Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.     

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.     

Effect of kilning on the antioxidant and pro-oxidant activities of pale malts

Pale malts were prepared using standard and rapid kilning regimes that differed in the temperature and moisture profiles in the kiln. Samples were taken over the last 9 h of kilning, that is, at 18, 20, 22, 25, and 27 h. Antioxidant activity, assessed by redox potential, scavenging of the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS*+), and ferric reducing/antioxidant power (FRAP), increased at moisture levels below 6.7% for both regimes. The 27 h malt exposed to the rapid regime (moisture content of 4.8%) had a higher activity than the 27 h standard regime sample (moisture content of 4.8%). None of the malts scavenged oxygen. Pro-oxidant activity profiles were different for the malts obtained using each regime and, at 27 h, the rapid procedure gave malt with higher activity. Levels of (+)-catechin and ferulic acid (the most abundant phenolic compounds identified) generally increased as the moisture content of malt fell below 6.7%. Differences in antioxidant and pro-oxidant activities of the 27 h malts are partly attributed to the Maillard reaction, as evidenced by lower L* and higher b* values and higher levels of Maillard-derived flavor compounds, in the sample obtained by the rapid procedure. Levels of lipid-derived flavor compounds were significantly higher after 27 h of kilning using the rapid procedure.     

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.