Enzyme Activity Profiles Research Articles

Evidence the Isc iron–sulfur cluster biogenesis machinery is the source of iron for [NiFe]-cofactor biosynthesis in Escherichia coli

[NiFe]-hydrogenases have a bimetallic NiFe(CN)2CO cofactor in their large, catalytic subunit. The 136 Da Fe(CN)2CO group of this cofactor is preassembled on a distinct HypC–HypD scaffold complex, but the intracellular source of the iron ion is unresolved. Native mass spectrometric analysis of HypCD complexes defined the [4Fe–4S] cluster associated with HypD and identified + 26 to 28 Da and + 136 Da modifications specifically associated with HypC. A HypCC2A variant without the essential conserved N-terminal cysteine residue dissociated from its complex with native HypD lacked all modifications. Native HypC dissociated from HypCD complexes isolated from Escherichia coli strains deleted for the iscS or iscU genes, encoding core components of the Isc iron–sulfur cluster biogenesis machinery, specifically lacked the + 136 Da modification, but this was retained on HypC from suf mutants. The presence or absence of the + 136 Da modification on the HypCD complex correlated with the hydrogenase enzyme activity profiles of the respective mutant strains. Notably, the [4Fe–4S] cluster on HypD was identified in all HypCD complexes analyzed. These results suggest that the iron of the Fe(CN)2CO group on HypCD derives from the Isc machinery, while either the Isc or the Suf machinery can deliver the [4Fe–4S] cluster to HypD.

Loss of testosterone induces postprandial insulin resistance and increases the expression of the hepatic antioxidant flavin-containing monooxygenases in mice exposed to intermittent hypoxia.

We tested the hypothesis that low testosterone alters the effects of intermittent hypoxia (IH) on glucose homeostasis, hepatic oxidative stress, and transcriptomic profile in male mice. We used sham-operated or orchiectomized (ORX) mice exposed to normoxia (Nx) or IH for 2 weeks. We performed fasting insulin and glucose tolerance tests and assessed fasting and postprandial insulin resistance with the HOMA-IR. The activity of hepatic prooxidant (NADPH oxidase-NOX), antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase-SOD, Cat, GPx), lipid peroxidation (MDA concentration), and the total concentration of glutathione (GSH) were measured under postprandial conditions. mRNA sequencing and pathway enrichment analyses were used to identify hepatic genes underlying the interactions between IH and testosterone. In Sham mice, IH improves fasting insulin sensitivity and glucose tolerance, while there are no effects of IH in ORX mice. In ORX mice, IH induces postprandial hyperinsulinemia, insulin resistance, and a prooxidant profile of enzyme activity (low SOD activity) without altering hepatic MDA and GSH content. ORX and IH altered the expression of genes involved in oxidoreductase activities, cytochromes-dependent pathways, and glutathione metabolism. Among the genes upregulated in ORX-IH mice, the flavin-containing monooxygenases (FMO) are particularly relevant since these are potent hepatic antioxidants that could help prevent overt oxidative stress in ORX-IH mice. Low levels of testosterone in male mice exposed to IH induce post-prandial hyperinsulinemia and insulin resistance and determine the mechanisms by which the liver handles IH-induced oxidative stress.

Enzyme activity and nutritional profile of different-sized carrot cubes treated with supercritical carbon dioxide

This study evaluated changes in the enzyme activity, color, carotenoids, sugar profile, individual phenolic compounds, and antioxidant capacity of supercritical carbon dioxide (SCCD)-treated carrot cubes of different sizes (1 cm and 2 cm). Results demonstrated that SCCD reduced the activities of polyphenol oxidase (PPO) and peroxidase (POD) more in 1 cm carrot cubes than in 2 cm carrot cubes. Nevertheless, the a* , b* , and ∆E values of 1 cm SCCD-treated were enhanced compared to 2 cm SCCD-treated samples. The carotenoids, phenolic compounds, and antioxidant capacity of 1 cm SCCD-treated samples were increased under moderate processing conditions. In contrast, the nutritional composition in 1 cm SCCD-treated samples was lower than in 2 cm SCCD-treated samples at harsh processing conditions. The principal component and correction analysis displayed a visual variation between the enzyme activities and nutritional composition of 1 cm and 2 cm carrot cubes after SCCD processing. According to the results above, 1 cm carrot cubes processed with SCCD at 10 MPa, 35 ℃, and 45 min are the most effective at preserving fresh-cut carrots with relatively high enzyme inactivation and the highest retention of bioactive compounds. This work provides a more comprehensive theoretical basis for optimizing SCCD processing for fresh-cut carrots and recommendations for industrial production.

