1 mo resulted in up to a 25% decrease in enzymatic activity in manatee plasma.     

Comparative Expression Analysis of Gametogenesis‐Associated Genes in Foetal and Adult Bubaline (Bubalus bubalis) Ovaries and Testes

This study was conducted to identify and analyse the expression of gametogenesis‐associated genes and proteins in foetal and adult buffalo gonads of both the sexes. Relative quantification of the genes was determined by qPCR and Western blotting. Immunohistochemistry was also performed for various gametogenesis‐associated proteins in foetal and adult gonads of both the sexes. We observed significantly (p < 0.05) increased expression of primordial germ cell‐specific, meiotic as well as genes associated with oocyte maturation and development in foetal ovaries as compared to the adult ones. However, significantly (p < 0.05) increased expression of proteins associated with oocyte maturation like GDF9 and ZP4 was found in adult ovaries, indicating temporal regulation of mRNA translation during oogenesis. Meiotic genes showed significantly (p < 0.05) increased expression in adult testes as compared to foetal testes and ovaries, indicating onset of meiosis at a later stage in spermatogenesis. In general, the expression of primordial germ cell‐associated as well as meiotic genes was higher in adult testes, indicating the increased biological activity in the organ. Immunohistochemistry revealed localized expression of gametogenesis‐associated proteins in ovarian follicles and seminiferous tubules of testes, while the surrounding somatic tissues were devoid of these proteins. The study gives an understanding of the sequential and temporal events of gene expression as well as mRNA translation during male and female gametogenesis. It could also be concluded that follicles and seminiferous tubules are the functional units of the female and male gonads, respectively, and their function could be enhanced by appropriate chemical and genetic intervention of the somatic tissue immediately surrounding them. This assumes importance in the context that buffalo attains sexual maturity at an older age of 2–3 years and have smaller ovaries with lesser number of primordial follicles in comparison with cattle, which is suggested to be the main reason of their poor breeding performance.     

Identification of active edge sites for electrochemical H2 evolution from MoS2 nanocatalysts

The identification of the active sites in heterogeneous catalysis requires a combination of surface sensitive methods and reactivity studies. We determined the active site for hydrogen evolution, a reaction catalyzed by precious metals, on nanoparticulate molybdenum disulfide (MoS2) by atomically resolving the surface of this catalyst before measuring electrochemical activity in solution. By preparing MoS2 nanoparticles of different sizes, we systematically varied the distribution of surface sites on MoS2 nanoparticles on Au(111), which we quantified with scanning tunneling microscopy. Electrocatalytic activity measurements for hydrogen evolution correlate linearly with the number of edge sites on the MoS2 catalyst.     

Penetration and establishment of Phakopsora pachyrhizi in soybean leaves as observed by transmission electron microscopy

For over 30 years, it has been known that Phakopsora pachyrhizi is unusual in that it penetrates from urediniospores directly through the leaf cuticle without entering stomates. This unusual mode of penetration suggests that disease resistance mechanisms might exist for soybean rust that do not exist for most rust diseases. As a result, we decided to conduct a histological study using transmission electron microscopy to further elucidate the mechanisms of penetration and early establishment of P. pachyrhizi in soybean leaves. Based on our study, it was concluded that P. pachyrhizi utilizes primarily mechanical force, perhaps with the aid of digestive enzymes, to penetrate the cuticle on the leaf surface. However, the lack of deformation lines in micrographs indicated that digestive enzymes, without mechanical force, are used by the penetration hypha to penetrate the outer and inner epidermal cell walls. Digestive enzymes, again indicated by the lack of deformation lines, are used by haustorial mother cells to breach the walls of mesophyll cells to form haustoria. The possibility exists for eventual determination of the precise roles of pressure and digestive enzymes in the development of soybean rust and elucidation of some of the determinants of resistance and susceptibility to this important plant disease.     

Arginine ammonification assay as a rapid index of gross N mineralization in agricultural soils

Seasonal dynamics of in situ gross nitrogen (N) mineralization rates were measured using the ¹⁵N-NH₄⁺ isotope dilution method in a Danish soil subjected to four different agricultural practices (set aside, barley, winter wheat and clover). Results were compared to arginine ammonification in the soil samples measured as NH₄⁺ production following addition of excess (1 mM) arginine. In the set aside, barley, winter wheat and clover soils the average annual rates of gross N mineralization (0.29, 0.60, 1.34 and 1.75 µg NH₄⁺-N g^-1 day^-1, respectively) and arginine ammonification activity (0.21, 0.55, 0.88, and 1.33 µg NH₄⁺-N g^-1 h^-1, respectively) were well correlated. Furthermore, the seasonal variations of gross N mineralization and arginine ammonification activities were very similar, showing rapid responses to rainfall and generally higher activities in wetted soils. As tested in the laboratory, the arginine ammonification activity correlated well with heterotrophic microbial respiration activity (CO₂ production) in soil samples and further displayed a simple, one-component Michaelis-Menten kinetics with a high affinity for arginine (K_m value of 48 µM LJ µM) as determined from non-linear parameter estimation. This indicated that arginine ammonification activity was primarily due to microorganisms, and the activity was also shown to be at a minimum in sterile soil samples. All evidence thus supported that our standard assay of arginine ammonification activity provides a good index of gross N mineralization rates by the microorganisms in soil under in situ conditions.