Exploring the hidden treasures: Deep-sea bacterial community structure in the Bay of Bengal and their metabolic profile

Deep sea bacterial communities demonstrate remarkable adaptability to high-pressure environments coupled with low temperatures which has sparked curiosity about their diversity and exceptional metabolic pathways. Additionally, bacteria in the deep sea exert a substantial influence over various biogeochemical processes. To date, we have relatively very little information about the deep-sea bacterial communities and, they remain largely unexplored. We investigated the variability in the physicochemical conditions, heavy metals and their influence on deep-sea bacterial community structure across three different depths in the Bay of Bengal. The structural and metabolic diversity of deep-sea sediment microbial communities were examined through culture-based sequencing of 16S rRNA genes, ecto-enzymatic studies, and community-level physiological profiling. Bacillota was the most dominant phylum representing 61% of the cultured bacterial isolates, while the remaining belonged to Actinomycetota and Pseudomonodata. Five potential novel species belonging to the genera Fictibacillus, Lysinibacillus, Salinicola, Robertmurraya and Blastococcus were identified. The extracellular enzymatic activity was positive for >50% of the bacterial isolates, wherein the genera Bacillus and Micromonospora exhibited versatile profiles. High metabolic diversity was recorded through the carbon substrate utilization profiles indicating that microbial communities are active participants in biogeochemical cycles in the deep sea. The most prominently utilized carbon substrates were α-cyclodextrin, glucose-1-phosphate, D-xylose, glycogen, and 2-hydroxy benzoic acid which serve as organic substrates for microbial metabolism, facilitating the decomposition of organic matter and, recycling carbon in deep-sea ecosystems. Multivariate statistical analyses confirmed that the environmental variables had a profound influence on the bacterial community. The findings shed light on spatial variability in the bacterial community structure, enzyme activity and metabolic profiles, and enhance our understanding of Bay of Bengal deep-sea sedimentary microbial ecology.

An eminent approach towards next generation solvents for sustainable packaging and stability of enzymes: a comprehensive study of ionic liquid and deep eutectic solvent mixtures.

Hybrid ionic fluids (HIFs) are newly emerging and fascinating sustainable solvent media, which are attracting a great deal of scientific interest in protecting the native structure of proteins. For a few decades, there has been a demand to consider ionic liquids (ILs) and deep eutectic solvents (DESs) as biocompatible solvent media for enzymes; however, in some cases, these solvent media also show limitations. Therefore, this work focuses on synthesising novel HIFs to intensify the properties of existing ILs and DESs by mixing them. Herein, HIFs have been synthesised by the amalgamation of a deep eutectic solvent (DES) and an ionic liquid (IL) with a common cation or anion. Later on, the stability and activity of hen's egg white lysozyme (Lyz) in the presence of biocompatible solvent media and HIFs were studied by various techniques such as UV-vis, steady-state fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and dynamic light scattering (DLS) measurements. This work emphasises the effect of a DES (synthesised using 1 : 2 choline chloride and malonic acid) [Maline], ILs (1-butyl-3-methylimidazolium chloride [BMIM]Cl or choline acetate [Chn][Ac]) and their corresponding HIFs on the structure and functionality of Lyz. Moreover, we also studied the secondary structure, thermal stability, enzymatic activity and thermodynamic profile of Lyz at pH = 7 in the presence of varying concentrations (0.1 to 0.5 M) of [BMIM]Cl and [Chn][Ac] ILs, Maline as a DES, and Maline [BMIM]Cl (HIF1) and Maline [Chn][Ac] (HIF2). Spectroscopic results elucidate that ILs affect the activity and structural stability of Lyz. In contrast, the stability and activity are inhibited by DES and are enhanced by HIFs at all the studied concentrations. Overall, the experimental results studied explicitly elucidate that the structure and stability of Lyz are maintained in the presence of HIF1 while these properties are intensified in HIF2. This study shows various applications in biocompatible green solvents, particularly in the stability and functionality of proteins, due to their unique combination where the properties counteract the negative effect of either DESs or ILs in HIFs.

Plant growth promoting potential of Arthrobacter spp., an early colonizer on the volcanic deposits of Mt. Merapi, Indonesia, as a restorative agent for degraded land

Exploration of novel and effective plant growth-promoting bacteria (PGPB) from unexplored environments is an intriguing option for restoring degraded land in Indonesia caused by mining activity. Due to their ability to develop in the absence of carbon, PGPB derived from volcanic environments may be a sustainable alternative. Previous research on the volcanic deposits of Mt. Merapi, Indonesia and Miyake-Jima, Japan, revealed that Arthrobacter spp. dominated the pioneer bacterial communities. Consequently, this research aimed to investigate the biochemical and physiological characteristics of volcanic Arthrobacter groups and assess their potential as PGPB. Using the conventional API system (20NE, 50CH, Zym), biochemical characteristics were evaluated. In vitro evaluations of plant growth-promoting activities such as siderophore production, phosphate solubilization, growth in media devoid of nitrogen, heavy metal resistance and weathering activity were conducted. The results showed broad substrate utilization and enzyme activity profiles of the Mt. Merapi isolates (BRU 37, BRU I.13, and BRU I.45). During 15 days of incubation on a minimal medium containing potassium feldspar, glucose, and NH4Cl, all of the isolates grew and produced acids. In nitrogen-free media, all of the isolates grew. These isolates showed positive reaction for the phosphate solubilization assay using Pikovskaya plates. After one day of incubation, all isolates produced yellow zones on the blue-green pigmented media (CAS-overlay media) and were therefore classified as potent siderophore producers. The heavy metal resistance assay showed that BRU37 grew in the heavy metal media. These results indicate the potential of selected indigenous Mt. Merapi Arthrobacter to enhance plant growth. Further characterization of plant growth-promoting traits is now in progress.

Blood cholinesterase enzyme activity profile of fogging officers at the Makassar health quarantine centre

Blood cholinesterase enzyme activity profile of fogging officers at the Makassar health quarantine centre

Quality properties and nutritional compounds of fresh-cut pumpkin treated with supercritical carbon dioxide

The work aimed to explore the effect of supercritical carbon dioxide (SCCD) treatment on the enzyme activity, color, carotenoid, sugar and phenolic profile, and antioxidant activity of fresh-cut pumpkins. The polyphenol oxidase (PPO) and peroxidase (POD) activities of SCCD-treated pumpkin cubes were reduced by 21% and 18%, respectively. The lightness value of SCCD-treated samples declined compared to the untreated samples, whereas the redness and yellowness values in treated samples were enhanced. The lutein, α-carotene, β-carotene and total carotenoids, glucose, sorbitol, and other polysaccharides in the SCCD-treated samples showed a fluctuation trend. Similarly, the treated sample's total phenolic contents (TPC), ABTS (2,2′- azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, DPPH (2,2-diphenyl-1-picrylhydrazyl), and superoxide radical scavenging activities were increased and then reduced. An enhancement was noted in the coumaryloquinic acid I and II, caffeic acid glucoside, 4-hydroxybenzoic acid, and p-coumaric acid of SCCD-treated samples. The work demonstrated that the SCCD technique at moderate conditions could obtain higher retention of nutritional composition in fresh-cut pumpkins.

DIMBOA-induced gene expression, activity profiles of detoxification enzymes, multi-resistance mechanisms, and increased resistance to indoxacarb in tobacco cutworm, Spodoptera litura (Fabricius)

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is one of the most destructive insect pests owned strong resistance to different insecticides. Indoxacarb as a novel oxadiazine insecticide becomes the main pesticide against S. litura. DIMBOA [2,4-dihydroxy-7-methoxy-2 H-1,4-benz-oxazin-3(4 H)-one] is involved in important chemical defense processes in corn plants. However, the insects’ adaptation mechanism to insecticides when exposed to defensive allelochemicals in their host plants remains unclear. Here, we assessed multi-resistance, and resistance mechanisms based on S. litura life history traits. After 18 generations of selection, indoxacarb resistance was increased by 61.95-fold (Ind-Sel) and 86.06-fold (Dim-Sel) as compared to the Lab-Sus. Also, DIMBOA-pretreated larvae developed high resistance to beta-cypermethrin, chlorpyrifos, phoxim, chlorantraniliprole, and emamectin benzoate. Meanwhile, indoxacarb (LC50) was applied to detect its impact on thirty-eight detoxification-related genes expression. The transcripts of SlituCOE073, SlituCOE009, SlituCOE074, and SlituCOE111 as well as SlGSTs5, SlGSTu1, and SlGSTe13 were considerably raised in the Ind-Sel strain. Among the twenty-three P450s, CYP6AE68, CYP321B1, CYP6B50, CYP9A39, CYP4L10, and CYP4S9v1 transcripts denoted significantly higher levels in the Ind-Sel strain, suggesting that CarEs, GSTs and P450s genes may be engaged in indoxacarb resistance. These outcomes further highlighted the importance of detoxification enzymes for S. litura gene expression and their role in responses to insecticides and pest management approaches.

Sublethal effects of chlorantraniliprole on biological characteristics, detoxifying enzyme activity and gene expression profile in the Allium mongolicum Regel leaf beetle Galeruca daurica (Coleoptera: Chrysomelidae)

AbstractAllium mongolicum Regel leaf beetle, Galeruca daurica (Coleoptera: Chrysomelidae) is rampantly harmful in Inner Mongolia grassland. However, the current management strategies for this pest still heavily rely on chemical control using traditional insecticides or those with novel action. In the study, we conducted an indoor bioassay to evaluate the sublethal effects of chlorantraniliprole on the biological characteristics. Additionally, we assessed the activity of detoxification enzymes, specifically the primary enzymes carboxylesterase (CarE) and cytochrome P450, and insect's secondary metabolic enzymes, namely glutathione S‐transferase (GST) and uridine diphosphate glycosyltransferase (UGT), as well as its gene expression profile. The developmental period of the 3rd instar larvae of G. daurica was significantly prolonged after treatment with chlorantraniliprole, which negatively affected the hatching, pupation and diapause rates as well as their body weight. The larvae showed different dynamics of the enzyme activities within 24 h of chlorantraniliprole treatment. Different sublethal concentrations of chlorantraniliprole showed an inducing effect on the activities, while no significant difference in activity was observed for P450. Expression profiling of detoxifying enzyme genes screened by transcriptome data has revealed that 6 CarE, 2 GST, 7 UGT and 31 P450 genes were up‐regulated. In conclusion, chlorantraniliprole's sublethal effect on 3rd instar G. daurica larvae resulted in a deceleration of their growth and developmental processes and significantly increased the activity levels of CarE, GST and UGT. The detoxifying proteins encoded by up‐regulation genes may involve in the detoxification metabolism of chlorantraniliprole. Our results provide a basis for understanding the molecular mechanisms of chlorantraniliprole action and detoxification in this key grassland insect pest.

Distinct laccase expression and activity profiles of Trametes versicolor facilitate degradation of benzo[a]pyrene.

A Trametes versicolor isolate from the Changbai Mountain showed promising activity in degrading benzo[a]pyrene (BaP), which is a high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH) compound. It was hypothesized that the T. versicolor isolate encode BaP-degrading enzymes, among which laccase is mostly sought after due to significant commercial potential. Genome of the T. versicolor isolate was sequenced and assembled, and seven laccase homologues were identified (TvLac1-7) as candidate genes potentially contributing to BaP degradation. In order to further identify the BaP responsive laccases, time-course transcriptomic and proteomic analyses were conducted in parallel on the T. versicolor isolate upon BaP treatment. Homologous laccases showed distinct expression patterns. Most strikingly, TvLac5 was rapidly induced in the secreted proteomes (secretomes), while TvLac2 was repressed. Recombinant laccase expression and biochemical characterization further showed corresponding enzymatic activity profiles, where TvLac5 was 21-fold more effective in BaP degradation compared to TvLac2. Moreover, TvLac5 also showed 3.6-fold higher BaP degrading activity compared to a commercial laccase product of T. versicolor origin. Therefore, TvLac5 was concluded to be a BaP-responsive enzyme from T. versicolor showing effective BaP degradation activity.

Coping with extremes: Alternations in diet, gut microbiota, and hepatic metabolic functions in a highland passerine

In wild animals, diet and gut microbiota interactions are critical moderators of metabolic functions and are highly contingent on habitat conditions. Challenged by the extreme conditions of high-altitude environments, the strategies implemented by highland animals to adjust their diet and gut microbial composition and modulate their metabolic substrates remain largely unexplored. By employing a typical human commensal species, the Eurasian tree sparrow (Passer montanus, ETS), as a model species, we studied the differences in diet, digestive tract morphology and enzyme activity, gut microbiota, and metabolic energy profiling between highland (the Qinghai-Tibet Plateau, QTP; 3230 m) and lowland (Shijiazhuang, Hebei; 80 m) populations. Our results showed that highland ETSs had enlarged digestive organs and longer small intestinal villi, while no differences in key digestive enzyme activities were observed between the two populations. The 18S rRNA sequencing results revealed that the dietary composition of highland ETSs were more animal-based and less plant-based than those of the lowland ones. Furthermore, 16S rRNA sequencing results suggested that the intestinal microbial communities were structurally segregated between populations. PICRUSt metagenome predictions further indicated that the expression patterns of microbial genes involved in material and energy metabolism, immune system and infection, and xenobiotic biodegradation were strikingly different between the two populations. Analysis of liver metabolomics revealed significant metabolic differences between highland and lowland ETSs in terms of substrate utilization, as well as distinct sex-specific alterations in glycerophospholipids. Furthermore, the interplay between diet, liver metabolism, and gut microbiota suggests a dietary shift resulting in corresponding changes in gut microbiota and metabolic functions. Our findings indicate that highland ETSs have evolved to optimize digestion and absorption, rely on more protein-rich foods, and possess gut microbiota tailored to their dietary composition, likely adaptive physiological and ecological strategies adopted to cope with extreme highland environments.

Oxidative stress associated with proteomic and fatty acid profiles of sperm from Nellore bulls at rest†.

Sexual rest is a transient condition, which compromises conception rates, characterized by large volumes of ejaculate with high percentages of dead sperm observed in bulls. The biochemical mechanisms leading to this ejaculate pattern are not fully understood. Six adult resting Nellore bulls were submitted to Breeding Soundness Evaluation by four consecutive semen collections through the electroejaculation method during a 30min period. Each ejaculate had its semen phenotypic parameters; morphology and physical aspects were evaluated. To assess enzymatic activity (superoxide dismutase, catalase, and glutathione S-transferase), lipid peroxidation (concentrations of malondialdehyde and nitric oxide), fatty acid, and proteomic profile aliquots of spermatozoa from the first and fourth ejaculates were used. All sperm parameters differed between the first and fourth ejaculates. Spermatozoa from the first ejaculate showed lower enzymatic activity and a higher concentration of lipid peroxidation markers. Among the 19 identified fatty acids, 52.7% are polyunsaturated. Relative abundance analysis showed that C12:0 and C18:0 fatty acids differed between the first and fourth ejaculates, being the fourth ejaculate richer in spermatozoa. The proteomics analysis identified a total of 974 proteins in both sample groups (first and fourth ejaculates). The majority of identified proteins are related to cellular processes and signaling. Quantitative proteomics showed 36 differentially abundant proteins, 6 up-regulated proteins in the first ejaculate, and 30 up-regulated proteins in the fourth ejaculate. Spermatozoa from bulls at sexual rest have less antioxidant capacity, causing changes in their fatty acid composition and protein profile, which generates the observed sperm pattern and lower fertilization capacity.

Biochemical evidence that the whole compartment activity behavior of GAPDH differs between the cytoplasm and nucleus.

Some metabolic enzymes normally occur in the nucleus and cytoplasm. These compartments differ in molecular composition. Since post-translational modification and interaction with allosteric effectors can tune enzyme activity, it follows that the behavior of an enzyme as a catalyst may differ between the cytoplasm and nucleus. We explored this possibility for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Homogenates of pristine nuclei and cytoplasms isolated from Xenopus laevis oocytes were used for whole compartment activity profiling in a near-physiological buffer. Titrations of NAD+ revealed similar whole compartment activity profiles for GAPDH in nuclear and cytoplasmic homogenates. Surprisingly however GAPDH in these compartments did not have the same behavior in assays of the dependence of initial velocity (v0) on G3P concentration. First, the peak v0 for nuclear GAPDH was up to 2.5-fold higher than the peak for cytoplasmic GAPDH. Second, while Michaelis Menten-like behavior was observed in all assays of cytoplasm, the v0 versus [G3P] plots for nuclear GAPDH typically exhibited a non-Michaelis Menten (sigmoidal) profile. Apparent Km and Vmax (G3P) values for nuclear GAPDH activity were highly variable, even between replicates of the same sample. Possible sources of this variability include in vitro processing of a metabolite that allosterically regulates GAPDH, turnover of a post-translational modification of the enzyme, and fluctuation of the state of interaction of GAPDH with other proteins. Collectively these findings are consistent with the hypothesis that the environment of the nucleus is distinct from the environment of the cytoplasm with regard to GAPDH activity and its modulation. This finding warrants further comparison of the regulation of nuclear and cytoplasmic GAPDH, as well as whole compartment activity profiling of other enzymes of metabolism with cytosolic and nuclear pools.

Versatile Biocatalytic C(sp3 )-H Oxyfunctionalization for the Site- Selective and Stereodivergent Synthesis of α- and β-Hydroxy Acids.

C(sp3 )-H oxyfunctionalization, the insertion of an O-atom into C(sp3 )-H bonds, streamlines the synthesis of complex molecules from easily accessible precursors and represents one of the most challenging tasks in organic chemistry with regard to site and stereoselectivity. Biocatalytic C(sp3 )-H oxyfunctionalization has the potential to overcome limitations inherent to small-molecule-mediated approaches by delivering catalyst-controlled selectivity. Through enzyme repurposing and activity profiling of natural variants, we have developed a subfamily of α-ketoglutarate-dependent iron dioxygenases that catalyze the site- and stereodivergent oxyfunctionalization of secondary and tertiary C(sp3 )-H bonds, providing concise synthetic routes towards four types of 92 α- and β-hydroxy acids with high efficiency and selectivity. This method provides a biocatalytic approach for the production of valuable but synthetically challenging chiral hydroxy acid building blocks.

Versatile Biocatalytic C(sp3)−H Oxyfunctionalization for the Site‐ Selective and Stereodivergent Synthesis of α‐ and β‐Hydroxy Acids

AbstractC(sp3)−H oxyfunctionalization, the insertion of an O‐atom into C(sp3)−H bonds, streamlines the synthesis of complex molecules from easily accessible precursors and represents one of the most challenging tasks in organic chemistry with regard to site and stereoselectivity. Biocatalytic C(sp3)−H oxyfunctionalization has the potential to overcome limitations inherent to small‐molecule‐mediated approaches by delivering catalyst‐controlled selectivity. Through enzyme repurposing and activity profiling of natural variants, we have developed a subfamily of α‐ketoglutarate‐dependent iron dioxygenases that catalyze the site‐ and stereodivergent oxyfunctionalization of secondary and tertiary C(sp3)−H bonds, providing concise synthetic routes towards four types of 92 α‐ and β‐hydroxy acids with high efficiency and selectivity. This method provides a biocatalytic approach for the production of valuable but synthetically challenging chiral hydroxy acid building blocks.

Maturity biomarkers predicting storage performance of early-harvested yellow-fleshed kiwifruit identified using integrated multi-omics analysis

Accurate determination of kiwifruit maturity in the first half of the harvest window is crucial in predicting storage risk, since early-harvested fruit are more susceptible to disorders and rots during extended cold storage. Maturity estimations are usually based on soluble solids content (SSC), but SSC alone is unreliable at early harvest times, necessitating the need for more precise markers. To address this, we performed multi-omics on harvested, yellow-fleshed kiwifruit at four chronological ages based on days after anthesis, namely early harvest (H1), early-mid (H2), late-mid (H3) and late harvest (H4), across two seasons from the same orchard. Analysis of phenotype, transcriptome, metabolome, proteome, hormone concentrations and enzyme activity profiles identified discriminating features within the datasets that separated harvest times, particularly in the first half of the harvest window. Strong seasonal variability was observed. The most reliable variates for maturity estimation were found in the transcriptome, and two transcripts with sharply decreasing abundance during early harvest (Aquaporin TIP4-1 and MYB10) and three transcripts with sharply increasing abundance during later harvest (β-Amylase 3.2, Pectinesterase inhibitor protein and α-Terpineol synthase) with relative seasonal stability were identified. In a different season we used these five potential biomarkers in combination with SSC as a predictive tool. This allowed precise assessment of the maturity stage of fruit in the first half of the harvest window from 12 orchards in four geographical regions, and accurate prediction of their storage outcome.

Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

Glucose-6-Phosphate Dehydrogenase Activity in Milk May Serve as a Non-Invasive Metabolic Biomarker of Energy Balance in Postpartum Dairy Cows

Negative energy balance (EB) postpartum is associated with adverse outcomes in dairy cows; therefore, non-invasive biomarkers to measure EB are of particular interest. We determined whether specific metabolites, oxidative stress indicators, enzyme activity, and fatty acid (FA) profiles in milk can serve as indicators of negative EB. Forty-two multiparous Holstein dairy cows were divided at calving into 2 groups: one was milked 3 times daily and the other, twice a day for the first 30 d in milk (DIM). Cows were classified retrospectively as being in either negative EB (NEB, n = 19; the mean EB during the first 21 DIM were less than the overall median of -2.8 Mcal/d), or in positive EB (PEB, n = 21; the mean EB was ≥-2.8 Mcal/d). The daily milk yield, feed intake, and body weight were recorded individually. Blood samples were analyzed for metabolites and stress biomarkers. Milk samples were taken twice weekly from 5 to 45 DIM to analyze the milk solids, the FA profile, glucose, glucose-6-P (G6P), G6P-dehydrogenase (G6PDH) activity, malic and lactic acids, malondialdehyde (MDA), and oxygen radical antioxidant capacity (ORAC). The NEB cows produced 10.5% more milk, and consumed 7.6% less dry matter than the PEB cows. The plasma glucose concentration was greater and β-hydroxybutyrate was lower in the PEB vs. the NEB cows. The average concentrations of milk glucose, G6P, malic and lactic acids, and MDA did not differ between groups; however, the G6PDH activity was higher and ORAC tended to be higher in the milk of NEB vs. the PEB cows. The correlation between milk G6PDH activity and EB was significant (r = -0.39). The percentages of oleic acid and total unsaturated FA in milk were higher for the NEB vs. the PEB cows. These findings indicate that G6PDH activity in milk is associated with NEB and that it can serve as a non-invasive candidate biomarker of NEB in postpartum cows, that should be validated in future studies.

Effect of thyroxine on Na+/K+-ATPase and SDH activities in gills and kidney during osmotic adjustments of Heteropneustes fossilis at higher salinity

Aim: The aim of the present study was to investigate the facilitatory role of thyroxine during adaptation process of catfish, Heteropneustes fossilis in higher salinity following its exogenous administration by monitoring changes in plasma osmolality, branchial and renal sodium-potassium dependent adenosine triphosphatase and succinic dehydrogenase enzyme activity profiles and study the effect of direct transfer of fish to higher salinities on plasma thyroxine levels. Methodology: Catfish were directly transferred from tap water to 30 and 35% sea water and plasma thyroxine profiles were analysed. Fish were injected with thyroxine at a dose of 2 and 5 µg g-1 b.wt. daily for five days and then transferred to tap water and 30% sea water. Fish were sampled after day 3 and day 6, post-transfer and plasma osmolality was measured, and the enzyme activities were determined in gills and kidney. Results: Higher levels of plasma thyroxine were observed after transfer of fish from tap water to higher salinities. Treatment of fish with thyroxine at higher dose (5 µg g-1 b.wt.) showed a significant increase in plasma osmolality in tap water (p<0.05) while the enzymes in gills were found to be significantly higher both in tap water (p<0.05; p<0.01for Na+/K+-ATPase and p<0.001 for SDH) and 30% sea water (p<0.001for Na+/K+-ATPase and p<0.01; p<0.001 for SDH). No significant changes were observed in any of the parameters analysed after exogenous administration of thyroxine at lower dose (2 µg g-1 b.wt.). Interpretation: Thyroxine affects the osmotic adjustment of fish following transfer to higher salinities and its exogenous administration at at a dose of 5 µg g-1 b.wt. may improve the hypoosmoregulatory ability. Key words: Hormonal control, Osmoregulation, Teleost, Thyroid hormones